Frans_M_Everaerts_Isotachophoresis_378342.pdf

Journal of Chromatogaphy Library - Volume 6
ISOTACHOPHORESIS
Theory, Instrumentation and Applications

JOURNAL OF CHROMATOGRAPHY LIBRARY
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by J. SevEik
Instrumental Liquid Chromatography. A Practical Manual on
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Isotachophoresis. Theory, Instrumentation and Applications
by F. M. Everaerts, J. L. Beckers and Th. P. E. M. Verheggen
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by J. F. Lawrence and R. W. Frei
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Journal of Chromatography Library - Volume 6
ISOTACHOPHORESIS
Theory, Instrumentation and Applications
Frans M. Everaerts
Department of Instrumental Analysis, Eindhoven University of Technology,
Eindhoven
Jo L. Beckers
Eijkhagen College, Schaesberg
The0 P.E.M. Verheggen
Department of Instrumental Analysis, Eindhoven University of Technology,
Eindhoven
ELSEVIER SCIENTIFIC PUBLISHING COMPANY
AMSTERDAM - OXFORD - NEW YORK 1976

ELSEVIER SCIENTIFIC PUBLISHING COMPANY
335 Jan van Galenstraat
P.O. Box 211, Amsterdam, The Netherlands
Distributors for the United States and Canada:
ELSEVIER/NORTH-HOLLAND INC.
52, Vanderbilt Avenue
New York, N.Y. 10017
Library of Congress Calaloging in Publication Data
Everaerts, Frans M 1941-
Isotachophoresis : theory, instrumentation, and
applications.
(Journal of chromatography library ; vo 6)
Includes bibliographies and index.
1. Electrophoresis. I. Beckers, Jo L., joint author.
11. Verheggen, Theo P. E. M., joint author. 111. Ti-
tle. TV. Series.
QD79.EaE93 543 ’ .087 7644834
fSBN 0-444-41430-4
Copyright 0 1976 by Elsevier Scientific Publishing Company, Amsterdam
All rights reserved. No part of this publication may be reproduced, stored in a
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mechanical, photocopying, recording, or otherwise, without the prior written
permission of the publisher,
Elsevier Scientific Publishing Company, Jan van Galenstraat 335, Amsterdam
Printed in The Netherlands

Dedicated to Pr0f.Dr.h. A.I.M. Keulemans, for
providing the possibility of developing this analytical
separation technique in his Department of Instrumental
Analysis, University of Technology, Eindhoven.

This Page Intentionally Left Blank

Contents
Preface ..................................................................... XI11
1.Historicalreview ........................................................... 1
Summary ................................................................ 1
1 . Historical review ......................................................... 1
References ............................................................... 4
THEORY
2 . Principles of electrophoretic techniques .......................................... I
Summary ........ ..................................................... 7
2.1.Introduction .......................................................... 7
2.2. Principle of zone electrophoresis ........................................... I
2.3. Principle of moving-boundary electrophoresis ................................. 9
2.4. Principle of isotachophoresis .............................................. 13
2.4.1. Introduction ..................................................... 13
2.4.2. Simplified model for isotachophoresis: ................................. 15
2.4.3. Concentration adaptation ........................................... 18
2.4.4. Some isotachophcrograms ........................................... 20
2.5. Principle of isoelectric focusing ............................................ 23
2.6.Discussicin ............................................................ 24
3.Conceptofmobility .........................................................
Summary ................................................................
3.1.Introduction ..........................................................
3.2. Interpretation of electrophoretic migration ...................................
3.3. Ionic mobility and ionic equivalent conductivity ...............................
3.4. Effective ionic mobility ..................................................
3.4.1. Partial dissociation ................................................
3.4.1.1. Protolysis ..................................... .......
3.4.1.2. Complex formation ..........................................
3.4.2. Relaxation and electrophoretic retardation ..............................
3.5. Determination of ionic mobilities ..........................................
3.5.1. Relationship between volume and ionic mobility .........................
3.5.2. Relationship between entropy and ionic mobility .........................
3.5.3.Discussion .......................................................
References ...............................................................
4 . Mathematical model for isotachophoresis .........................................
Summary ................................................................
4.1.Introduction ..........................................................
4.2. General equations ......................................................
4.2.1. Equilibrium equations ..............................................
4.2.2. Electroneutrality equations ..........................................
4.2.3. Mass balances for all ionic species ...........
4.2.4. Modified Ohm’s law ......................
4.2.5. Parameters and equations ...........................................
4.3. Mathematical model for the steady state in isotachophoresis .....................
4.3.1. Concept of isotachophoretic separation .................................
4.3.2. Mathematical model of isotachophoresis ................................
4.3.2.1. Equilibrium equations .......................................
4.3.2.2. The isotachophoretic condition ................................
21
21
21
21
29
31
32
33
33
36
31
31
39
40
40
41
41
41
43
45
41
48
51
51
55
55
58
58
58

VIII CONTENTS
4.3.2.3. Mass balance of the buffer ....................................
4.3.2.4. Principle of electroneutrality ..................................
4.3.2.5. Modified Ohm’s law ....
4.3.3. Computer program for calculation of the steady state ......................
4.3.3.1. Computation procedure ......................................
4.3.3.2. Iteration procedure .........................................
4.3.3.3. Discussion .....................................
4.4. Validity of the isotachophoretic model ......................................
4.4.1. Introduction .....................................................
4.4.2. Influence of diffusion on the zone boundaries ...........................
4.4.3. Influence of axial and radial temperature differences ......................
4.4.4. Influence of activity coefficients .....................................
4.5. Check of the isotachophoretic model .......................................
References ...............................................................
5 . Choice of electrolyte systems .................................................
Summary ................................................................
5.1.Introduction ..........................................................
5.1.1. General remarks ..................................................
5.2. Choice of the solvent ...................................................
5.2.1. Methanol as a solvent ..............................................
5.2.1.1. Comparative behaviour with water ..............................
5.2.1.2. Determination of pK values in methanolic solutions ................
5.3. Choice of the buffering counter ionic species .................................
5.4. Choice of the pH of the leading electrolyte ...................................
5.5. Choice of the terminating and leading ionic species .............................
5.6. Additions to the electrolyte solutions ...............................
5.6.1. Stabilizers .......................................................
5.6.2. Surface-active chemicals ............................................
5.6.3. Reference materiall~foridentificationandi~e~ifi~atio~~~ calibration of concentrations .......
5.6.4. Spacers and carriers ................................................
5.7.Discussion ............................................................
5.8.Examples ............................................................
References . ..........................................................
INSTRUMENTATION
6 . Detection systems ................................................
Summary ................................................................
6.1. Introduction ...............................
6.1 . 1. Universal detectors ................................................
6.1.2. Specific detectors .......................................
6.1.3. Combinations of universal and specific detectors .........................
6.2. Thermometric recording .................................................
6.2.1. Introduction .....................................................
..................................................
6.2.3. Experimental ..........................................
6.2.4. Resolution ......................................................
6.2.5. Conclusion ............................... ...................
6.3. High-frequency conductivity detection .............. ...................
6.3.1. Introduction .....................................................
6.3.2.Construction .....................................................
6.4. Conductivity detection ......... .................................
6.4.1. Introduction ... .................... ........................
59
60
61
62
62
62
69
69
69
74
75
76
76
81
83
83
83
84
84
87
87
89
92
93
96
99
99
99
99
99
100
100
113
117
117
117
118
118
119
119
119
119
125
126
129
130
130
131
133
133

CONTENTS IX
6.4.2. The d.c. method of resistance determination .............................
6.4.3. The d.c.-a.c. converter .............................................
6.4.4. The a.c. method of resistance determination .............................
6.4.5. Conductivity probe with equiplanar-mounted sensing electrodes .............
6.5. UV absorption meter ....................................................
6.5.1. Introduction .....................................................
6.5.2. Construction of the UV source ...................................
6.5.3. UV detector in combination with a non-modulated UV source ...............
6.5.4. UV detector in combination with a modulated UV source ..................
6.5.5.UVcell .........................................................
6.5.6. Experimental ....................................................
6.6. Additives to the electrolytes ..............................................
6.6.1. Introduction ......................................................
6.6.2. Effect of additives on the electroendosmotic flow .........................
6.6.3. Effect of additives on the micro-sensing electrodes ........................
6.6.4.Additives ........................................................
6.7. Coating of the micro-sensing electrodes ..................................
6.7.1. Introduction .................................................
6.7.2. Experimental ............. ..................................
6.8. Detection limits .......................................................
6.8.1. Introduction .....................................................
6.8.2.Experimental ....................................................
6.9.Conclusion ...........................................................
References ...............................................................
7 . Instrumentation ...........................................................
Summary ................................................................
7.1.Introduction ..........................................................
7.2. Injection systems ...................... .............................
7.2.1. Introduction .....................................................
7.2.2.Four-way tap .....................................................
7.2.3. Six-way valve ............ ....................................
7.2.4. Injection block .................. ..............................
7.2.5. Simplified injection block . . ....................................
7.3. Counter electrode compartments ..........................................
7.3.1. Introduction .....................................................
7.3.2. Cylindrical counter electrode compartment .............................
7.3.3. Counter electrode compartment with flat membrane .......................
7.4.Equipment ...........................................................
7.4.1. Introduction ....................................................
7.4.2. Narrow-bore tube surrounded with a water-jacket .........................
7.4.3. Narrow-bore tube thermostated with an aluminium block ...................
7.4.4. Equipment with high-resolution detectors ...............................
7.5. Counter flow of electrolyte ...............................................
7.5.1. Introduction .....................................................
7.5.2. Counter flow with level regulation .....................................
7.5.3. Counter flow with light-dependent resistor regulation ......................
7.5.4. Counter flow with direct control on the pumping mechanism via the
power supply .....................................................
7.5.5. Counter flow with no regulation ......................................
7.5.6. Counter flow regulated by the cur -stabilized power supply; the
membrane pump ............. ..................................
135
140
143
143
153
153
155
159
161
164
165
171
171
171
174
180
191
191
191
193
193
196
199
201
203
203
203
203
203
204
205
208
211
211
211
213
215
217
217
219
221
224
230
230
231
233
237
238
24 1

X CONTENTS
APPLICATIONS
8.Introduction ...............................................................
Summary ................................................................
8.Introduction ............................................................
9 . Practical aspects ............................................................
Summary ................................................................
9.1.Introduction ..........................................................
9.2. Disturbances caused by hydrogen and hydroxyl ions ............................
9.2.1. Disturbances from the terminator zone in unbuffered systems ...............
9.2.1.1. HI-MI boundary ...........................................
9.2.1.2. MI-MI, boundary ... ....................................
9.2.2. Disturbances from the leading zone in unbuffered systems ..................
9.2.3. Disturbances due to the presence of hydrogen and hydroxyl ions in buffered
systems .........................................................
9.3. Disturbances due to the presence of carbon dioxide ............................
9.4. Enforced isotachophoresis ...............................................
9.4.1. Disc electrophoresis ................................................
9.5. Water as terminator .....................................................
9.6. Purification of the terminator .............................................
9.7. Conversion of data measured with different detectors ...........................
References ...............................................................
10 . Quantitative aspects ........................................................
Summary ...............................................................
10.1.Introduction .........................................................
10.2.Theoretical ..........................................................
10.3. Thermometric measurements ............................................
10.3.1. Reproducibility .................................................
10.3.2. Calibration constant .............................................
10.4. Conductimetric measurements ...........................................
10.4.1. Reproducibility .................................................
10.4.2. Calibration constant .............................................
10.5.Conclusion ...........................................................
References ...............................................................
11 . Separation of cationic species in aqueous solutions ................................
Summary ................................................................
11.1. Separation of cationic species in aqueous solutions using a thermocouple as detector . .
11.1.1. The system WHCl ...............................................
11.1.2. The system WHIO ..............................................
11.1.3. The system WKAC ...............................................
11.1.4. The system WKCAC .............................................
11.1.5. The system WKDIT ..............................................
11.2. Separation of cationic species in water and deuterium oxide using a conductivity
detector (a.c. method) and a UV absorption detector (256 nm) ...................
12 . Separation of anionic species in aqueous solutions .................................
Summary .................................................................
12.1. Separation of anionic species in aqueous solut'ions using a thermometric detector .....
12.1.1. Operational system histidine/histidine hydrochloride (pH 6) ...............
12.1.2. Operational system imidazole/imidazole hydrochloride (pH 7) .............
12.2. Separation of anionic species in aqueous solutions using a conductivity detector (a.c.
method) and a UV absorption detector (256 nm) .............................
12.2.1. Introduction ...................................................
249
249
249
253
253
253
253
253
254
254
257
260
263
264
265
267
268
210
271
273
213
273
214
215
275
215
219
279
280
281
282
283
283
283
285
286
288
289
293
293
295
295
295
295
296
300
300

CONTENTS XI
12.2.2. Applications ........... .....................................
13 . Amino acids, peptides and proteins .......................................
Summary ....................... .....................................
13.1.Amino acids .........................................................
13.1.1. Introduction. ...................................................
13.1.2. Separation at low pH values in aqueous systems ........................
13.1.3. Separation at high pH values in aqueous systems ........................
13.1.4. Separation by use of complex formation ...........................
13.1.5. Separation in aqueous propanal solutions .............................
13.2. Separation of proteins in ampholyte gradients ...............................
13.2.1. Introduction ...................................................
13.2.2. Experimental ...................................................
13.3. Separation of small peptides .............................................
13.3.1. Introduction ...................................................
13.3.2. Experimental ..................................................
References ...............................................................
14 . Separation of nucleotides in aqueous systems .....
............................
Summary ................................................................
14.1.1ntroduction .........................................................
14.2. Separation using a thermometric detector ...................................
14.3. Separation using a conductivity detector (ax . method) and a UV absorption detector
(256 nm) ............................................................
15 . Enzymatic reactions ........................................................
Summary ...................................................... ......
15.1.Introduction .........................................................
15.2. Enzymatic conversion of glucose (fructose) into glucose-6-phosphate (fructosed-
phosphate) with hexokinase from yeast .....................................
15.3. Enzymatic conversion of pyruvate into lactate with lactate dehydrogenase from
pigheart ............................................................
References ...............................................................
301
311
311
311
311
312
312
318
319
322
322
325
335
335
336
336
337
337
337
337
342
347
347
347
16 .Separations in non-aqueous systems ................. ....................... 361
Summary .................................... ................ 361
16.1.Introduction ......................................................... 361
16.2. Separation of anionic species in methanol using a thermometric detector ........... 362
16.3. Separation of cationic species in methanol using a thermometric detector ............ 364
16.3.1. The operational system MHCl ...................................... 365
16.3.2. The operational system MKAC ..................................... 367
16.3.3. The operational system MTMAAC .................................. 373
16.4. Experiments in aqueous methanolic systems using a conductimetric detector
(a.c. method) and W absorption detector (256 nm) ......................... 373
17 . Counter flow of electrolyte ..................................................
Summary ...........................................................
17.1. lntroduction .........................................................
17.2.Experimental .........................................................
375
375
375
378
17.3.Conclusion ..........................................................
References ...............................................................
384
384
APPENDICES
A . Simplified model of moving-boundary electrophoresis for the measurement of effective
mobilities ................................................................ 387
348
355
360

XI1 CONTENTS
A.1.inrroduction ..........................................................
A.2. Model of moving-boundary electrophoresis ..................................
A.2.1. Electroneutrality equations .........................................
~
A.2.2. Modified Ohm’s law ...............................................
A.2.3. Mass balances for all cationic species ...............
A.3. Procedure of computation ...............................................
A.4.Experimental ................................................
A.5.Discussion ............................................................
References ...........................................................
B . Diameter of the narrow-bore tube, applied for separation ...............
C . Literature .........................................................
Symbols and abbreviations .....................................................
Symbols .................................................................
Subscripts ...............................................................
Superscripts ..............................................................
Examples ................................................................
Abbreviations .............................................................
Subjectindex ...............................................................
387
387
388
388
388
389
390
392
394
395
397
409
409
410
411
411
411
413

It is very well known that charged particles move under the influence of an electric
field. Because the final velocity of such particles depends on numerous parameters, many
scientists through several decades have applied ths phenomenon to the characterization
and separation of a variety of charged particles, with a wide range of molecular weights,
both for analytical and preparative purposes.
Because the vital components of electrophoretic equipment need to be made of
insulating materials, in the early days it was a handicap for the further development of
electrophoretic instrumentation that modern insulating materials such as Perspex, PTFE
and fluoroethylene polymer were not available. Moreover, sensitive detection systems
had not been developed, so that the minimum detectable amount was rather high in
comparison with some other separation techniques.
After World War 11, the chemical industry began to show considerable interest in the
development of chromatographic separation techniques for the analysis of hydrocarbons
and other (complex) organic compounds. It may have bezn due to this development that
the materials for the construction of the vital parts of the electrophoretic equipment,
including the detectors, rapidly became available. Moreover, in the same period the
electronics industry also underwent a phenomenal expansion.
Although this book is devoted mainly to isotachophoresis, with which all kind of
charged molecules can be separated (as is shown in the Section Applications), the instrument
described can be used for other types of electrophoretic separations. Three main aspects
of isotachophoresis are covered, in three different sections.
In the first section, the theory of the isotachophoretic separation technique is given,
and other electrophoretic techniques are briefly described. For isotachophoresis, both a
simplified and a more complicated model are given. The latter model results in a computer
program suitable for the qualitative and quantitative interpretation of the analytical results.
In the Section Instrumentation, several detectors and the “isotachophoretic” equip-
ment are described. Also, a means is described of formulating a simplified model rapidly,
because for many problems simplified equipment is adequate. Moreover, not much cheap
equipment is commercially available yet.
In the last section, possible fields of application are considered. Analytical conditions
(so-called operational systems) are presented and results are given in the form of both
automatically recorded isotachopherograms and tables. The data in these tables can be
used for the qualitative interpretation of isotachophoretic analyses. Because all of the
values given were derived directly under the operational conditions considered, they
cannot be used for the calculation of, for example, mobilities at infinite concentration.
All of the isotachophoretic zones have a well defined temperature, pH and composition of
the electrolytes present, and these are constant in a chosen operational system but are
different from each other. For further theoretical approaches, corrections need to be made.
In the Appendices, a method is described for mobility determinations, the influence of
the diameter of the narrow-bore tube is dealt with and a list of relevant papers concerning
isotachophoresis is given.
Each section can be used almost independently by scientists interested in fundamental
aspects, by research groups who intend to construct an instrument and by scientists
whose main interest is in the analytical aspects.

XIV PREFACE
In this book, most of the results given summarize about 12 years research, performed
with a variety of instruments. Not only the authors but also the research students, who
worked in the electrophoresis research group contributed to the development of this
technique. In particular, we thank Ir. M. Geurts, who developed most of the electronic
circuits described, and Ir. F. Mikkers, who helped greatly in the collection of many of the
data presented and in the work with tKe instrument equipped with the UV absorption
detector and the a.c. conductivity detector.
Eindhoven
Schaes berg
Eindhoven
FRANS M. EVERAERTS
JO L. BECKERS
THEO P.E.M. VEI~HEGGEN

Chapter 1
Historical review
SUMMARY
Mens ugitat molem*
1. HISTORICAL REVIEW
In the middle of the nineteenth century, Wiedeman [1,2] and Buff [3] reported on
the phenomenon that charged particles migrate in a solution when an electric field is
applied. Later experiments, carried out by Lodge [4] and Whetham [S, 61 , were the
basis on which Kohlrausch [7] developed a theory of ionic migration. With the equation
that he derived, all electrophoretic principles can be described**, including zone electro-
phoresis, moving-boundary electrophoresis and isotachophoresis.
The discovery by Hardy [8,9] that many biocolloids, such as proteins, show
characteristic mobilities that depend largely on the pH of the electrolyte solution in
which the analysis is performed, greatly stimulated interest in electrophoretic work. The
characterization of such substances on the basis of their electrophoretic properties,
especially the pl points, increased interest in electrophoretic separation techniques.
As an early example, the work of Michaelis [ 101 can be considered. He found that
enzymes can be characterized on the basis of their isoelectric points, measured in
migration experiments performed at various pH values; this work, of course, was carried
out before pure enzymes were available.
Although at first the terms cataphoresis and electrophoresis were introduced in order
to indicate the migration of charged colloidal particles and the term ionophoresis was
reserved for substances of lower molecular weight, nowadays most workers use the term
electrophoresis to describe the migration of charged particles in aqueous and non-aqueous
stabilized and free solutions.
Perhaps owing to the major interest in compounds such as proteins and enzymes, or
because high-resolution detectors had not been developed, most attention was paid to
only one of the basic principles, as already described by Kohlrausch [7] , namely zone
electrophoresis and few reports dealing with the other principles were published. It is a
fact that substances such as proteins need appropriate stabilization by electrolytes, as
discussed in Chapter 13.
It was not until about 1923 that a principle of electrophoresis other than zone
electrophoresis was described. Kendall and Crittenden [ 1 I] succeeded in separating rare
*Motto, University of Technology, Eindhoven.
** In Chapter 2, isoelectric focusing is also briefly described because it is a separation technique that
has many similarities with electrophoretic techniques, although once separated the charged particles
do not migrate if the amphiprotic compounds have reached their isoelectric points (the overall
charge is zero).
1

2 HISTORICAL REVIEW
earth metals and some simple acids by, as they called it, the ‘ion migration method’,
which was, in fact, isotachophoresis.
He stated that the ions not only separate, but also adapt their concentrations to the
concentration of the first zone according to the Kohlrausch [7] regulating function, the
‘beharrliche Funktion’. It was also Kendall [ 12) who considered that it is necessary to
be able to follow the separation in some convenient way and suggested that a coloured
ion, with an effective mobility intermediate between those of the ions of interest, could
be used. The end of the experiment could, by the addition of this coloured ion, easily
be determined without the need for a detector. Kendall also suggested that other
detection methods are possible, e.g., utilizing thermometric and conductivity detectors,
and pointed out that, especially when analyzing metals, spectroscopic detection can easily
be used. Finally, when appropriate, the measurement of the radioactivity can be used to
obtain qualitative and quantitative information. The experiments in which he attempted
to separate 35 Cl from 37Cl, as proposed by Lindemann [ 131 , failed, even when very long
analysis times were used. Other isotopes also could not be separated. The ‘movingboundary
method‘ of MacInnes and Longsworth [ 141 , which was used for the determination
of transport numbers, was also based on the Kohlrausch [7] theory.
For about 10 years, little relevant work was carried out on electrophoretic techniques
other than zone electrophoresis, then in 1942 Martin [ 151 separated chloride, acetate,
aspartate and glutamate by isotachophoresis, which he called ‘displacement electrophoresis’,
because it was so similar to the displacement technique in chromatography.
There was a further gap until 1953, when a paper was published by Longsworth [16],
who realized the importance of Kendall’s work. In a Tiselius moving-boundary apparatus,
he introduced a mixture of cations (Ca2’, Ba” and Mg2+) between two other zones,
called the leading solution and the trailing solution. Once separated, the effective
mobilities decrease on going from the leading solution towards the trailing solution.
Detection via Schlieren scanning patterns showed very clearly the sharpness of the
boundaries between the consecutive zones. Longsworth introduced a counter flow of
electrolyte, because the separation chamber in a Tiselius apparatus is very short, and
adjusted it in such a way that the zones remained in the detection region until they were
separated. He also found that a steady state was reached, once the components were
separated. The importance of the pH of the trailing solution was recognized.
The work of Poulik [17] is important, although he was not aware that he was working
along the lines of the Kohlrausch [7] regulating function. Kaimakov and Fiks [ 181
reported on experiments carried out in an electrophoretic equipment, the separation
chamber being filled with quartz sand so as to eliminate convection problems. The
separation chamber was initially filled with an electrolyte, called an indicator electrolyte,
that was different from the test solutions. Again, their results showed that a decreasing
sequence of mobilities was obtained once the steady Ftate had been reached. They also
used a counter flow of electrolyte. Transport numbers were measured by Kaimakov
[I91 and Konstantinov and Kaimakov [20]. Konstantinov et al. 121,221 extended the
work of Hartley [23] and Gordon and Kay [24].
Konstantinov and Oshurkova [25,26] in 1963 described an analytical application
based on the ‘moving-boundary method’. Their separation chamber was a narrow-bore
tube of I.D. 0.1 mm and a wall thickness of 0.05 mm. Measurements of the refractive
index of the various zones by photographic methods gave a recording of the zone
boundaries.

HISTORICAL REVIEW 3
Independently in 1963, Everaerts [27] started, together with Martin, work that finally
resulted in the appearance of this book on isotachophoresis.
As Martin [ 151 , he used the term ‘displacement electrophoresis’. He performed the
analysis in a narrow-bore tube of Pyrex glass of I.D. 0.5 mm and an O.D. of 0.8 mm.
In order to prevent hydrodynamic flow between the two electrode compartments
through the narrow-bore tube, caused by differences in levels, this tube was filled with
an electrolyte the viscosity of which was increased up to 100 CP by addition of a watersoluble
linear polymer, e.g., hydroxyethylcellulose. This polymer was purified by
shaking it with a mixed-bed ion exchanger. A thermocouple (30-pm copper-25-pm
constantan) was used as the detector. Independently, and unaware of this work, Kaimakov
and Sharkov [28] reported on the use of microthermistors to detect zone boundaries.
In 1964, Ornstein [29] and Davis [30] introduced disc electrophoresis. They placed
a protein mixture between an electrolyte with an anion of low effective mobility and
an electrolyte with an anion with a high effective mobility. Owing to the concentration
phenomenon of isotachophoresis, the proteins are stacked in narrow zones between the
two electrolytes (‘steady-state stacking’). The zones, however, are so narrow that even
a high-resolution detector cannot detect them. Therefore, in the second stage of the
analysis, the principle of zone electrophoresis was used, which allowed every protein to
move at a different velocity. Cross-linked polyacrylamide was used as a stabilizing
medium and as a molecular sieve. The mobilities of the proteins could be controlled,
moreover, by varying the pore size in the gel. Ornstein [29] derived several equations,
with which it was possible to calculate the mobilities and the pH values of the electrolyte
systems.
In 1966, Vesterrnark [31] introduced a new term, ‘cons electrophoresis’, for the
electrophoretic technique that makes use of Kohlrausch’s regulating function [7] .
Vestermark also used the spacer technique.
In 1966, Preetz [32] gave a theoretical treatment of the use of a counter flow of
electrolyte in isotachophoretic systems. In 1967, Preetz and Pfeifer [33] described an
instrument that was specially designed for measurements of potential gradients and ion
concentrations. Preetz also performed analyses in narrow-bore tubes. A further development
was the continuous counter flow equipment described by Preetz and Pfeifer
[34]. Based on work of Everaerts [35] and Martin and Everaerts [36], Verheggen and
Everaerts built an instrument and introduced the technique in Bergstr6m’s department
at the Karolinska Institute, Stockholm, Sweden, in 1968. This was the basis of the
commercial production of isotachophoretic equipment, produced by LKB Produkter AB
in Bromma, Sweden. In 1969, Everaerts and Verheggen introduced the technique at the
Charles University in Prague, Czechoslovakia, in Vacik’s research group.
Up to 1970, several names had been used for similar electrophoretic techniques,
including ion migration method, Kendall [ 121 (1928); moving-boundary method,
MacInnes and Longsworth [ 141 (1932); displacement electrophoresis, Martin [ 151 (1942)
and Everaerts [27] (1964); steady-state stacking, Ornstein [29] (1964); cons electrophoresis,
Vestermark [31] (1966); and ionophoresis, Preetz [32] (1966). Together with
Haglund [37], a group of research workers in the field introduced a new name, based
upon an important phenomenon of the electrophoretic technique, namely the identical
velocities of the sample zones in the steady state: isotacho-electro-phoresis* , or
isotachophoresis for short.
*r
LOO = equal; ~01x0~ = velocity; @peeueorc = to be dragged.

4 HISTORICAL REVIEW
It is very difficult to summarize the individual contributions of the various scientists
to the development of the technique after 1970. In Appendix B, we give an almost
complete list of the relevant papers on the subject. Of all the papers, special note is made
of two, in which new types of operational detector were described, representing land-
marks in the development of isotachophoresis. In 1970, Arlinger and Routs [38]
introduced an operational UV absorption detector, and in 1972, Verheggen et al.
[39] introduced an operational conductivity detector.
REFERENCES
1 G. Wiedeman, Pogg. Ann., 99 (1856) 197.
2 G. Wiedeman, Pogg. Ann., 104 (1858) 166.
3 H. Buff, Ann. Chem. Pharm., 105 (1858) 168.
4 0. Lodge, Brit. Ass. Advan. Sci., Rep., 56 (1886) 389.
5 W.C.D. Whetham, Phil. Trans. Roy. SOC. London, Ser. A, 184 (1893) 337.
6 W.C.D. Whetham,PhiI. Trans. Roy. SOC. London, Ser. A, 186 (1895) 507.
7 F. Kohlrausch,Ann. Phys. (Leipzig), 62 (1897) 209.
8 W.B. Hardy, Proc. Roy. Soc. London. 66 (1900) 110.
9 W.B. Hardy, J. Physiol. (London), 33 (1905) 251.
10 L. Michaelis, Biochem. Z., 16 (1909) 81.
11 J. Kendall and E.D. Crittenden, Proc. Nut. Acad. Sci. US., 9 (1923) 75.
12 J. Kendall, Science, 67 (1928) 163.
13 A. Lindemann, Proc. Roy. Soc., Ser. A, 99 (1921) 102.
14 D.A. MacInnes and L.G. Longsworth, Chem. Rev., 11 (1932) 171.
15 A.J.P. Martin, unpublished results, 1942.
16 L.G. Longsworth, Nut. Bur. Stand. (US.). Circ., No. 524 (1953) 59.
17 M.D. Poulik, Nature (London), 180 (1957) 1477.
18 E.A. Kaimakov, and V.B. Fiks, Rum. J. Phys. Chem., 35 (1961) 873.
19 E.A. Kairnakov, Russ. J. Phys. Chem., 36 (1961) 436.
20 B.P. Konstantinov and E.A. Kaimakov, Rum. J. Phys. Chem., 36 (1962) 437.
21 B.P. Konstantinov, E.A. Kaimakov and N.L. Varshovskaya, Russ. J. Phys. Chem., 36 (1962) 535.
22 B.P. Konstantinov, E.A. Kaimakov and N.L. Varshovskaya, Russ. J. Phys. Chem., 36 (1962) 540.
23 G.S. Hartley, Trans. Faraday SOC., 30 (1934) 648.
24 A.R. Gordon and R.L. Kay, J. Chem. Phys., 21 (1953) 131.
25 B.P. Konstantinov and O.V. Oshurkova, Dokl. Akad. Nauk. SSSR, 148 (1963) 11 10.
26 B.P. Konstantinov and O.V. Oshurkova, Sov. Phys.-Tech. Phys., 11 (1966) 693.
27 F.M. Everaerts, Graduation Rep., University of Technology, Eindhoven, 1964.
28 E.A. Kaimakov and V.I. Sharkov, Russ. J. Phys. Chem., 38 (1964) 893.
29 L. Ornstein, Ann. N. Y. Acad. Sci., 121 (1964) 321.
30 B.J. Davis, Ann. N. Y. Acad. Sci., 121 (1964) 404.
31 A. Vestermark, Cons Electrophoresis: An Experimental Study, unpublished results, 1966.
32 W. Preetz, Tdanta, 13 (1966) 1649.
33 W. Preetz and H.L. Pfeifer, Talanta, 14 (1967) 143.
34 W. Preetz and H.L. Pfeifer, Anal. aim. Acta, 38 (1967) 255.
35 F.M. Everaerts, Thesis, University of Technology, Eindhoven, 1968.
36 A.J.P. Martin and F.M. Everaerts,Anal. Chim. Acta, 38 (1967) 233.
37 H. Haglund, Sci. Tools, 17 (1970) 2.
38 L. Arlinger and R.J. Routs, Sci. Tools, 17 (1970) 21.
39 Th. P.E.M. Verheggen, E.C. van Ballegooijen, C.H. Massen and F.M. Everaerts, J. Chromatogr.,
64 (1972) 185.

THEORY

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Chapter 2
Principles of electrophoretic techniques
SUMMARY
The principles of the four main types of electrophoresis, viz., zone electrophoresis,
moving-boundary electrophoresis, isotachophoresis and isoelectric focusing, are
described and a simplified mathematical model for isotachophoresis is given. The
characteristics of these four main types are compared.
2.1. INTRODUCTION
As already described in the Preface, ionic species will move, under the influence of
an applied electric field, E, with a velocity, v, of
v=mE (2-1)
where m is the effective mobility of the ionic species, which depends on several factors
that will be discussed in Chapter 3. Differences in effective mobilities cause differences
in velocities and, by utilizing, this effect, the ionic species can be separated. Separation
techniques based on this principle are called electrophoretic techniques, which can be
divided into three main types, viz., zone electrophoresis, moving-boundary electrophoresis
and isotachophoresis.
In isoelectric focusing, ionic species are not separated according to differences in
mobilities, differences in PI values determining whether they can be separated. In the
steady state, ionic species do not migrate. Because the ionic species migrate electro-
phoretically in order to attain that steady state, isoelectric focusing can also be
considered to be an electrophoretic technique; hence four main types of electrophoresis
can be distinguished.
In principle, all of these electrophoretic techniques can be carried out in any electro-
phoretic equipment. Such an instrument generally consists of five units, viz., the anode
and cathode compartments, the separation chamber, the injection system and the detector.
In this chapter, the principles of the four main types of electrophoresis are discussed
briefly, the most attention being paid, of course, to isotachophoresis.
2.2. PRINCIPLE OF ZONE ELECTROPHORESIS
For the description of the principle of zone electrophoresis, we shall consider a narrow-
bore tube as the separation chamber, which is connected with the anode and cathode
compartments. The distinguishing feature of all zone electrophoretic systems is that the
whole system (anode and cathode compartments and the separation chamber) is filled
with one electrolyte, the so-called background or supporting electrolyte, which carries the

8 PRINCIPLES OF ELECTROPHORETIC TECHNIQUES
electric current and generally has a buffering capacity. The sample (a mixture of
anionic and cationic species) is introduced into the system in this background
electrolyte. In general, the concentration of this background electrolyte is high
compared with that of the sample ionic species and therefore it provides a constant pH
and voltage gradient in the whole system.
The ionic species of the background electrolyte have a certain effective mobility
and, when an electric current is passed through the system, these ionic species will
migrate with specific velocities, cations migrating towards the cathode and anions
towards the anode. The sample ionic species also migrate under the influence of the
applied electric field, each ionic species having a characteristic velocity, depending on the
conditions chosen. Because of the high concentration of the background electrolyte,
the influence of the sample ionic species on the voltage gradient and pH is negligible and
therefore all sample ionic species migrate with constant velocity in time, resulting in a
flow of ions of the background electrolyte accompanied by a flow of sample ions. The
ionic species of the sample will be separated after some time if the differences in
effective mobilities are sufficiently great. Owing to the diffusion, the peaks are wide
(tailing), and adsorption phenomena can cause further tailing. Often detection is effected
by a specific method, e.g., measurement of the colour of the sample ionic species.
Quantitative information can be obtained by measuring the intensity of the colour due
to the reaction products, while qualitative information (identification) is obtained from
the migration distance. Just as in paper chromatography, for example, here the R, values
can be used for identification in standardised systems, where R, is defined as the
migration distance (Z) of the ionic species in question related to the migration distance of
a standard ionic species:
R, = 'ionic species
'standard
A disadvantage of such a detection method is that the detection takes place after the
separation procedure. In addition to the extra steps required and the long time involved,
disturbances such as diffusion in the various zones often occur.
In Fig.2.1, the separation of a mixture of anionic species A, B and C and a cationic
species D is shown. The background electrolyte consists of an anionic species Q and a
cationic species P. In Fig.2. la, the whole system is filled with the background electrolyte
and the sample is injected. In Fig.2.lb, all ionic species of the sample are separated. The
migration distances are lA, I, I, and I, respectively. The R, values relative to the
anionic species C would be R,(A) =- 'A and R,(B) = IB
'C
In Fig.2.2, the voltage gradients, temperatures and pH values for some zones are shown.
The background electrolyte containing sample ionic species with low effective mobilities
shows a higher voltage gradient over the zone than that for rapid ionic species. This
influence is small for high concentrations of the background electrolyte and nearly
constant pH and voltage gradients can be expected in practice. In this instance, a specific
means of detection must be used (see Chapter 6).
This method can be compared with elution chromatography.

MOVINGBOUNDARY ELECTROPHORESIS
a S
b
0
/ P O \
I
I
I
I
L /a _I I
I I
I
L _ / A
I
J
Fig. 1. Electrophoretic separation of the anionic species A, B and C and -..e cationic species D
along the lines of zone electrophoresis. All compartments are filled with the electrolyte PQ-. The
distances lA, Ig, lc and I,, can be used for the determination of the ionic species A, B, C and D.
(a), Sample injection; (b), all ionic species of the sample are separated.
2.3. PRINClPLE OF MOVING-BOUNDARY ELECTROPHORESIS
S
We shall first consider the separation of anionic species according to the method of
Tiselius. In Fig.2.3a, the anionic species to be separated, mixed with a buffer solution,
fill the lower part of a U-tube while the upper part is filled with the buffer solution. If
an electric current is passed through such a system, the anionic species migrate in the
direction of the anode and, after some time, a partial separation occurs. Two series of
mixed zones are obtained; in front of the original zone are present the zones A and A+ B
and behind the original zone are present B+C and C (see Fig.2.3b), if the effective
mobilities of the ionic species A, B and C decrease in the order mA > mB > mc.
It is also possible to carry out moving-boundary electrophoretic experiments in the
following way. Anionic species can be separated by using a narrow-bore tube as the
separation chamber, connected with anode and cathode compartments. The anode
compartment and narrow-bore tube are filled with an electrolyte, the anionic species of
which is chosen to be more mobile than the anionic species to be separated. The sample
is introduced into the cathode compartment (see Fig.2.4a). The anionic species migrate
towards the anode, and the sample anions can never pass the anionic species of the
leading electrolyte because its effective mobility is higher. The mobilities of the
9

10
I E
l T
I I
PH
PRINCIPLES OF ELECTROPHORETIC TECHNIQUES
Fig.2.2. Electric field strength (E), temperature (T) and pH in the different zones of the zone
electrophoretic separation procedure. Theoretically, the zones show small differences in the electric
field strength and temperature. The dotted lines are exaggerated. No general means of detection can be
applied, e.g., conductimetric or thermometric. X refers to, the position in the separation chamber.
anionic species of the sample differ, however, so that some of them will migrate forward.
Thus a situation as shown in Fig.2.4b will be obtained after some time. Substance A,
which is more mobile than the other substances of the sample, is partially separated from

MOVINGBOUNDARY ELECTROPHORESIS
0
iuf ter
n
0
buffer
A
A+B
A+B+C
r
0
butter
a b
0
buffer
-C
- B+C
Fig.2.3. Separation according to the Tiselius moving-boundary principle. In (a), the lower tube is
filled with a mixture of the anions A, B and C. Specific buffers need to be applied for optimal
separation. In (b), it is shown that zones exist on both the front and rear sides, vii., A, A+B and
A+B+C, and A+BtC, BtC and C, respectively.
A+B+C
B and C. Substance B, mixed with A, forms the second sample zone after the pure A
zone. The third zone contains the mixture At B+C.
This method can be compared with frontal analysis in chromatography. In moving-
boundary electrophoresis, the zones generally contain more ionic species of the sample.
The composition of the sample plays an important role in the determination of the
concentrations, pH values and conductivities of the different zones. This situation
contrasts with that in isotachophoresis, where all of these quantities are independent of
the quantitative composition of the sample. A quantitative description of this method
is given in Section 4.2. As will be clear after the description of isotachophoresis, the first
zone in moving-boundary electrophoresis has a self-correcting effect, so that the first
boundary will be sharp. All other zones are not sharp, although this influence is generally
smaller than in zone electrophoresis. In Appendix A, a method is given with which
effective mobilities can be measured by using moving-boundary electrophoresis. Fig.2.4
shows the temperature, voltage gradients and electrical resistances for the different zones.
All of these quantities show similar relationships.
11

12 PRINCIPLES OF ELECTROPHORETIC TECHNIQUES
1
t-
X
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
L A A+B A+B+ C
I
1 U
I
I
I
t E, 1, R.
Fig.2.4. (a) Separation according to the moving-boundary principle. The sample anions A+B+C are
introduced into the cathode compartment. The separation chamber and the anode compartment
are filled with a leading electrolyte, a suitable choice of counter ion needs to be made, because it
determines the pH at which the analysis is performed. After some time, a partial separation is
obtained, which is shown schematically in (b). The electric field strength Q, electric resistance (R)
and temperature (r) are shown schematically for the different zones.

ISOTACHOPHORESIS 13
2.4. PRINCIPLE OF iSOTACHOPHORESIS
2.4.1. Introduction
We shall consider here the separation of anionic species in narrow-bore tubes. For
the separation of anionic species, the narrow-bore tube and anode compartment are
filled with the so-called leading electrolyte, the anions of which must have a mobility
that is higher than that of any of the sample anionic species. The cations of the leading
electrolyte must have a buffering capacity at the pH at which the analyses will be
performed. The cathode compartment is filled with the terminating electrolyte, the
anions of whch must have a mobility that is lower than that of any of the sample
anionic species. The sample is introduced between the leading and terminating
electrolyte, e.g., by means of a sample tap or a micro-syringe.
#en an electric current is passed through such a system (see Fig.2.5a), a uniform
electric field strength over the sample zone occurs and hence each sample anionic species
will have a different migration velocity according to eqn. 2.1. The sample anionic species
with the highest effective mobility will run forwards and those with lower mobilities will
remain behind. Hence, both in front of and behind the original sample zone, the moving-
boundary procedure results in two series of mixed zones (comparable with the Tiselius
method). In the series of mixed zones, the sample anionic species are arranged in order
of their decreasing effective mobilities (see Fig.2.5b),
The anionic species of the leading electrolyte can never be passed by sample anions,
because its effective mobility is chosen so as to be higher. Similarly, the terminating
anions can never pass the anionic species of the sample. In this way, the sample zones
are sandwiched between the leading and terminating electrolyte. In the mixed zones of
the sample (see Fig.2.5b), the separation continues and, after some time, when the
separation is complete, a series of zones is obtained in which each zone contains only one
anionic species of the sample if no anionic species with identical effective mobilities are
present in the sample. Of course, this series of zones is still sandwiched between leading
and terminating electrolyte (see Fig.2.5~).
The first sample zone contains the anionic species of the sample with the highest
effective mobility, the last zone that with the lowest effective mobility. After this stage,
no further changes to the system occur and a steady state has been reached. In such a
case, we can speak of an isotachophoretic separated system. (Of course, one or more
unmixable ‘mixed zones’, i.e., zones that contain one or more anionic species with
identical effective mobilities, may still be present.) In this state, all of the zones must run
connected together, in contrast to zone electrophoresis, where all zones release. Here the
zones cannot release as there is no background electrolyte that can support the electric
current (a requirement for the solvent is that its self-conductance must be negligible; see
section 5.2 .)* .
*If it is assumed that the zones release, then the concentration of the ionic species at that position will
decrease, the electric field strength will increase (working at a constant current density) and hence the
migration velocity of the ionic species involved will be higher. Therefore, finally these ionic species will
reach the preceding zone.

14 PRINCIPLES OF ELECTROPHORETIC TECHNIQUES
b
0 L
C
, conatant ,,,
Fig.2.5. Separation of a mixture of anions according to the isotachophoretic principle. The sample
A+B+C is introduced between the leading anionic species L and the terminating anionic species T.
A suitable cationic species is chosen as the buffering counter ion. The original conditions are shown
in (a). After some time (b), some mixed zones are obtained according to the moving boundary
principle. Finally (c), all anionic species of the sample are separated and all zones contain only one
anionic species of the sample (‘ideal case’).
For this steady state, all zones must have identical migration velocities, determined
by the migration velocity of the anionic species of the leading electrolyte. Considering
the zones L, A, B, C and T (see Figure 2.5~):
v, = VA = V B = vc = VT
or
m,E, = mAEA = mBEB = m,Ec = mTET (2.4)
Eqn. 2.4 will be called the ‘isotachophoretic condition’ and it is characteristic of
isotachophoretic separations.

ISOTACHOPHORESIS 15
As the anionic species are arranged in order of decreasing effective mobilities*, i.e.,
mL > mA > mg > mc > mT, the electric field strengths increase on the rear side.
Working at a constant current density, the product EI (a measure for the heat production)
also increases on the rear side and therefore the temperatures increase in the preceding
zones. In Figs.2.6~ and 2.6d, the electric field strengths and temperatures are shown for
the zones of Fig.2.6a. In Fig.2.6b, the variation of potential with position in the tube is
shown.
The increase in the voltage gradients in the consecutive zones induces two important
characteristics of isotachophoretic systems.
The first characteristic is the ‘self-correction’ of the zone boundaries. When a zone
has attained the steady state, the boundary will not broaden further, which again is in
contrast to zone electrophoresis, where the peaks are unsharp and broad owing to
adsorption and diffusion phenomena. This effect can easily be understood. If an ion
remains behind in a zone with a higher electric field strength, then it will acquire a hgher
migration velocity according to eqn. 2.1, until it reaches its own zone. If it diffuses into
a preceding zone, where the electric field strength is lower than the value that corresponds
to its velocity, its velocity will decrease and it will be overtaken by its proper zone*. The
second characteristic is the increase in temperature in the preceding zones, and by this
feature the zones can be detected with a thermometric detector.
In order to obtain a better understanding of isotachophoresis, we now give a much
simplified model and subsequently some isotachopherograms, obtained with several
detection systems, are shown and discussed in order to facilitate the understanding of
later chapters.
2.4.2. Simplified model for isotachophoresis
Let us first consider a boundary between two connected zones, containing anionic
species A and B with mA > mB. The influence of diffusion, etc., will be neglected.
Suppose the counter ionic species Q are similar in both zones and have a constant mobility
mQ , all ions are monovalent and fully ionized and the influence of the presence of H and
OH- ions can be neglected. Working at a constant current density, the following equations
can be derived:
(a) According to the principle of electroneutrality, the amounts of positive and
negative ions in both zones must be identical, so
The subscripts indicate the ionic species and the zone, e.g., cA,l represents the concen-
tration of anionic species A in the first zone.
(b) According to the isotachophoretic condition, the zones must have identical
*If not too large pH shifts occur in the consecutive zones considered (see Chapter 9).

16 PRINCIPLES OF ELECTROPHORETIC TECHNIQUES
V
d l T
/
I
Fig.2.6. Graphical representation of potential V (b), electric field strength E (c) and temperature
T (d) for the different zones, moving in the steady state of an isotachophoretic analysis (a). X =
Position in the narrow-bore tube where the analysis is performed; s = position of introduction of
sample.
I
\
X-
X-

ISOTACHOPHORESIS
velocities, so that
v1 =v2
(c) According to Ohm’s law:
I= constant =El hl = E2 h2
and the conductivities of the zones can be written as
+ mQ)
A1 =CA,~~AF+CQ,~~QF=~A,~F(~A
Replacing El lE2 with mBlm, according to eqn. 2.9:
17
(2.10)
(2.1 1)
(2.12)
(2.13)
(2.14)
(2.15)
From eqn 2.15. it can be seen that the concentration of all zones is determined by the
concentration of the leading electrolyte, and depends on the mobilities of the ionic
species concerned.
In Chapter 4, a more accurate model will be derived, with corrections for the influence
of pH, different temperatures in the zones, buffering counter ionic species, the pK values
of the ionic species present, etc.
Although the model here is greatly simplified, it can be stated that the concentrations
in the zones are constant in a given system and that the ionic concentrations decrease to
the rear side. The concentrations do not depend on the composition of the sample. If
the sample is very dilute, then during analyses by other techniques (e.g., zone electro-
phoresis and gas chromatography), the concentrations will be further decreased. In
isotachophoresis, however, the concentration always attains a value fixed by the
composition of the leading electrolyte. Therefore, isotachophoresis is sometimes used
with other techniques in order to concentrate the sample in narrow zones. For example,
in disc electrophoresis, the first stage in the analysis involves concentration of the sample

18 PRINCIPLES OF ELECTROPHORETIC TECHNIQUES
by the so-called ‘stacking electrophoresis’*. The importance of this phenomenon is
clear when it is realized that, because the concentrations in the zones are constant, the
length of a zone (the distance between two differential signals) is a direct measure of
the concentration of the sample ionic species.
2.4.3. Concentration adaptation
If zones migrate, they must have a concentration that is fixed by the preceding zones
according to Ohm’s law, and we call them ‘adapted zones’. The effects on changes in
concentration during an isotachophoretic analysis are shown in Figs.2.7a-2.7f for the
separation of a mixture of anionic species A and B, introduced between the leading
electrolyte L and the terminating electrolyte T.
In Fig.2.7a, the original situation is shown. A mixture of A and B (Ao +Bo) is
introduced between the leading ions L and the terminating ions TI. Of course, the
zone A. +Bo is not adapted to L according Ohm’s law, and nor is zone TI. In Fig.2.7b,
the situation is shown after a certain time, where the leading zone L has migrated over a
certain distance, its concentration remaining constant, however. According to all movingboundary
procedures, a zone containing the anionic species A is formed and the concentration
in this zone Al is adapted to zone L. The mixed zone A+B that has passed the
original boundary is also adapted. But behind the original boundary, the originzl mixture
A. +Bo is present, still not adapted. Behind that zone A. +Bo, a zone B1 is formed
that contains only the anionic species B and this zone is adapted to the zone A. + Bo.
Also, the migrated zone Tz is adapted to zone A. +B,. In Fig.2.7c, the original mixture
A. +B, has disappeared, but now there are two zones B, one adapted to the leading
zone L(Bz) and one still adapted to the non-existing original zone A. + Bo. In Fig.2.7d,
the terminator has passed the original boundary and from this time also a zone T3 exists,
already adapted to the leading zone L. At this moment, three T zones exist, viz., a zone
T3 adapted to the leading zone L, a zone T2 adapted to the non-existing zone A. +Bo
and the original zone TI **. In Fig.2.7e, the same situationisshown, the mixed zone A + B
being much smaller. In Fig.2.7f, the mixed-zone A+B has disappeared, ie., anionic species
A and B are separated. Three T zones still exist, marking the spot where the sample was
introduced.
It is important to understand this procedure, although we shall not take this effect into
account, because it is of no importance at the position of detection, as the original
boundaries do not move and never reach the position of detection. In fact, we can never
detect these zones with electrophoretic equipment, as will be discussed later***. These
three zones do not remain sharp, because the ‘self-correcting’ effect, characteristic of
isotachophoresis, does not occur in these zones.
*It should be noted that the proteins in the ‘stack‘ can be easily denatured, because the conditions
are not ideal for proteins, as indicated in Chapter 13 where the separation of proteins is considered.
**In the separation chamber, all zones are now adapted to the composition of the leading zone,
although mixed zones are still present. If a counter flow of electrolyte (as described in section 7.5.5)
is to be applied, it should be applied at this moment, because the zone of the terminating electrolyte,
which has passed the boundary occupied originally by the sample, has already attained its isotacho-
phoretic velocity. Even if 100% counter flow of electrolyte occurs, neither the sample zone nor the
zone of the terminating electrolyte is flushed back.
***No scanning device has yet been constructed.

ISOTACHOPHORESIS 19
d.t
’.t
I
Fig.2.7. Changes in concentrations for the different zones in an isotachophoretic analysis. The
sample is a mixture of A+B (original concentration A,+B,). The sample is introduced between the
leading electrolyte L and the terminating electrolyte T. Theoretically, three different zones, marking
the terminator concentrations T,, T, and T,, are finally obtained, in addition to the zones of the
sample species to be analyzed. For further explanation, see text. s = position of introduction of sample;
R = increasing electric resistance; X = position in the separation chamber.
X
X-

20 PRINCIPLES OF ELECTROPHORETIC TECHNIQUES
2.4.4. Some isotachopherograms
In order to detect the zones in isotachophoretic separations, several detection methods
can be used, some of which are described in the section Instrumentation (Chapters 6
and 7). In order to understand the isotachopherograms shown in later chapters, some of
them are discussed here, although only a brief description will be given.
The first isotachopherograrn (see Fig.2.8) was obtained by means of a thermocouple.
As explained in section 2.4, the temperatures of the proceeding zones increase and these
temperatures can be measured by means of a thermometric detector, e.g., a thermocouple
(made of 15-pm constantan and 25-pm copper wire). A signal as shown in Fig.2.8 is
obtained. The construction of the thermocouple is described in the section Instrumentation.
In Fig.2.8, the differential of the linear trace is also given. This signal marks the zone
boundaries more clearly. The distance between two differential signal peaks is a measure
of the zone length and hence it is a measure of the amount of the ionic species in that
zone, because the concentration of the ionic species in that zone is constant for a given
operational system. The step heights to be measured on the linear trace of the thermo-
couple signal are a measure of the conductivity in that zone and are also a measure of
the effective mobilities of the ionic species in the zones. Hence the step height can be
used for the identification of the ionic species in the sample.
For recording the isotachopherogram shown in Fig.2.8, a potential recorder with
zero suppression was used, which is advantageous for the accurate determination of the
various step heights, but may confuse the information if one is not familiar with it.
Under the conditions chosen, the step heights hl and h2 are characteristic of the tetra-
methylammonium and the ammonium ion, respectively. It should be noted that hl and
h2 refer to the temperature of the chloride zone, which is also constant under the condi-
tions chosen. The data presented later (Chapters 11 and 12) are referred to the thermo-
couple signal at 0 PA. [Note that, e.g., in gas chromatography, the distances are a measure
of the identification (retention times) and the peak areas are commonly a measure of
the amounts present.]
Fig.2.9 shows an isotachopherogram for the separation of some anions. The experi-
ments were carried out in the operational system at pH 6 (Table 12.1). The analyses
were performed in equipment that is described in section 7.4.4, using the two high-
resolution detectors: a conductimeter (a.c. method)* and a UV absorption detector
(256 nm).
The linear trace from the conductivity detector, as in the linear trace from a thermo-
couple detector, is a measure of the conductivities of the zones. Hence it is a measure of
the effective mobilities of the ionic species in the zones and characterizes the ionic
species. The ‘step heights’ that can be found in the linear trace can be used for the
identification of the various ionic species in a well defined operational system. All of
the step heights, as described in the section Applications (Chapters 8-17), refer to the
conductivity of the zone of the leading electrolyte, which is-adjusted to ‘zero’ with the
electronic device described in Chapter 6 (Fig.6.18). The differential of the linear signal
*For the difference between the as. method, using a conductivity detector, and the d.c. method, using
a potential gradient detector, see Chapter 6.

ISOTACHOPHORESIS 21
t
Fig.2.8. Isotachopherogram of the separation of some cations in the operational system listed in
Table 16.1 (methanol was used as the solvent), obtained by using a thermometric detector. For further
explanation, see text. 1 = H’ (leading ion); 2 = (CHJ,N+; 3 = NH:; 4 = K+; 5 = Na+; 6: Li,; 7 = Mn2+;
8 = Cuz+; 9=CdZ’ (terminating ion). h,=Step height (qualitative information) of the tetramethylammonium
ion; h, = step height of the ammonium ion. These step heights are referred to the
‘temperature’ of the zone of the leading ion and x, and x , are a measure of these quantities.
T = temperature; i = time.
of the conductivity detector is also given in order to mark the zones, as the zone length
is a measure that can be used for quantitative determinations.
.4s the signals from W absorption detector depend on the absorption properties of
the ionic species in the zones (not depending on the conditions of the leading electrolyte),
one cannot always obtain quantitative and qualitative information from whch the ionic
species can be defined. It will be clear that the combination of two high-resolution
detectors will give the maximum amount of information in isotachophoretic separations.

22
4-
__ - .
I 3
i
PRINCIPLES OF ELECTROPHORETIC TECHNIQUES
’7 -
I
0
n-
a , 3
?

ISOELECTRIC FOCUSING 23
The four isotachophoretic separations shown in Fig.2.9. were obtained under identical
conditions, Le., stabilised electric current (70 PA), operational system, thermostating
(22°C) and speed of the recorder chart paper (6 cm/min). The isotachopherograms show
that the step heights in the linear traces from the conductivity and UV absorption
detectors are not influenced by the sample size, that a quantitative determination of the
ionic species is possible and that the mutual influence of the various ionic species is zero.
In Fig.2.9A, the difference in zone lengths is due to the fact that the pyrazole-3,5-
dicarboxylate has a greater electric charge than the acetate at the pH of the operational
system chosen. In Fig.2.9B, it can be seen that both zone lengths increase as the amounts
of the components increase.
2.5. PRINCIPLE OF ISOELECTRIC FOCUSING
Amphiprotic substances (e.g., proteins), which contain acidic and basic groups in their
molecules, have a so-called isoelectric point, pl, which is the pH value at which they have
no net charge. At this pH, they are present mainly in the form of a zwitter-ion. In
solution, with a pH equal to the pl value of the amphiprotic substances, they do not
migrate when they are placed in an electric field. At higher pH, they lose protons and
become negatively charged, so that they will then migrate towards the anode if an
electric field is applied. At lower pH, their net charges will be positive and consequently
they 4 1 migrate towards the cathode.
The basic principle of isoelectric focusing is that a buffer gradient is used such that
the pH in the separation chamber increases from one side to the other, the lowest pH
being obtained at the anode and the highest pH at the cathode. When a mixture of
amphiprotic substances is introduced into such a system, all substances d 1 acquire
different net charges according to their p1 values and hence will have different mobilities.
On applying an electric field across the system, each substance will migrate towards that
position where the pH is equal to its pI value. For example, if a protein is introduced at
a pH higher than its p1 value, it becomes negatively charged, migrates towards the anode,
in which direction the pH decreases, and reaches finally the position where the pH is equal
to its p1 value. At this position, its net charge is zero and its velocity decreases to zero.
By isoelectric focusing, all substances in the sample will be concentrated into narrow
zones. Because plvalues are characteristic of, for instance, proteins, they can be
separated by this means; proteins with pldifferences of 0.02 pH unit have been successfully
separated.
Fig.2.9. Isotachopherogram of the separation of some anions in the operational system at pH 6
(Table 12.1). R =increasing electric resistance; A =increasing UV absorption; ?=time. 1 =Chloride;
2=pyrazole-3,5dicarboxylate; +acetate; 4=glutamate. A, 10 nmole of acetic acid and 10 nmob
of pyrazole-3,5-dicarboxylate injected (note the difference in step length, due to the difference in
charge of the ionic species): B, 20 nmole of acetic acid and 20 nmole of pyrazole-3,5-dicarboxylate
injected; C, 10 nmole of acetic acid and 20 nmole of pyrazole-3,5dicarboxylate injected; D, 20 nmole
of acetic acid and 10 nmole of pyrazole-3,5-dicarboxylate injected. A conductimetric (ax. method)
and a UV absorption (256 nm) detector were used. The step heights are constant (qualitative informa-
tion), while the distance between the peaks (= length of the corresponding step) varies (quantitative
information).

24 PRINCIPLES OF ELECTROPHORETIC TECHNIQUES
a
0
s
pH IffCREASlNO
4 5 6 7 8
L
c
pH INCRPASINO
Fig.2.10. Separation of the amphiprotic substances A, B and C with PI values of 4, 6 and 8,
respectively, by isoelectric focusing. The amphiprotic substances are eventually concentrated into
narrow zones where the pH of the buffer gradient is equal to the PI value of the amphiprotic substance
in each instance. (a), Sample introduction; (b), separation of A, B and C. s = position of introduction
of sample.
In Fig.2.10, ths situation is shown for the separation of three substances with pf
values of 4,6 and 8, respectively. The substances A, B and C are introduced at a pH of 6,
Le. the net charge of A is negative (pH is higher than its pZ value), the net charge of B
is zero (pH = pf) and the net charge of C is positive (pH is lower than its pf value).
Substance A will migrate towards the anode until it reaches a pH of 4, substance B stays
at the position where pH = 6 and substance C migrates towards the cathode until it
reaches a pH of 8. After a certain time, A, B and C are separated and concentrated into
narrow zones with pH values of 4,6 and 8, respectively.
Further information on equipment, performances, carrier ampholytes, etc. is given in
detad in the literature.
2.6. DISCUSSION
In the preceding sections, the four main types of electrophoresis have been described,
and in Fig.2.11 their characteristics are summarized. In each instance a sample is
introduced (at an injection point X) that consists of two anionic species B and C and one

DISCUSSION 25
Fig.2.11. Survey of the four main electrophoretic techniques: (a) zone electrophoresis; (b) moving-
boundary electrophoresis; (c) isotachophoresis; (d) isoelectric focusing. X indicates the position where
the sample is usually introduced. For further information, see text.
cationic species D. h Fig.2.11 a, the situation in a zone electrophoretic system after a
certain time is shown. The background electrolyte AE is present in the whole system,
and the anionic species B and C have migrated in the direction of the anode. Anionic
species B, which has a higher mobility, has covered a greater migration length. Cationic

26 PRINCIPLES OF ELECTROPHORETIC TECHNIQUES
species D has migrated in the direction of the cathode. Fig.2.1 lb shows the moving-
boundary procedure. The sample mixture is introduced into the cathode compartment
and the leading electrolyte AE fills the separation chamber and the anode compartment.
The cationic species D thus remains in the cathode compartment and the anionic species
B and C, partially separated, migrate behind the leading electrolyte AE. Note that the
pure zone B and the mixed zone B+C have the counter ionic species of the leading
electrolyte E. The first boundary (with a velocity V,) is sharp (according to the isotacho-
phoretic condition), whereas the separation boundary (with a velocity V*) is not sharp,
as in zone electrophoresis. The zone velocities v* and V1 are different. Fig.2.1 lc shows
the separation procedure for an isotachophoretic system. The sample is introduced
between the leading electrolyte AE and the terminating solution TE. The anionic species
B and C are separated and migrate between the terminator T and the leading ion A. All
zones have equal velocities, and contain the same counter ionic species E. The cationic
species D of the sample has migrated to the cathode. In Fig.2.1 Id, the sample is intro-
duced in an isoelectric focusing system. The point of injection is not important. The ionic
species are eventually separated according to their plvalues and are concentrated on
spots where the pH values are identical with their pl values, their net charges and
velocities then being zero. B and C have migrated towards the anode (lower pH) while
the cationic species D has migrated towards the cathode (higher pH). Only amphiprotic
substances can be separated by this method.

Chapter 3
Concept of mobility
SUMMARY
Electrophoretic migration is discussed and the ionic mobility is defined. The relation-
ship between equivalent conductance and ionic mobility is shown, the concept of
effective mobility is described and the influence of partial dissociation, relaxation and
the retardation effect on the effective mobility is discussed. Some approaches are
suggested for the determination of unknown mobilities.
3.1. INTRODUCTION
The concept of mobility plays an important role in electrophoretic techniques, as
differences in effective mobilities determine whether or not ionic species can be separated.
The concentrations and the voltage gradients of the different zones, related to the
parameters of the leading zone, are also fixed by the effective mobilities. In this chapter,
the concept of mobility is discussed.
We do not intend to give here a complete survey of all of the mathematical theories
proposed and experiments carried out on this subject, which have been described in
various papers. We will consider mobility only so far as is necessary in order to understand
and use the theory of electrophoresis. Further, some approaches will be given with which
unknown ionic mobilities can be estimated by relationships with other parameters.
3.2. INTERPRETATION OF ELECTROPHORETIC MIGRATION
If an electric field is applied to an electrolyte solution, charged particles will move
and a stationary state will be reached in which the velocity of the particles, in the
direction of the field, is constant with time. In this state, there are four different forces
acting on a particle, called FI, F2, F3 and F4 (see Fig.3.1).
F1 is a force exerted on the charge of the particle and can be denoted by
F, =qE (3.1)
where E is the electric field strength.
F2 is a friction force, which Stokes determined for a rigid spherical particle as
F2 = -f,v = -6nqrv (3 -2)
where v is the electrophoretic velocity, r is the radius of the particle, q is the viscosity of
the solvent andf, is the friction factor. For a non-spherical particle,f, is still propor-
tional to Q, but a correction factor has to be introduced so as to allow for-the sizeTshape
and orientation of the particle.
21

28 CONCEPT OF MOBILITY
Fig.3.1. Forces acting on a positively charged particle, which moves under an electric field, E, can be
represented by F, , Fz , F3 and FA. The originally symmetrical, in this instance negatively charged,
ionic atmosphere(1) is shifted due to the electric field E(2). For further explanation, see text.
The forces F3 and F4 are due to the presence of oppositely charged particles,
forming a so-called ionic atmosphere. For F3, the electric field exerts a force on the
ions of the ionic atmosphere, which is transferred to the molecules of the solvent. The
particles considered do not move through a stationary solvent, but through a solvent
flowing in the opposite direction, so that the net velocity is decreased. This effect is
called the ‘electrophoretic retardation’. F4 represents the ‘relaxation effect’. The
distribution of ions in the vicinity of the particles is deformed when an electric field is
applied, because the particles move away from the centre of the ionic atmosphere. The
Coulomb forces between the ions tend to re-build the ‘atmosphere’ in its ‘proper’ place,
which takes a finite time called the relaxation time. Hence, the centre of the ionic
atmosphere of the particle constantly lags behind the centre of the particle in the
stationary state, resulting in an electrical force on the charge of the particle. This force
is called the relaxation effect.
For the stationary state, the sum of these forces must be zero, so that
FI +F2 +F3 +F4 =O (3 -3)
01
or
From eqn. 3.5, the influence of electrophoretic retardation, the relaxation effect, the
shape, charge and radius of the particle and the influence of the solvent on the electro-
phoretic migration velocity can be understood. In the next section, the relationship
between ionic conductance and migration velocity is considered and the absolute ionic
mobility is defined.

IONIC MOBILITY AND IONIC EQUIVALENT CONDUCTIVITY
3.3. IONIC MOBIUTY AND IONIC EQUIVALENT CONDUCTIVITY
We speak of an 'equivalent weight' of an electrolyte if, for complete dissociation,
the total amounts of positive and negative charges are eN and -eN respectively, where
N is Avogadro's number and e is the electronic charge. For example, one equivalent
weight of potassium fluoride gives, for complete dissociation, one Avogadro's number
of K’ and of F ions.
The conductance of such an amount of electrolyte is the conductance measured in a
conductance cell with electrodes 1 cm apart and with such cross-sections that the volume
of solution between the electrodes will contain exactly one equivalent of the electrolyte.
This conductance is known as the 'equivalent conductance' and is denoted by A*.
Kohlrausch showed that at a fned temperature the relationship between the equivalent
conductance of an electrolyte and the square root of the concentration is nearly linear,
especially for very low concentrations and strong electrolytes. At infinite dilution, the
equivalent conductances can be interpreted in terms of ionic contributions, whereby the
contribution of an ion is independent of the other ionic species of the electrolyte (the
influence of retardation and relaxation effects can be neglected, as no ionic atmosphere is
present at infinite dilution). At infinite dilution, we can therefore write
A: = Ax’ + Ax- (3.6)
where Ax’ and Ax- are the equivalent ionic conductivities of the anions and cations,
respectively, and A: is the equivalent conductance, all at infinite dilution.
If a voltage V is applied to a cell as mentioned above (see Fig.3.2) a current I flows
through the cell:
I= V/R or I= VA* (3.7)
Assuming that such a cell contains one equivalent of the electrolyte, N/z' positive and
N/z- negative ions are present, where z’ and z- are the valences of the positive and
negative ions, respectively. If the velocities of the ions are represented by v’ and v-,
respectively, the positive ions present in volume B and the negative ions present in
volume C (see Fig.3.2) will have passed the cross-section A in 1 sec. Because the cell is
I cm in length, this means that the volumes B and C will contain v+/l and v-/l parts of
the total amount of the positive and negative ions of the cell, whch is
v’(N/z’) positive ions and v-(N/z-) negative ions (3.9)
The currents corresponding to these flow rates are obtained by multiplying by the ionic
charges ez' and ez- and by this:
r' = ez' v’ (IV/z+) = e Nu+ = Fv'
r = ez- v- (N/z-) = eNv- = Fv-
At infinite dilution, combination of eqns. 3.8 and 3.10 gives
29
(3.10a)
(3. lob)

30 CONCEPT OF MOBILITY
Fig.3.2. Conductance cell with electrodes 1 cm apart. For further explanation, see text.
The average velocity with which an ion moves under the influence of a potential of
1 V is called the ionic mobility, and the ionic mobility at infinite dilution is called the
absolute ionic mobility. Thus,
(3.1 la)
(3.1 Ib)
(3.12)
It can be concluded from eqn. 3.12 that absolute ionic mobilities can be calculated by
dividing the equivalent ionic conductivities at infinite dilution by the Faraday constant.
The equivalent ionic conductivities can be obtained measuring the transport numbers.
As the transport numbers give the fractions of the total current carried by each ion,
ie., the fraction of the total conductance that each ion contributes, we can write
hg+ = t,'A,* (3.13a)
and
A,*- = ti A: (3.13b)
where t = transport number. Data for conductances and transport numbers in order to

EFFECTIVE IONIC MOBILITY 31
obtain A*" and A*- for the calculations of the ionic mobilities at concentrations other
than infinite dilution cannot be properly used, because the law of independent
migration of the ions is invalid and the conductance is really a property of the electrolyte
rather than of the individual ions of the electrolyte. This means that the ionic conduc-
tivities (and hence ionic mobilities) of a chloride ion in 1 N calcium chloride solution
and in 1 N sodium chloride solution are different.
In such instances a correction must be made for the influence of relaxation and
retardation effects and for incomplete dissociation (ion pair formation). Also, for
"weak" electrolytes it is sometimes very difficult to obtain correct values for the
equivalent ionic conductances at zero concentration (infinite dilution). For such
solutions, we can calculate the correct values from the ionic contributions of strong
electrolytes at infinite dilution. For example:
A,*(HAc) = A,*(NaAc) + A: (Ha)- A,* (NaCl)
because the right-hand side can be interpreted as
AX+ (N2) + A:- (Ac- ) + Ag"(H) + A:- (GI-) -Ax' (Na') -A,*- (a- )
= Ar(H+) + A:- (Ac-) = A;f' (HAc)
(3.14)
(3.15)
This procedure is not valid at concentrations other than zero, but in practice it can be
used in order to obtain conductivities and mobilities at concentrations other than zero.
In fact, corrections for the differences in relaxation and retardation effects and ion pair
formation in electrolytes are neglected and it can be used only as a rough approximation.
3.4. EFFECTIVE IONIC MOBILITY
The absolute ionic mobility, m:, is defined as the average velocity of an ion per unit
of electric field strength at infinite dilution. This absolute ionic mobility is a characteristic
constant for every ionic species in a certain solvent and is proportional to the equivalent
conductance at zero concentration:
A,*=AE'fX:- =(m,'+m;)F (3.16)
In practice, we are not working at infinite dilution and the influence of other ionic species
present in an electrolyte solution cannot be neglected. The effective mobility of an ionic
species is related to the absolute mobility. Corrections have to be made for influences
such as the electrophoretic retardation and the relaxation effect, as described by Onsager
(see ref. 1). By using the Onsager equation, a correction can be made for ion-ion
interactions. Another influence is the effect of partial dissociation. Tiselius [2] pointed
out that the effective mobility is the sum of all products of the degree of dissociation and
the ionic mobilities:
meff.= 7 aimi (3.17)
where meff. is the effective mobility, ai is the degree of dissociation and mi is the ionic
mobility.

32 CONCEPT OF MOBILITY
To summarize, we can state that the effective mobility of an ionic species depends
on several factors such as the ionic radius, solvation, dielectric constant and viscosity of
the solvent, shape and charge of the ion, pH, degree of dissociation and temperature. It is
very difficult to give a precise mathematical expression for the effective mobility. When
speaking about effective mobilities, we shall use the expression
meff. = C cui rimi
i
where cti is the degree of dissociation, yi is a correction factor for the influence of
relaxation and retardation effects and mi is the absolute ionic mobility. The correction
factors c+ and yi will be described in more detail.
3.4.1. Partial dissociation
(3.18)
If an ion does not exist in the free form, but is in an equilibrium with the undissociated
form, its effective mobility is smaller than its ionic mobility. For example, acetate, in
water, is always in equilibrium with acetic acid according to the equation
HAc + Hz 0 * H3 O+ + Ac-
and the equilibrium constant is
As the degree of dissociation is defined as
cu=
[Ac-]
[HAc] + [Ac-]
then, during time t, the ionic species exists in the form of acetate during time cut.
Therefore, the migration distance in time f is
(3.19)
(3.20)
(3.2 1)
s=vat = cumEt (3.22)
Normally, the migration distance of an ion is
s =v t = m Et (3.23)
From eqns. 3.22 and 3.23, it can be concluded that the effective mobility can be
calculated as
meR. =am (a < 1)
Tiselius [2] pointed out that a substance consisting of several forms with different
mobilities in equilibrium with each other will generally migrate as a uniform substance
with an effective mobility of
meff.= F aimi
(see eqn. 3.17) provided that the time of existance of each ionic species is small in

EFFECTIVE IONIC MOBILITY 33
comparison with the duration of the experiment. As the equilibrium adjustments are
very slow, the ionic species seems to consist of two components (an example is the
esterification of oxalic acid in methanol, see section 16.2, Fig.16.1) and sometimes
disturbances can be expected (see Chapter 9).
For the equilibrium states, we shall distinguish two types of interactions, viz.,
protolysis and complex formation.
3.4.1.1. Protolysis
Here a proton takes part in the dissociation reaction, as shown in the dissociation of
acetic acid (see eqn. 3.19). The degree of dissociation depends on the pH and the
equilibrium constant.
The relationship between pH and pKa and the degree of dissociation is given by the
Henderson-Hasselbalch equation:
(3.24)
(positive for anionic species and negative for cationic species). Also, for ionic species
with more than one pK value, thls equation can be used if the differences between the
pK values are not too small. In Fig.3.3, a nomogram is given by which the degree of
dissociation can be obtained for given pH and pK values.
The relationship between the degree of dissociation and the pH for some anionic
species is shown in Fig.3.4, from which it can be concluded that changes in pH are
important between k 2 pH units from the pK value. Between these values, the degree
of dissociation changes from about 1% to 99%, and hence the effective mobility changes
from 1% to 99% of the absolute ionic mobility, neglecting other influences on the
absolute ionic mobility.
In Fig.3.5, some relationships between effective mobility and pH are shown for
anionic species, cationic species and amphiprotic substances. Of course, the mobilities
depend on the absolute ionic mobility chosen. Fig.3.5 shows clearly that differences
in pH have a great effect on the effective mobility near the pK values.
3.4.1.2. Complex formation
Now a particle different from a proton takes part in the dissociation reaction, e.g.
Pb(CH3C00)2 =+ Pb(CHjCOO)++ CH3COO- (3.25)
The degree of complex formation depends mainly on the partial concentrations and the
complex constant. Corrections can be made for this effect in a manner similar to that
described above. Often, both types affect the effective mobility, e.g., for Al*:
Al(H20)F* Al(OH)(H,O)T+ H+
AI(CH3COO)3 =+ Al(CH3COO)T + CH3COO-
Further dissociations are possible.
(3.26)
(3.27)

34 CONCEPT OF MOBILITY
14
13
12
-
11
10
9
8
7
6
5
4
3
2
1
0
pKa
PH
Fig.3.3. Nomogram giving the relationship between pH, pKa and the rate of dissociation (a).
C
A
T
I
0
N

EFFECTIVE IONIC MOBILITY
Fig.3.4. Relationship between CY and pH for some anionic species.
Fig.3.5. Relationship between the effective mobility, meff., and pH. (a) Cationic species with an
absolute ionic mobility m, = 40 and pK = 3; (b) cationic species with ma = 20 and pK = 3; (c) cationic
species with ma=40 and pK =5; (d) anionic species with ma = 40 and pK = 10. (e) amphiprotic
compound with tn; = 30 and mi = 30 and pK = 3 and 11.
If the dielectric constant decreases, the interionic forces increase. This effect,
especially for cationic species with hgh charges, results in stronger complex formation.
The pK values of the dissociation also depend on the dielectric constant of the solvent.
35

36 CONCEPT OF MOBILITY
3.4.2. Relaxation and electrophoretic retardation
as
According to Hiickel, Debye and Onsager (see ref. I), the conductance can be written
A= '0 - Arel. - 4et. (3.28)
where A,,. and he, are corrections for the decreasing effects on the conductivity due
to the relaxation and retardation effects, respectively.
s= ___ lz+l iz- I - A: + A;
IZ+l+ lz- I 1.2- I A: + 12' I A,
Substitution of eqns. 3.29, 3.30, 3.31 and 3.32 into eqn. 3.28 gives for the molar
conductivity
A=A~-~&
with
In a similar way [ 11, for the equivalent conductance we can derive
A* = A* 0 -a* ,/ (k+I + 12- I)c*
with
(3.29)
(3.30)
(3.31)
(3.32)
(3.33)
(3.34)
(3.3 5)
(3.36)
(3.37)
In order to show the importance of the influences for different solvents and for different
charges of the ions, we calculated the effective mobilities according to these expressions
for monovalent and divalent cations in water and methanol for a hypothetical value
of the absolute mobility of 50 - lo-' cm*/V - s at a concentration of 0.01 N. The results
are shown in Table 3.1.

DETERMINATION OF IONIC MOBILITIES 37
TABLE 3.1
TIIEORETICAL EFFECTIVE MOBILITIIiS OF MONO- AND DIVALENT CATIONS IN WATER
AND METHANOL (9570, w/w)
In both instances the counter ions are monovalent.
Water Methanol
105 meff.-1O5 m,.105 ineff; lo5
Univalent cations and anions 50 46 50 31.5
Divalent cations and univalent anions 50 43 50 25
For water as solvent at 25°C:
For methanol as solvent at 25°C:
(3.38)
2s
(Y * = 1.1 5 ___ - (z' z- 1 A8 + 55.3 (IZ’ I + 1z-I) (3.39)
1 +&
The effects discussed above are even stronger for solvents with lower dielectric
constants and for cations with higher charges.
3.5. DETERMINATION OF IONIC MOBILITIES
As already mentioned in preceding sections, ionic mobilities can be calculated from
equivalent conductivities and corrections can be made for the influence of concentration,
relaxation and retardation effects. As the exact data for many ionic species are unknown,
many workers have sought correlations between ionic mobilities and parame ten such as
the radius of the molecule, ionic volume and the entropy of the ions. Some of these
approaches are considered here and may be useful in estimating unknown mobilities.
3.5.1. Relationship between volume and ionic mobility
In general, it is said that for a 'steady flow' of molecules, Stokes' law can bc applied in
order to calculate the resistance force (assuming a spherical particle in an infinite fluid
[3]). If the ionic radius is not too small, the following equations can be deduced [4-61 :
v * = (qE+F,,, +Fre,.)/fc
(see eqn. 3.5) so that at infinite dilution
vd =4Elfc
mi = v d/E= q/fc =z'e/6nqr
For smaller particles (3-5 A) [7, 81, a modified expression can be used, viz. :
(3.40)
(3.41)
mi= z'el [5nvrCf/fo)] (3.42)

38 CONCEPT OF MOBILITY
where flfo is a correction factor for non-spherical particles. For water (21 C), this means
that
mf= 1.14. 10-3~f/[TCflf~)] (3.43)
From this equation, it can be concluded that the ionic mobility is a function of the shape,
charge and radius of the ion and the viscosity of the solvent. Edward [7] and Bondi [9]
calculated the contribution of different groups in a molecule to the volume of the
molecule (and hence to the radius) from the covalent radius according to Pauling and
the Van der Wads’ radii and angles [ 101 .
Perrin [ 111 derived equations for friction factors from the ratio of the axes of prolate
and oblate ellipsoids. Edward and Waldron-Edward [12] showed the possibility of
calculating friction factors from diffusion constants.
In the papers mentioned, reasonable results were obtained for the calculated values
in comparison with the experimental values, deviations being found for small ions and
strongly polar groups. Values for non-spherical and nonellipsoid ions, such as the
“knobby shape” ions, can also be calculated. Very irregular ions cannot be treated
Fig.3.6. Relationship between entropy (S) and ionic mobility (m) for some cations (a) and anions (b).
The values correspond to those given in Table 3.2.

DETERMINATION OF IONIC MOBILITIES 39
TABLE 3.2
IONIC MOBILITY AND ENTROPY OF IONIC SPECIES
~ ~~~~~
Ionic species m - lo5 S Ionic species m lo' S
NH:
cs+
Li+
K+
Ag+
Tl+
Na+
Rb+
HCO;
10;
HC, 0;
HSO;
HSO;
CHOO-
BrO;
Cl0;
Cl0;
NO;
74
78
38.7
73.5
62
76
50.5
76.5
44.5
41
40.2
50
50
54.6
56
65
68
71.5
27
31.8
3.4
24.5
17.7
30.4
14.4
29.1
22.7
27.7
36.7
31.6
30.3
21.9
40.9
39
43.5
35
Ba2+
CdZ+
Ca'+
co2+
Ni"
cuz+
Fez+
Znz+
Pb"
Mgl+
sr2+
Mn'+
Br-
c1-
F-
I-
CN-
63.8
54
59.3
51
50.5
54.5
54
54
70-73
53
60
52
78.4
76.5
54.7
77
78
because their friction factors are unknown and, in general, they have lower mobilities
than spherical ions of the same volume.
3.5.2. Relationship between entropy and ionic mobility
3
-14.8
-13.2
-37.1
-38.2
-23.6
-27.1
-25.5
5.1
-28.2
-9.4
-20
19.2
13.2
-2.3
26.1
28.2
Zolotarev [13] tried to relate entropy to ionic mobility in aqueous solutions.
Combination of the equations derived by Kapustinskii [ 141 :
s= (A/r)+B (3.44)
and
m f = z'e/6n qr
(see eqn. 3.41) gives
S=(K1 mf)+Kz (3.45)
This equation is valid for a series of equally charged substances.
In Table 3.2, the entropies and absolute ionic mobilities of some ionic species are
given. The relationship between entropy and mobility is shown graphically in Fig.3.6.
In Fig.3.6a, the relationship for mono- and divalent cations can be seen to be linear,
according to eqn. 3.45. A similar relationship for anionic species, however, is less evident
(see Fig.3.6b).

40 CONCEPT OF MOBILITY
3.5.3. Discussion
In this section, some approaches for the estimation of ionic mobilities have been
considered. A different approach was used by Lindemann [ 1 51, based on the kinetic
theory of gases, in which the ions are supposed to suffer repeated collisions with solvent
molecules, at each of which it retains a certain fraction of its velocity depending on the
relative mass. Between collisions, it moves freely under the influence of the electric field.
A relationship is thus established between the mobility of the ion and its mean free path
between collisions.
Although some effects that affect the effective mobility can be described mathematically
and although the ionic mobilities can be treated theoretically from data such as entropy
and Stokes' law, the estimation of mobilities in practice is difficult. The mathematical
models cannot be applied to all ionic species and specific interactions give differences
between experimental and theoretical values, especially when non-aqueous solvents are
used. Experimental measurements often have to be carried out for the determination of
the ionic mobilities.
REFERENCES
1 H. Falkenhagen, Elektrolyte, Verlag von S. Hirzel, Leipzig, 1932.
2 A. Tisellus, Nova Acta Regiae soc. Sci. Upsal., Ser. 4, 4 (1930) 7.
3 J.T. Edward, Advan. Chrornutogr., 2 (1 966) 64.
4 P.V. Chengand H.K. Schachrnan, J. Polym. Sci., 16 (1955) 19.
5 W. Weidel and E. Kellenberger, Biochim Biophys. Acru, 17 (1955) 1.
6 J.T. Edward, J. Polym. Sci., 25 (1957) 483.
7 J.T. Edward, Chcm Ind (London), (1956) 714.
8 J.T. Edward, Sci. Proc. Rqv. Dublin Soc., 9 (1956) 273.
9 A. Bondi,J. Phys. Chem, 68 (1964) 441.
10 L. Pauling, Nature sf'thc Chtmical Bond, Cornell Univ. Press, Ithaca, N.Y., 2nd. ed., 1948.
11 F. Perrin, J. Phys. Radium, 7 (1936) 1; 5 (1934) 497.
12 J.T. Edward and D. Waldran-Edward, J. Chromarogr., 24 (1966) 125.
13 E.K. Zolotarev, Russ. J. Phys. Chcnz., 39 (1965) 573.
14 A.F. Kapustinskii, Acfu Physicochim. URSS, 14 (1941) 508.
15 F.A. Lindemann, 2. Phys. Chem (Leipzig], 11 0 (1924) 394.

Chapter 4
Mathematical model for isotachophoresis
SUMMARY
In isotachophoresis, as already described in Chapter 2, the sample, whch is a mixture
of anionic and cationic species, is introduced between a leading electrolyte and a
terminating electrolyte. For the separation of anionic species, the leading anionic species,
A,, is chosen such that its effective mobility is higher than those of all other anionic
species, whereas the terminating anionic species, A,, is chosen with a mobility lower
than those of all other anionic species. As an electric current is passed through such a
system (see Fig.4.1), in the first instance all ionic species will migrate with a velocity
determined by, e.g., the actual pH, ionic strength, the absolute mobility and the potential
gradient.
In fact, this first stace in the electrophoretic separation procedure is a moving-
boundary separation. After this stage, in which the anionic species of the sample are to be
separated according to differences in effective mobilities, a ‘steady state’ will be reached
in which all zones migrate with a velocity equal to that of the leading anionic species.
Each zone will contain only one anionic species.
Only in this case can we speak of isotachophoresis proper*. The first sample zone
contains the sample anionic species with the highest mobility, and the last zone that with
the lowest mobility.
In section 4.2, the general equations for electrophoretic separations are derived and
applied to a model of moving-boundary electrophoresis. In section 4.3, these equations
are applied to a model of isotachophoresis, with which all quantities, such as concentra-
tions, conductivities of the zones and pH values of the zones, can be calculated. The
model is subsequently verified (section 4.4).
4.1. INTRODUCTION
Experiments based on the principle of electrophoresis have been carried out for many
years and theoretical models have already been described by several workers [l-171.
In 1897, Kohlrausch [ 121 gave a mathematical model for electrophoretic processes.
Using the divergence theorem, the continuity equations can be derived and, using the
principle of electroneutrality and assumptions such as constant relative mobilities, he
formulated the so-called ‘beharrliche funktion’:
C. 4 e=
constant
*Although ideal mixed zones can always be present, they do not influence the other zones.
41

42
0
MATHEMATICAL MODEL FOR ISOTACHOPHORESIS
Fig.4.1. The original situation for the separation of anionic species. A mixture of anionic and
cationic species has been introduced between a leading electrolyte and a terminating electrolyte.
AL, leading anionic species; A, . . Ir sample anionic species; AT, terminating anionic species; BL,
buffering counter ionic species of the leading electrolyte; B, . . ,., counter ionic species of the sample;
BT, counter ionic species of the terminating electrolyte.
This regulating function prescribed that at any point the sum of concentrations divided
by the mobilities must be constant for all ionic species. In practice, all theoretical models,
which have been described in several papers, are essentially based on this principle,
although the situation is complicated by the use of different names and approaches. Also,
often no clear distinction has been made between the different performances of the
electrophoretic separations.
In this chapter, a theoretical model is described for isotachophoretic separations and
a number of experiments are described with which the model was verifie'd. A clear
distinction is made between the first stage of the separation by isotachophoresis, which
can be compared with moving-boundary electrophoresis, and isotachophoresis proper, i.e.,
at the steady state if all ionic species of the sample are separated.
For a model as general as possible, all substances will be regarded as amphiprotic
polyvalent molecules, so that the molecules can contain different chemical groups with
different chemical equilibrium constants. For such a molecule, the following equilibria
can be set up:
(In this model, only proton interactions are taken into account. Equilibria referring to other
dissociations, complex formation etc. are neglected.) The symbol A represents an anionic
species and the subscript r characterizes that anionic species. The superscript zA, indicates
the anionic form of the anionic species A,, i.e., it refers to the charge of that ion. The

GENERAL EQUATIONS 43
anionic species A, has n A, pK values, ordered according to increasing pK values. The
particle A?*r, i.e., the ionic form with the highest charge ZA~ is taken as the
reference in all calculations.
Although the difference between anionic and cationic species disappears when this
notation is used, we still use the notation A and B for anionic and cationic species for
the sake of clarity and in order to reduce the number of indices used. Whether a particle
is an anion or a cation depends on its pK values and the pH in the zones.
The electrolyte system has to be chosen such that one of the ionic species acts as the
leading ionic species (anionic for the separation of anions) and another acts as a
buffering counter ion (cationic for the separation of anions) at the chosen pH. The way
in which an appropriate choice can be made will become clear in Chapter 5.
In section 4.2, the equations that describe the first stage in the separation, in fact a
kind of moving-boundary electrophoresis, are derived.
4.2. GENERAL EQUATIONS
For the derivation of the general equations in electrophoretic processes, we shall
consider the formation and movement of zone boundaries when an electric field is
applied over an existing zone boundary between two electrolyte solutions (see Fig.4.2).
On one side of the boundary, a mixture of several anionic and cationic species is
present, and on the other side a 'single electrolyte'. The anode is placed in the single
electrolyte. Only the migration of the anionic species is considered, and the effective
mobility of the anionic species of the single electrolyte is assumed to be higher than
that of any of the anionic species in the mixture. After some time, all of the anionic
species wd1 have the same counter ion B, because the cationic species B1 . . .r are moving
in the opposite direction. The anionic species migrate in the direction of the anode, which
results in a partial separation (moving-boundary electrophoresis). A number of boundaries
will be formed and a situation as shown in Fig.4.3 will be the result.
The anionic species Al, with the highest effective mobility in the mixture, has the
highest migration velocity and will be partially separated from the other anionic species.
It creates its own zone, Al, which becomes elongated in time. In the next zone, the
anionic species Az is separated from A3. . . in a similar way and, together with A1, it
forms the zone Al +Az. Each subsequent zone will contain one anionic species more
- -B
1.. .r
BL
0
'I.. .r-
m < m
A ~ . .r .
AL
Fig.4.2. A zone boundary between a mixture of several anionic and cationic species and a single
electrolyte.

44 MATHEMATICAL MODEL FOR ISOTACHOPHORESIS
B
t t t t t t t ! t
Concentration r-I separation Boundary
boundary boundaries l‘. e. -A I
< r n < .......... ern i rn < rn
AP-l A2 Al A1
Fig.4.3. Zone boundaries formed when an electric current is passed across a zone boundary as shown
in Fig.4.2.
from the mixture, viz., that anionic species with the highest effective mobility of the
anionic species that remain.
The last boundary created is the boundary A,. . . JAl . . . (r- I). The last zone boundary is
the original boundary Al.. .,-/Al . . .r, where an adaptation in concentrations according
to Ohm’s law takes place. This concentration boundary can be considered as stationary
[6]. Two types of boundaries have to be distinguished, viz., the concentration and the
separation boundaries. For the concentration boundary, the number of anionic species
is identical on both sides of the boundary, whereas for the separation boundaries one
particular ionic species is present on one side of the boundary only. In general, rt 1
boundaries will be present if an electric field is passed across the original boundary as
shown in Fig.4.2, considering the separation of anionic species, viz., one concentration
boundary (the original boundary), r-1 separation boundaries and the boundary
between the single electrolyte and the zone containing the anionic species with the
highest effective mobility in the anionic mixture (see Fig.4.3).
The velocity of the boundary A,/A, is equal to the velocity of the anionic species
A, and 4,. The velocities of the separation boundaries are equal to the velocities of the
ionic species with the lowest effective mobilities in those zones (see section 4.2.3).
These anionic species are not present in the preceding zones.
For the derivation of the general equations, the following assumptions are made: the
electric current is constant; the cross-section of the tube is constant; the influence of
diffusion, hydrostatic flow and electroendosmosis is neglected. The activity coefficients
and the influence of the radial temperature differences can be neglected.
Further, only those boundaries that are formed between the original zone boundary
and the anode are considered. The general equations describing electrophoretic processes
are: the equilibrium equations; the electroneutrality equation; the mass balances for all
ionic species; and the modified Ohm’s law. These equations are considered in more detail
in sections 4.2.1-4.2.5.
0

GENERAL EQUATIONS
4.2.1. Equilibrium equations
The thermodynamic equilibrium constant for an equilibrium
d
A-B+C
can be defined as
aBaC
K, =-
aA
where aA, aB and ac denote the activities of substances A, Band C, respectively. Often
the concentration equilibrium constant is used, defined as
The concentration equilibrium constant can be calculated from K, by correcting for the
activity coefficients, which are dependent on the ionic strength. For the sake of clarity
we shall use K, in all derivations. Considering reaction 4.1, the general expressions for
the equilibrium constants will be
45
(4.3)
(4.5a)
(4.5b)
(4.5c)
The indices A,,, U and i indicate that the equilibrium constant refers to the ith ionization
equation of the anionic species A, and is valid for the Uth zone. An indication of the
zone is needed, because all zones have, for example, different temperatures and concentrations
so that each substance will have different equilibrium constants in each of the
zones.
With eqn. 4.5, the concentrations of each ionic form can be expressed as the concen-
tration of the ionic form with a higher charge. In this way, we can write for the
relationship between the concentrations of the ionic forms with charges of 2%-i andz+:
and, in a similar way:
(4.6a)

46 MATHEMATICAL MODEL FOR ISOTACHOPHORESIS
(4.6b)
(4.6~)
Replacing the ionic concentration on the right-hand side with the concentration of the
higher charged ionic forms, we find the relationship with the concentration of the highest
charged ionic form. Eqn. 4.6~ gives
or
i
In this way, all concentrations of the ionic forms can be expressed as the concentration
of the ionic form with the highest charge by means of the equilibrium constants and the
concentration of the hydrogen ions. These equations will be used later to derive the
expressions for pH-dependent quantities such as the effective mobilities.
The total Concentration of an ionic species is
&,U = “A,., V,
+C +. . .
zAr ‘“A,,, U, zA,l A,., V, ~ ~ - 2
Substitution of eqn. 4.7 gives
or
(4.7)

GENERAL EQUATIONS 41
i
Similar equations can be derived for all ionic species in all zones.
Combining eqns. 4.7 and 4.9, the ionic concentration c u, zAr-i, can be expressed
Ar,
as the total concentration of A,.:
c~R, V,zA -
R
This equation wil be used in the following sections
4.2.2. Electroneutrality equations
(4.10)
(4.1 1)
In accordance with the principle of electroneutrality, the arithmetic sum of all
products of the concentrations of all forms for all ionic species and the corresponding
valences, present in each zone, must be zero. While the first zone contains one ionic species
of the sample, each following zone always contains one ionic species more, viz., that
ionic species with the highest effective mobifity of the ionic species that remain. The
Uih zone will consequently contain Uionic species of the sample. For one ionic species,
the sum of all products of the concentrations and the corresponding valences for the
different ionic forms is
(4.12)
This is the total amount of charge present per volume for this ionic species. If the ionic
species are numbered in order of decreasing effective mobilities, for the Uth zone we can
write as ‘electroneutrality equation’:
[ 2 U
CH,rJ-COH, U+ 2 [(‘Ar-’) ‘Ar,U,zAr-i]) +~~(‘B-‘) ‘B,U, zg-i] = (4.13)
r= 1

48 MATHEMATICAL MODEL FOR ISOTACHOPHORESIS
Substitution of eqns. 4.7 and 4.9 into 4.13 for both the sample ionic species and
counter ions gives
r= 1
4.2.3. Mass balances for all ionic species
-+
(4.14)
In the stationary state (N.B., the ‘steady state’ is not meant here), if all zone boundaries
are formed and migrate, some ionic species will migrate more rapidly and others more
slowly than a particular zone boundary, and ionic species will therefore pass continuously
those zone boundaries that have a lower velocity. For the ionic species that pass zone
boundaries, mass balances can be formulated.
In order to decide which ionic species will pass zone boundaries and their amounts,
we shall consider the velocities of the zone boundaries. The velocity of the concentration
boundary can be neglected, so that for constant effective mobilities the ratios of the
concentrations on the two sides of the concentration boundary are identical for all ionic
species (see eqn. 4.18).
The velocity of a separation boundary, S, is equal to the migration velocity of the
ionic species with the lowest effective mobility in that zone (see Fig.4.4).
The velocity of the zone boundary (U--l)/(U-2) is equal to the migration velocity
of the (U-1)th anionic species, which is not present in the preceding zone U-2.
Similarly, the velocity of the zone boundary U/(U-1) is equal to the velocity of the
anionic species A,. If the electric field strengths in the zones are E,, Eup1 and E,-,,
respectively, the migration velocities of those boundaries are E,-l mA and E, MAu,
U- 1
respectively. In these terms, the quantities indicated with a bar (m) do not apply to ions,
but to the equilibrium mixtures of all forms of the constituent; consequently, FI
represents the effective mobilities of the ionic species. As the boundary velocity is
determined by the llth ionic species, the subscript r in M is replaced with U. For the
effective mobility, Tiselius 1161 pointed out that a substance which consists of several
forms with different mobilities in equilibrium with each other will generally migrate as a
A,

GENERAL EQUATIONS 49
Uthzone (U-llthzone (U-Zjthzone
Fig.4.4. The Uth zone contains Uanionic species of the sample (A,, . u). The voltage gradient in the
Uth zone is Eu: The (U-1)th and (U-2)th zones contain one and two anionic species less of the
sample, respectmly. The migration velocity of the zone boundary U/(U-1) is determined by the
migration velocity of the anionic species Ay which is not present in the (U-1)th zone.
uniform substance with an effective mobility given by
ii = aimi = $ /cimi/ct> (4.1 5)
i= 0 i =O
provided that the time of existence of each ionic species is small in comparison with the
duration of the experiment. In this effective mobility, factors such as the relaxation
effect, the electrophoretic effect and the influence of temperature are neglected.
Substituting eqns. 4.7 and 4.9 into eqn. 4.1 5, we can write for the effective mobility
the expression
(4.16)
For the separation boundary U/(V-l), this means that the ionicspecies A1 to AUp1
can continuously pass this boundary, as their migration velocity is higher than
EufiA (fiA, > fiA2 > . . . . > fiA > r71 ). For the anionic species A,-,, for
u-1 Au
exampye, we have the following situation (see Fig.4.5). An ion at point P (at time t = 0)
can just reach the separation boundary S within one unit of time (at time t= 1). In this
time, the separation boundary has moved from So to S1. An ion at point So (at
time t=O) can just reach point M (at time r= 1). This means that all ions of the anionic
species AU-l present between points P and So (at time t=O) will be found again between

50 MATHEMATICAL MODEL FOR ISOTACHOPHORESIS
Fig.4.5. Migration paths for the different ionic species over a separation boundary S. For furthe1
explanation, see text.
the points S1 and M (at time t = 1). The distance SoSl is equal to EUriiAp u. The ion
present at point P (at time t = 0) migrates in the zone U, so the distance PS1 is equal to
EUfiAu-l, U and the ion present at point So (at time t = 0) migrates in the (U- 1)th zone,
so the distance SoM is equal to EU-l~Au-l, The amount of ions between P and So
(at time t=O) is
oc~u-l, U (psI-sO sl) = ocAu-l, U(EUriiAu-l, U-EUriiA, U)
where 0 is the cross-sectional area of the narrow-bore tube and ci is the total
U-l’U
concentration of the anionic species A,, in the Uth zone.
The amount of the anionic species A,, between the points S1 and M (at time
t= 1) will be
The amount of the anionic species between P and So (at time r = 0) reaches the
separation boundary within one unit of time and the amount between S1 and M passes
the separation boundary within one unit of time. These amounts must be identical for
a stationary state, so we can write for the mass balance of the anionic species A,, :
‘kU-,,U (EUmAu-,,TEUfiAu,U)= c~~-,,U-i (EU-l fiAu-l,U-l-EUfiAu,U)
The general expression for the mass balance of an anionic species is
‘A,., U WEUfiAu, U) = A,., U-1 (EU-l fiA,., U-l-EUmAu, U) (4.19)
In a similar manner, the following expression for the counter ions can be derived:
‘B, t U- 1 (EU-l ‘B , U-1 -k EU fiAu, U) = ‘i, U (EUfiB, U -k EUriiAu, U)
(4.18)
(4.20)

GENERAL EQUATIONS
4.2.4. Modified Ohm’s law
Working at a constant current density:
i/C = constant = E h,
(The Faraday constant is included in G). The overall electrical conductivity of a zone is
the sum of the values cinz,lzj\, and consequently
i
u nAY
I/G=EU coH,u’ptoH,u+cH,umH,u + C 2 (~zA;ilcA,.,u, zALi mAr,u, zA -i) +
r=O i=O
J
Substitution of eqns. 4.7 and 4.9 into eqn. 4.22 gives for the modified Ohm’s law:
4.2.5. Parameters and equations
51
(4.21)
(4.22)
1
(4.23)
The general equations given above describe the moving-boundary model. If the
compositions of the leading electrolyte and of the sample are known, all parameters
can be calculated. In Table 4.1, all parameters, known parameters and equations are
listed for all zones. For each ionic species, both n + 1 ionic concentrations and n
equilibrium equations are present. Using eqns. 4.7 and 4.9, all ionic concentrations can
be expressed as the total concentration for each type of ion. In this way, both the number

52
TABLE 4.1
MATHEMATICAL MODEL FOR ISOTACHOPHORESIS
NUMBER OF PARAMETERS, KNOWN PARAMETERS AND EQUATIONS FOR THE DIFFERENT
ZONES IN MOVINGBOUNDARY ELECTROPHORESIS
Zone Parameters and equations
Leading Parameters: EL, pHL, pOHL, ng + 1 ionic concentrations of c %, nAL + 1 ionic
concentrations of eA L
Known parameters and equations: c&, ckL, pK,, Ohm's law, electroneutrality
equation, nBL + nAL equilibrium equations
First
Second
Uth
Parameters: E,, pH,, pOH, , n + 1 ionic concentrations of cB, , nA, + 1 ionic
Bl
concentrations of c
A,
Known parameters and equations: pK,, Ohm's law, electroneutrality equation, buffer
mass balance, mass balance of A,, nB, + nAl equilibrium equations, isotachophoretic
condition
Parameters: E,, pH,, pOH, , ng, + 1 ionic concentrations of c B,, nA, + 1 ionic
concentrations of cA, , nA, + 1 ionic concentrations of c
Known parameters and equations: pK,, Ohm's law, electroneutrality equation, mass
balance of the buffer, mass balances of A, and A,, ng, + "A, + nA,equifibnum
equations
Parameters; Ey pHU, pOHv n + 1 ionic concentrations of the buffer, and
nA, + 1, nA, + 1, nA, + 1, . . ., :f + 1 ionic concentrations of the anionic species
Known parameters and equations: %ass balance of the buffer, U mass balances of the
anionic species, Ohm's law, electroneutmlity equation, pK,, "B + nA, + . . . + nAu
equilibrium equations
Terminating Parameters: ET, pHT, pOHT, ng + 1 ionic concentrations of the buffer, and nA, + 1,
nA, + 1, nA, + 1, . . ., n + 1 ionic concentrations of the anionic species
AU
Known parameters and equations: Ohm's law, electroneutrality equation, pK,, ciT,
t t t
CA,, CA,. . . ., CA~, ng+ nAl + . . . + "Au equilibrium equations
of parameters is reduced by n and the number of equations is reduced by n. Further, the
pOH value can be expressed as pH by using the pK, value.
In Table 4.2, the reduced number of parameters and equations is given. It can be seen
that the first zone has a surplus of one equation, whereas the terminating zone has a
shortage of one equation. Note the unusual part of the terminating zone, ci in this zone
being taken as unknown. It might be thought that ci could be determined by the choice
of the pH in the terminating zone, the type of buffer ionic species and its concentration,
but this is not so. Of course, the pH and the concentration of the counter ion can be
fixed, but if the stationary state is reached and if all original counter ions have migrated
to the cathode, the counter ions will be replaced with those of the leading electrolyte,
A2

GENERAL EQUATIONS
TABLE 4.2
REDUCED NUMBER OF PARAMETERS, KNOWN PARAMETERS AND EQUATlONS FOR THE
DIFFERENT ZONES IN MOVINGBOUNDARY ELECTROPHORESIS
Zone Parameters and equations
Lea ding Parameters: ifL, PH~, ck, cx,
Known parameters and equations: ck, ci, Ohm’s law, electroneutrality equation
Number of known parameters and equations is equal to the number of parameters
First Parameters: E,, pH,, ci, c$,
Known parameters and equations: Mass balances of B and A,, electroneutrality
equation, Ohm’s law, isotachophoretic condition
Surplus of one equation
Second Parameters: E,, pH,, c fj, ca, I cL2
Known parameters and equations: Mass balances of B, A, and A,, Ohm’s law,
electroneutrality equation
Number of known parameters and equations is equal to the number of parameters
In all other zones, the number of anionic species is increasing. Consequently, the
number of unknown parameters increases, but the number of mass balances also
increases, and the number of unknown parameters and equations and number of
parameters become equal again
Uth Parameters: Eu pHU, cfj, c a t
. . .
Known paramefers and equations.’ Mass balances of B, A,, A,, . . ., AU, Ohm’s law,
electtoneutrality equation
Number of known parameters and equations is equal to the number of parameters
Terminating Parameters: ET pH.,., ck, ci , . . ., ciu
1
Known parameters and e9Uan’onS: cir, . . ., ci, Ohm’s law, electroneutrality
equation
Shortage of one equation
and the pH and the concentration of the counter ions in the terminating zone are
determined by the buffer mass balance of the leading electrolyte zone (the surplus of
one equation). This indicates the problem or calculations with the moving-boundary model.
A surplus of one equation in the first zone determines the pH in the last zone, whereas
the pH in the last zone determines the effective mobilities of the anionic species in that
zone and hence the mass balances of those anionic species which determine the situation
in the first zone. Thus the pH, requires a calculation from the first zone in accordance
with the buffer mass balances, whereas the composition of the first zone is determined
by calculations from the last zone in accordance with the mass balances of the anionic
species.
For nearly all calculations in moving-boundary electrophoresis, simplifications have to
be made in order to avoid this difficulty. A mdel suitable for calculations on strong
electrolytes (for which the mobilities are independent of the pH), neglecting the
53

Fig.4.6. Simplified model for the formation of the zonesin an isotdchophoretic separation of a five-component sample (A, -As). The sample is introduced
already sandwiched between the leading and terminating electrolytes. At various times, mixed zones disappear, as a function of the effective mobilities of
the ionic species and their actual concentrations. Such a figure can only be realized in practice if a fast scanning device is available. At the time r=8 (x),
the steady state has been reached and the sample zones will not broaden further, assuming that the electrolyte is of constant composition and the cross-
section of the separation chambe1 is constant. The zone lengths are also not influenced by variations in the electric current, assuming that the pK values of
the ionic species present are not influenced by the temperature. In this sample, the ‘dilution’ effect of isotachophoresisis shown. If one compares this figure
with Fig.2.7, it should be noted that the situationgiven in the present figure can be obtained only if a counter flow of electrolyte is present, because for a
complete adaptation of all concentratidns the terminator ion must have passed the position where the sample is introduced, although we recommend
to start the counter flow of electrolyte as soon as the terminating zone has passed the injection point.

MATHEMATICAL MODEL FOR THE STEADY STATE
presence of hydroxyl and hydrogen ions, was given by Brouwer and Postema 161. This
model describes the first stage in the Isotachophoretic separation, which is a movingboundary
system.
In Appendix A, a simplified model for moving-boundary electrophoresis is described,
for measuring the effective mobilities of strong electrolytes.
4.3. MATHEMATICAL MODEL FOR THE STEADY STATE IN ISOTACHOPHORESIS
4.3.1. Concept of isotachophoretic separation
In the previous section, the first stage of isotachophoretic separations was discussed.
The formation and migration of zones were described for the case when a stabilize<
electric current is passed across a zone boundary (see Fig.4.2) between a mixture of
anionic and cationic species on one side and a single electrolyte on the other side. In
general, r+ 1 zone boundaries will be obtained for the separation of anionic species
(see Fig.4.3).
No complete separation of the anionic species can be obtained in this way, however.
In the model discussed only those zone boundaries which are formed between the original
boundary and the anode were considered. Essentially, this means that the amount of the
mixture in the cathode compartment is taken to be unlimited (exhausting phenomena
being neglected), and no attention is paid to the influence of the counter ions. For an
isotachophoretic separation, however, a limited amount of sample ions is introduced
between a leading and a terminating electrolyte. The terminating ionic species can never
pass the sample ionic species as its effective mobility is chosen so as to be lower than
those of the sample ions, and hence all sample ionic species will migrate between the
terminating and leading anionic species.
In front of the original sample zone, a series of zones will be formed, as described in
the previous section, but behind the original sample zone a series of zones will now also
be formed, because sample anionic species also remain behind according to their lower
effective mobilities. The last zone formed will contain one anionic species of the sample,
viz., that with the lowest effective mobility of the sample. The last zone but one will
contain two anionic species of the sample with the lowest effective mobilities and each
subsequent zone contains one anionic species more of the sample, in accordance with
their increasing effective mobilities. Analogous to the formation of a series of mixed zones
in front of the original sample, a series of mixed zones will also be formed behind the
original sample, divided into two series of mixed zones.
If an adaptation in concentration according to Ohm’s law has taken place, the whole
system migrates through the capillary tube, during which the separation of the anionic
species continues until a steady state is reached, i.e., all anionic species of the sample
are separated and all sample zones contain only one anionic species of the sample. Only
in this instance can we speak of an isotachophoretic separation.
In Fig.4.6, a very simplified model for the formation of the zones for an isotacho-
phoretic separation of a five-component sample is shown. At zero times, a mixture of
Al . . . is introduced between A, and A,. After a certain time, two series of mixed
55

56 MATHEMATICAL MODEL FOR ISOTACHOPHORESIS
zones are obtained. The original sample zone length has been shortened. The whole
system migrates between A, and A,, whereas the mixed zones elongate with time until
the anionic species of the sample are separated. The developed zones are shown that are
present in the narrow-bore tube at different times. As the amount of the sample is
limited, the sample anionic species Al and, for example, As will be separated completely
at a given moment. The two anionic species A, and A5 are separated completely if the
last ion of the ionic species with the highest effective mobility (Al) has overtaken the first
ion of the slowest ionic species (A5).
The length of the original sample zone is taken as Al and the length of the capillary tube
needed for a separation is I (the distance after which Al and AS are separated) (see
Fig.4.7). This means that the time needed for the separation of Al and A5 is obtained as
follows.
Distance covered by Al :
Eml t = li-Ai
Distance covered by A5 :
Em5 t=l
Thus :
A1 = Et (ml -m5)
or
Al
t= -
EAm
(4.24)
After this time 1, the anionic species A, and A5 are not present together in one mixed
zone. The time of separation depends on several factors (see eqn. 4.24) such as differences
in effective mobilities, the concentrations and the amount of the sample. At a certain
moment, each ion of the anionic species Al will have migrated from all other zones and can
be found only in its own zone Al . From this moment, this zone does not elongate further
with time. This procedure occurs for all ionic species and in Fig.4.6, for example, all
ionic species are separated except one mixed zone of A3 +Az at time t =7.
Fig.4.7. The last ion of the anionic species A, will have overtaken the fust ion of anionic species A,
in the original sample zone at point P. The separation time will be t = Al/E Am.

MATHEMATICAL MODEL FOR THE STEADY STATE
If all anionic species are separated, the zones are constant in length and number and all
zones, containing only one ionic species of the sample, migrate through the capillary tube
with a velocity identical with that of the leading zone. We can then speak of a ‘steady
state’ and this situation is called an ‘isotachophoretically separated system’.
Before discussing the equations that describe this steady state, we shall consider the
number of parameters and equations. Only the mass balance of the buffer will be used,
as the anionic species of the sample are not present in other zones and wd1 never pass
zone boundaries. However, each zone must conform to the isotachophoretic condition.
In Table 4.3, all of the relevant parameters and equations are given. In Table 4.4, a
reduced number of parameters and equations are given, obtained by expressing all ionic
concentrations of one type as the total concentration of that ionic species using the
equilibrium equations. Further, the pOH value can be expressed as pH by using the pK,
value.
In Table 4.4, it can be seen that for the leading zone two unknown parameters and
two equations remain, by means of which all parameters can be calculated. For all other
TABLE 4.3
NUMBER OF PARAMETERS, KNOWN PARAMETERS AND EQUATIONS FOR THE
DIFFERENT ZONES IN ISOTACHOPHORESIS
Zone Parameters and equations
~
Leading Parameters: EL, pOHL, pHL, n + 1 ionic concentrations of the buffer, nAl + 1 ionic
concentrations ofAL
Equations: ng+n equilibrium equations, pK, electroneutrality equation, Ohm’s law
AL
Known parameters: ck and ct
AL
First
TABLE4.4
Parameters: El, pOH, , pH,, nB+ 1 ionic concentrations of the buffer, nA, + 1 ionic
concentrations of A,
Equations: n +nA equilibrium equations, pK, buffer equation, Ohm’s law, electro-
neutrality equation, isotachophoresis condition
As all other zones (including the terminating 73ne) have only one anionic species and
the same buffer ionic species, all other zones have an equal number of parameters and
equations
REDUCED NUMBER OF PARAMETERS, KNOWN PARAMETERS AND EQUATIONS FOR THE
DIFFERENT ZONES IN ISOTACHOPHORESIS
Zone Parameters and equations
Leading Parameters: EL. pHL, ck, caL
Other
Known parameters: ct and ct
3 AL
Equations: Ohm’s law, electroneutrality equation
Parameters: El , pH,, c:, C:
Equations: Electroneutrality equation, Ohm’s law, buffer equation, isotachophoretic
condition
51

58 MATHEMATICAL MODEL FOR ISOTACHOPHORESIS
zones, four unknown parameters and four equations are obtained, and these parameters
can also be calculated.
AU zones are correlated with the leading zone by means of the buffer balances, in
which all zones are determined by the conditions of the leading zone and do not affect
each other. Each zone can be calculated in relation to the leading zone directly. The
equations needed for the calculations are described in section 4.3.2.
4.3.2. Mathematical model of isotachophoresis
By analogy with the general equations and with the same assumptions, we give here
the equilibrium equations, the mass balance of the buffer, the electroneutrality equation
and the modified Ohm's law, whch, in combination with the isotachophoretic condition,
describe the isotachophoretic separation. The equations are valid for separations of
anionic species, while analogous equations can be derived for separations of cations. The
computer program discussed in section 4.3.3 can be used for both anionic and cationic
species.
4,3.2.1. Equilibrium equations
In a similar manner to that described in section 4.2, we can derive for the equilibrium
constant, ionic concentrations and total concentration of an ionic species the following
equations*
KAv,i=
cAv.zAv-icH, V
c~ v, zA v-(i.- 1)
These equations are valid for molecules with equilibria according to eqn. 4.1.
4.3.2.2. The isotachophoretic condition
In the steady state, all zones move with a velocity identical with that of the leading
zone and therefore
*In eqn. 4Sa, the concentration has been characterised by the subscripts A, (indicating the anionic
species), II (indicating the zone) and z 1 (indicating the ionic form), because all anionic species
can be present in all zones. Here, in a zone 4-
V, only one anionic species can be present, called Av
The second subscript, V, indicating the zone, is superfluous. An exception must be made for the
hydroxyl and hydrogen ions, present in all zones: for these ions, the zone must be indicated.
(4.25)
(4.26)
(4.27)

MATHEMATICAL MODEL FOR THE STEADY STATE 59
EL rEAL = EvfiAv (4.28)
where fi and fiA are the effective mobilities of the leading ion in the leading zone and
AL . 1.I
the sample ions A, in the Vth zone, respectively.
(4.29)*
For all other ionic species, a similar expression for the effective mobilities can be derived.
The isotachophoretic condition is the essential difference between isotachophoresis and
other electrophoretic methods.
4.3- 2.3. Mass balance of the buffer
The movements of the zone boundaries LV and VW per unit of time are equal (AX;
see Fig.4.8):
AX=ELfiAL=E V fi Ay =E W fi AW
A buffer ion Pin the Vth zone (at t=O) can just reach the zone boundary VW at t= 1 if
the distance over which it moves during one unit of time is
B2X = Ev%v
and a buffer ion Q (at t=O) can just reach the zone boundary LV if
(4.30)
(4.3 1 )
B1X = ELMB. (4.32)
This means that the amounts of the buffer that pass the zone boundaries LV and VW are
the amounts of the buffer present in the volumes A, and A2 at time t=O. The amounts
of the buffer entering and leaving a zone must be equal in the steady state, and therefore
OAIC&= O A ~ V C ~
or
O(AX+BlX) cr = O(AX+B2X) 4
BL
V
*Compare with eqn. 4.16.
(4.33)

60 MATHEMATICAL MODEL FOR ISOTACHOPHORESIS
t. = 1
zone t +p
WI
I I
I I
I I
I
I
I I
I !
;zone
vw LV
1 1
I I
2.e.
Rg.4.8. Migration paths of the ionic species and movement of the zone boundaries in an isotacho-
phoretic system.
Combining eqns. 4.30 and 4.33, we obtain
ci, (1 + fiJj,/fi*,) = c& (1 + fiBV/fiAV)
This equation is the mass balance of the buffer, valid for all zones. All zones are directly
related to the leading zone by the mass balance.
4.3.2.4. Bnciple of electroneutrality
In accordance with section 4.2.2, we can write for the electroneutrality the equation
".[ . ipl i KAv,i]
2 ('Av-'>
i= 1 ('H, V)'
+ZAV
%, V*OH, V -k
n A ~ If KAv,j I+C i=1
i=l ( c H , ~ ) ~
(4.34)

MATHEMATICAL MODEL FOR THE STEADY STATE
r- 1
4.3.2.5. Modified Ohm ’s law
Working at a constant current density:
IIG = constant = EL hL = E, X,
The overall electrical conductivity of a zone is the sum of the values ci mi bil
and consequently
Substitution of eqns. 4.26 and 4.27 in eqn. 4.37 gives
COH,L %H,L +CH,L~H,L +cAL
r 1
61
(4.3 5)
(4.36)
(4.37)
(4.38)

62 MATHEMATICAL MODEL FOR ISOTACHOPHORESIS
A similar expression can be obtained for the sample zone. Calling the right-hand side of
eqn. 4.38, QL and Q, for the leading and Vth zones, respectively, the function RFQ
defined as
must be zero according to eqn. 4.36.
4.3.3. Computer program for calculation of the steady state
4.3.3. I. Compu ration procedure
(4.39)
If all mobilities and pK values are known, and suitable values for the total composition
and pH of the leading electrolyte are chosen, the effective mobilities and the products of
the equilibrium equations (which are constant at a given pH) of both the leading ions and
the buffer ions in the leading zone can be calculated. From an equation similar to eqn.
4.27 9 ‘AL, zAL can be calculated from the total concentration, and with eqn. 4.26 all
partial ionic concentrations of the ionic species A, can be found. With eqn. 4.35, the
total buffer concentration in the leading zone can be obtained and with equations
similar to eqns. 4.26 and 4.27 the partial ionic concentrations of the buffer ions can be
found. Further, QL and the term on the left-hand side of the buffer eqn. 4.34 can be
obtained.
All parameters of the leading zone are now known. Assuming a certain pH for the
following zones, in a similar manner to that indicated for the leading zone, we can
obtain the effective mobilities and products of the equilibrium equations. With eqn. 4.34,
the total concentration of the buffer in the following zones can be found and with
eqns. 4.26 and 4.27 all other partial concentrations. With eqn. 4.35, the total concen-
tration of the sample anionic species in the zones can be obtained. With eqn. 4.38, QV
can be obtained and eqn. 4.39 gives the value of the function RFQ for the assumed pH.
This value must be zero for the correct pH,- In fact, several zero points will be possible,
and the method of finding the correct pH, zero points is dealt with in the next section.
4.3.3.2. Iteration procedure
As mentioned in section 4.3.2.5, the function RFQ must be zero for the correct pH,
value. For several cases this function is calculated as a function of the pH. In Fig.4.9,
the function is plotted for the separation of univalent cations and anions, the buffering
counter ions also being univalent.
In Fig.4.10, the function is plotted for polyvalent sample ionic species and buffer ions
and in Fig.4.11 the function is shown for a system in which, in the leading zone, the
leading ion acts as a buffer instead of the counter ions. Only in the sample zones do the
counter ions act as a buffer and in general this means that there is a large difference in
pH between pHL and pH,. This effect is used in disc electrophoresis according to
Ornstein [18] and Davis [7]. In Figs.4.9,4.10 and 4.1 1, anionic and cationic separations
are indicated by the symbols 8 and @, respectively. The functions are indicated by numbers

MATHEMATICAL MODEL FOR THE STEADY STATE 63
3
2
1
0
2
1
-1
t@
~
16 7
5 10
pH =3
L
A
- PH"
t e
-1 I 1
-1
pHL= 11
5 10 - PH"
91 I
Fig.4.9. (Continued on p. 64)
B

64
-1
3
2
1
0
2
t
1
I
t
-1
5
Fig.4.9 (continued).
13
pHL= 10
G
10 - P S
MATHEMATICAL MODEL FOR ISOTACHOPHORESIS
3
2
-1
1
i!
t
2
1
i
t
-1
e pHL= 6
12
0 pHL = 4

MATHEMATICAL MODEL FOR THE STEADY STATE
i
@
I
2
pHL= 11
pH =12
L
3
2
1
:
1-
3
le I
1
e
pHL= 2
pHL= 3
I
i
I
I L
Fig.4.9. Relationship between the function RFQ and pH in the zones for several isotachophoretic
systems. For A-L, see Table 4.5.
65

66
1
I
E
t
-1
-1
co ! I i'i
.I I I 1
- 1 I / /
;n
w
pH =6
L
\ I
MATHEMATICAL MODEL FOR ISOTACHOPHORESIS
2
1
z
1
-1
-2
3
2
1
0
2
t
-1
8 pHL= 6
Fig.4.10. Relationship between the function RFQ and pH in the zones for several isotachophoretic
systems. For A-D, see Table 4.5.

MATHEMATICAL MODEL FOR THE STEADY STATE
-2
PHL’ 4.7 5
Fig.4.11. Relationship between the function RFQ and pH in the zones for a disc electrophoretic
system.
representing the pK values of the sample ionic species. All assumed pK values and ionic
mobilities for the leading electrolyte and sample ionic species are given in Table 4.5.
For all of these electrolyte systems, different functions were obtained, some of which
show no real zero points, two zero points and with some discontinuities occur. All of
these effects depend on quantities such as pK values and mobilities.
Although not all possible functions have been calculated, we can conclude that all
systems have one common property, viz., in a cationic separation the correct zero point
is always the transition between a negative and positive value of the function RFQ in
the direction of higher pH values and for an anionic separation it is a transition between
a positive and a negative value of RFQ (for the false zero points, negative concentrations
were obtained).
The method of finding the correct zero point is therefore as follows. In the computer
program, a pH, is first searched for with a positive (or negative) value of RFQ and then
a pH, with a negative (or positive) value of RFQ for an anionic (or cationic) separation.
The correct pH, at whch the function RFQ is zero, within certain limits, is obtained by
iterating these two values. If no pair of positive-negative or negative-positive pH, values
can be obtained in a range of six pH units from the pH, value, then 'NO REAL ZERO
POINTS will be printed out by the computer. The iteration procedure is shown in
Fig.4.12.
61

68 MATHEMATICAL MODEL FOR ISOTACHOPHORESIS
TABLE 4.5
pK VALUES AND IONIC MOBILITIES OF THE IONIC SPECIES USED FOR THE CALCULATIONS
OF THE RELATIONSHIP BETWEEN THE FUNCTION RFQ AND THE pH VALUES IN THE ZONES
~~ ~ ~~
Fig. Leading zone
4.9A
4.9B
4.9c
4.9D
4.9E
4.9F
4.9G
4.9H
4.91
4.9J
4.9K
4.9L
4.10A
4.10B
4.10C
4.10D
4.1 1
~
Buffer ionic species
Leading ionic species
rn. 105 PK n z Concn. m-105 pK n z pHL
(cm2/V. s) (molell) (cm’/V. s)
0.50
19,O
030
30,O
0,50
19,O
0,50
30,O
0,50
30,O
0,50
30,O
50,0,50,70
0,40
19,O
19,O
19,O
3 1 0 0.01
11 1 1 0.01
4 1 0 0.01
10 1 1 0.01
6 1 0 0.01
6 1 1 0.01
10 1 0 0.01
4 1 1 0.01
11 1 0 0.01
3 1 1 0.01
12 1 0 0.01
2 1 1 0.01
2,4,8 3 1 0.01
4.75 1 0 0.01
6 1 1 0.01
6 1 1 0.01
8 1 1 0.01
75,O
0,76.5
75,O
0,76.5
75,O
0,76.5
75,O
0,76.5
15,o
0,76.5
75,O
0,76.5
75,O
75,o
0,763
0,76.5
0,40
Fig. Sample ionic species Fig * PK
m.105 PK n Z
(cm2/V. s)
4.10A 50,O
50,O
50,O
70,30,0,30
4.10B 70,30,0,30
70,50,0
50,O
4.10C 0,50
0,50,70
50,0,30,60
4.10D 50,0,30,60
50,0,50
70,70,0,50,70
4.11 30,0,30
1
1
1
3
3
2
1
1
2
3
3
2
4
2
1
1
1
2
2
2
1
0
0
0
1
1
2
1
14 1 1 3
-2 1 0 11
14 1 1 4
-2 1 0 10
14 1 1 6
-2 1 0 6
14 1 1 10
-2 1 0 4
14 1 1 11
-2 1 0 3
14 1 1 12
-2 1 0 2
14 1 1 5
14 1 1 5
-2 1 0 6
-2 1 0 6
4.15 1 0 4.75
4.9A
4.9B
4.9c
4.9D
4.9E
4.9F
4.9G
4.9H
4.91
4.95
4.9K
4.9L
3,s ,6J
9,10,11,12
3,4,5,6,8,10,12
1-6,9,12
3,5,7,9,13
1,6,10,11,12
4,8,10,13
1,4,5,10
2,4,8,10,11
4,598
2,4,8,12
1,4,5,8
*Because in this instance the assumed mobilities for the monovalent cations and anions were 50,O
and 0,50, respectively, only the pK values of the sample ionic species are given.

VALIDITY OF THE ISOTACHOPHORETIC MODEL
j pHy=pllv+0.2
rp = [H+L)/2
PHV = Q c-
if( IH-L~

MATHEMATICAL MODEL FOR ISOTACHOPHORESIS
LIST
0010 'REG IN'
0100
01 10
0120
'REQL' PHLJCSTLI~MI~LI 9 M~HL~I~~HL,M~~LS~HPLJQHL,TITU!~J
KTL I , KMI- I J KI LI J K I ML I 9 KTtn-J KML3 J K I u3 , KIMLQJ H, BH J
CUL I , WUL I, MLI 1 , c ULH, csm, NF.ULU, wii 1 , QC IM , KI IL,
0125 S,(JHI QL,
0130
01 50
01 70
0172
K ULT Hb-il;
' 1VTE:GER ' 4, NL I > I I J, il, NLR , Z I 3 ZY J L t Vt Mi
'HEAL' ' AiXR4Y 'PKLI > MLI JPKD , P I , KL I , KLB, MH, MBH, Mll I , MBR CO : 10 1 ,
CSTn JCST I JCOI ,CBU, NEU9 ,NEU I , PH, C~HI HPI MI 1 3 M3 1 J KTB.
0173 KI~~KIR~KP~~"RJKI~I~KII~KMIJKTICO:~OI~KI~KR~
0180 PKI JKIJPK:3rTUHEO: 10~0: 10 1;
0190
0195
0200
020 1
' INTEGER' 'AR%4Y' NItWi,ZIN,ZFjNCO: 10 I;
s : =READ ;
PHL: =IIEAD;A:=HEAD;CSTLI:=FIEAD;NLI:=READ;MdLI:=HEAD;
ZI : =READ;
0210
0220
'F0H'I:=l'STEP9 I'UNTIL'NLI'DB'
'RF:GIiV' PKI-I C I 1: =READ ; MLI C I 1 : =READ; ' EN!O' ;
0230 MCIHL: =READ; MHL: =READ; NLF3: =!?EAD;MOLS: =READ;
023 1 Z9 : =READ ;
02 40
0250
'FOii'I:=l'STEP'l'UNTIL'NLF3'DB'
'REG IN' PKLB C I 1 :=REAL,; Mi-V C I 1 : =READ; 'EN>' ;
0% 60
0270
0275
0280
0290
0300
'FLlil'I:=l'STEP'I'UNTIL'A'DB'
'BEGIN' NI C I 1 : =READ; MHC I 1 : =READ;M0HC I 1: =IIEAD;MBI C I 1: =REAP;
ZINC 11: =ilEAD;
' FOR ' J: =1 'STEP ' 1 ' UNT IL ' NI C I 1 ' Dl3'
'REGIN'PKI C 1, J 1: =READ; MI C I, J 1 : =READ; 'END' 5
NF3 C I 1 : =READ; M09 C I 1 : =ilEAD;
0305
0310
0320
0330
ZRN C I 1 : =READ;
'FBR * J: =1 'STEP' 1 'UYTIL'N5 C I 1 ' DO '
' BEGIN' PKl3 L: I , J 3 : =REAL); M3 C 1, J 1 : =READ; 'END' J
' END ;
0400'CBMPENT' qalculation first zone:
0410 HPL: =lo T ( -PHL) j UHL: =1 Ot ( -1 4+PHL) ;
OM0 KLI t 0 1 : = 1 ; KTL I : =KMLX : =KI LI : =K I I% I : =O;
0430
0440
0450
Fig.4.13.
'FkJR'I:=l'STEP'l'UNTIL'NLI'DB'
'REGIN'KLI CI I: =KLI [I-1 1*10t( -PKLI CI l)A-IPL;QR:=ZI-I;
KTLI : =KTLI +KLI C I 3; KILI : =KILI +KL I C I l*QH;

VALIDITY OF THE ISOTACHOPHORETIC MODEL
0460
0470
0480
0490
0500
0510
0520
0530
0535
0540
0550
0560
0 570
0580
0590
0600
0610
0620
0630
0640
0650
0660
0670
0680
0690
0700
07 LO
0720
0730
KMLI:=KMLI+KLICI WMLICI 3*SIGN+KMLH )/R;
BCBR:=(l+ARS(MLBl )/ABS(MLIl ))*CSTLB;
T: k0LI * CARS ( Z I ) *MBLI +K I MLI 3
TUW: =C0LB*(AW( ZR) *MQLR+KIMU3 1 ;
KBL: =HPL*MHL+VIHL*M")HL+T+~~~~;
PR INTTEXTC ’ ( ’ M0F3LE = ’ ) ’ 1 ;F IXT (61 4r MLB 1 1 i NLCR;
’ FBR ’ I : =O * STEP ’ 1 ’ UNTIL * NLR ’ DO ’
’BEGIN’FIXT~SrOrZR-I~~F’L0T~5r2rKlECII*CBLR~~NLCK~’END’~
PRINTTEXT(’

72 MATHEMATICAL MODEL FOR ISOTACHOPHORESIS
1000
1010
1020
1030
KPBEV l:=KM3CVl+KBCVr I l*M3CVr I I*SIGNCQR);
K IB C V 1 : =K IB C V 1 +KB C V r I I*QR 5
KI("BCVI:=KItvACVl+KBCV~I l*ADSCQH)*IVBCVrI I;
’END’;
1040
1050
1060
1070
1080
1090
1100
1110
M31 CV 1: =CMB CV ]*SIGN( ZBNCV I>+KW CV I)/( 1 +KTBCVI );
CSTB CV 1 : =BCOR/C 1 +ABSC PB 1 CV 1 )/ARSC MI 1 CV 1) 1 ;
CBM C V l : =CSTR EV 1 /C 1 +KTR C V 1 1 ;
NEUR C-V 1 : =< ZBN CV 1 +KIB C V 1 1 *CBQ CV 1 ;
T : =CBO C V I* C ABS t ZRN CV 1 1 *YOB CV l+K I M3 CV 1 1 ;
NEUI CV 1 : =C Z INCV l+K I I CV 3 1 1 +KTI CV 1 ) ;
CSTI CV 1 : =- CNEUR cv ]+HP[V 3-dH CV I) /NEUI CV 1;
CBI CVI: =CSTI CV I/< l+KTI CVI);
1120
1130
11-40
mid: ;NLCII;NLCR;
1170 ’GWTU’SYSTEMC8 I;
1200 L2 : ’ IF ’ -S*FIFO>=O ’ THEN’ ’BEG IN’ QL: =PP CV 1; K: =3;PHCV 1 : =PHCV 1+0 -2;
1210 K:=l;’GGlTO’SYSEMCl ];’END’
1220 ’ELSE ’ ’REG IN ’FH CV I : =PHCV l-S*O .2; K: =K+ 1 ; ’ IF’ KC30’ THEN’
1222 ’GBTB’SYSTEMC 1 ]’ELSE’ ’G(ZITO’SYSTEMC7 1; ’END’ ;
1230 L3: ’ IF’-S~~FQ~~O’THEN’’BEGIN’Qt~:~PHCV1~~~:~4~PHCVl~~CG\H+QL~/2~
1240 K: =1; ’GUTB’SYSTEMC 1 1; ’END’
1250 ’ELSE’ ’BEGIN’PHCVI: =PHCV 1+0*2;K:=K+l$ ’ IF’K=O’THEN’ ’REGIN’QL:=PHCVl; ’GBTB’SYSTEMC51; 'EIUD'
1270
’ELSE’ ’REtiIN’OH: =PHCV I; ’GUTB’SYSTEMCS I; ’END’;
1280 L5: * IF’AP,SCQH-CL)

VALIDITY OF THE ISOTACHOPHORETIC MODEL
HUN
WAIT
?enera1 inqornation
-1 ~4.8>3>0.02>
an ionic
lrO>0>4.7S>41>200~350~ .~.ea~ 19>1,8>0>
lr350>200r0>0>7~30> , anionic
*Second zone
1~19rl rH>O>
!J Y cationic
2~350~200~0~0~7~20~8~30~ ) Third zone
1>19>1>8>0>
i. anionic
\ cationic
A anionic
2>350>200>30> 1 >2.2>0>9
1 > 19>1>8>0> cationic
Leadinp zone
PHL= +4-800 MIlDLI =
0 +.94250’- 2
-1 +.10575’- 1
CSTLI= +-2OOOO'- 1
M0BLf3= +18 9880
+1 +.10559’- 1
0 +.66624’- 5
CSTU3= +. 10566’- 1
LAPBDA= + 63975 ’ + 0
?!ext zone +1
PHV= +6.880 MmI=
0 +.10276’- 1
-1 +.77890’- 2
CSTI= +.1806S’- 1
M0BU = +17 661 3
+1 +.77889’- 2
0 +.59041’- 3
csm= +.83794’- 2
IArSDA +.38172’+ 0 HFO
Yext zone +2
PHV= +6.734 MWI=
0 +.91648’- 2
-1 +.49642’- 2
-2 +*26889’- 3
CSTI= +.14398’- 1
MUBRR = + 1 8.0237
+1 +.55019’- 2
0 +.29802’- 3
csm= +.57999’- 2
Fig.4.13 (continued).
-21 -6788
-12.9352
-. 13039’- 6
-7.4560
73
(Conrinued on p. 74)

74
LAlvBDA +.22003'+ 0
"Text zone +3
PHV = +8 707 MPBI=
+1 +.50943'- El
0 +.16384'- 1
-1 +.13924'- 2
CSTI= +.17776'- 1
MOBB = +3.1162
+1 +.13975'- 2
0 +.71232'- 2
CSTl3= +.85206'- 2
LAM3DA +.69343'- 1
END OF JDB
GO AHEAD
MATHEMATICAL MODEL FOR ISOTACHOPHORESIS
HFQ= -.57742'- 7
-2.3498
RFQ= +.35763'- 6
Fig.4.13 Computer program for the calculation of concentrations, conductivities, pH values and
effective mobilities, useful for the steady state in isotachophoretic separations. For computations,
the following input is required: -I or I (anionic or cationic separations, respectively), pH the
L'
number of zones, ciL, nAL, mA all pK values and ionic mobilities of the ionic species
L' AL' zAL'
4, moH,L, myL, nBL, mgLJ zBL and all pK values and ionic mobilities of BL. For the following
zones we require nAv, mY v, 3' v, mA
V' Ay' zAVi
, zBV and all pK values and ionic mobilities of B,.
nBy, %v$z~~
all pK values and ionic mobilities of A v ,
the concentrations of sample and buffer ionic species, electrical conductivities of the
zones, the pH values of the zones and effective mobilities of the ionic species in the zones
during the steady state. For the calculations, the composition of the leading electrolyte
zone and the ionic mobilities and pK values of all ionic forms must be known.
In this model, the activity coefficients, influence of temperature (different in
each zone), relaxation and electrophoretic effects, diffusion, hydrostatic flow and
electroendosmosis were neglected*. In this section, some of these factors are discussed. For
some of them, corrections are made in the calculations and the results of these cgcula-
tions are compared with the results of some experiments in order to check the validity
of the model. Factors that affect the effective mobility have already been discussed in
Chapter 3.
4.4.2. Influence of diffusion on the zone boundaries
In the model of isotachophoresis, the influence of diffusion was neglected, although
it affects the sharpness of the boundary, giving a finite width to the zone boundary. This
*For some of these effects, corrected values can be introduced in the computer program by repeated
calculation.

VALIDITY OF THE ISOTACHOPHORETIC MODEL 15
effect can be neglected only if the zone boundary width that results from it is very small
in comparison with the zone length.
Several workers [ 10, 13, 15, 201 have given an approximation for this effect and
showed that the width of the zone boundary due to diffusion is less than 0.1 mm; for
long zone lengths, this can be neglected.
4.4.3. Influence of axial and radial temperature differences
During electrophoretic experiments, radial differences in temperature exist in the
zones and axial differences in temperature between the different zones. Several quantities,
such as mobilities and pK values, depend on temperature and the concentrations and pH
values of the zones are also affected by temperature. HjertCn 1211 and Routs [I51 studied
the influence of temperature in the radial direction and found that a parabolic shape of the
zone boundary can be expected. Another important point is the difference in pK values
of the ionic species due to the different temperatures of the zones. In Fig.4.14, the pK
values of some ionic species are shown as a function of temperature.
From Fig.4.14, it can be concluded that particularly the positively charged ionic
species such as imidazole, tris and histidine, which are used as buffering counter ions for
T 1 80
60
40
20
Fig.4.14. Relationship between temperature (r) and pK values of some ionic species. 1 = pK, of
glutamic acid; 2 = pK, of glycine; 3 = pK of formic acid; 4 = pK, of glutamic acid; 5 = pK, of
oxalic acid; 6 = pK of acetic acid; 7 = pK, of histidine; 8 = pK of imidazole; 9 = pK, of citric acid;
10 = pK of tris (hydroxymethy1)aminomethane; 11 = pK of 2-amino-2-methyl-l,3-propanediol;
12 = pic of orthoboric acid.
PK

76 MATHEMATICAL MODEL FOR ISOTACHOPHORESIS
the separation of anionic species, show a strong temperature dependence. Therefore, it
is to be expected that for the separations of anions of very low effective mobility this
influence cannot be neglected. In the computer program, different mobilities and pK
values can be entered for the different zones and corrections for this effect can be made.
4.4.4. Influence of activity coefficients
#en an equilibrium
is considered, the pH and pK values are defined as
pH = -log aH
and
pK=-log K= pH +log (uAH/uA)
and the activities are defined as
-
‘A - ‘A TA
where a,is the activity, cAis the molar concentration and yAis the activity coefficient
of component A. These activity coefficients can be calculated from the Debye-Huckel
limiting law as
log yA= Kz’fl
where K is a constant, z is the valence and Z is the ionic strength of the solution. Although
it is possible to compute all activity coefficients and to develop a computer program that
includes these coefficients, they are neglected in our isotachophoretic model. This means
that the following definitions are used:
pH = - log [H+]
and
PK= PH + log ([A] / [AH1 )
Interpreting all pH values as -log [W] and all pK values as p&, correct computations
can be carried out, The p& values can be calculated from the p& values by correction
for the activity coefficients and repeated calculations give the exact values.
4.5. CHECK OF THE ISOTACHOPHORETIC MODEL
When working with a stabilized electric current, the conductivity of a zone determines
the characteristic potential gradient over the zone. The heat produced per unit volume
corresponds to ZE and determines the temperature of the zone in the steady state.
For a check of the theory, the difference between the temperature inside the
capillary tube and the temperature of the air (air cooling) should be known. The
difference in temperature measured with a thermocouple (dT,,) is different from the

CHECK OF THE ISOTACHOPHORETIC MODEL 77
true value, but there is a linear relationship between dTth and the real difference in
temperature [22] (see Chapter 6). As a linear relationship between the conductivity of a
zone and the temperature inside the capillary tube can be expected over a limited traject,
a linear relationship can also be expected between the conductivity of the zones and the temperature
detected by means of a thermocouple. This relationship is used to check the theory.
In this section, some calculations of the parameters of the different zones are made
and the results are compared with those obtained in some experiments. Calculations
were made both for anions and cations, correcting for the influence of activity
coefficients, relaxation and electrophoretic effects and different temperatures in the
zones. The temperatures in the zones were estimated from the thermocouple signals
and the temperatures in the capillary tube [22] .
Calculations were made for the cations BaZ+, Caz+, Mg2+, Fez+, k?, Ag and Na+. These
cations were chosen because the slope of the function A. = KJc* agrees reasonably
well with the expected slope according to the Onsager relationship. If other influences
such as complex formation occur, the decreasing effect on the mobility should be greater
and the calculations would not be valid as the computer program does not deal with
effects such as complex formation. For the anionic calculations, acids were chosen for
which data such as ionic mobilities and pK values were readily available [ 13, 19,23,24] .
The concentrations, pH values, step heights and zone resistances are given in
Table 4.6 for cations in the system WKAC (see Table 11.3) and in the Tables 4.7 and 4.8
for anions in the systems histidine hydrochloride and imidazole hydrochloride (see Tables
12.1 and 12.2) respectively.
Firstly, calculations were made with no corrections. The relationship between the
experimentally measured step heights and the uncorrected calculated conductivities of
the zones are given in Figs.4.l5a, 4.16a and 4.17a. Although one continuous relationship
would be expected, two distinguishable curves are obtained for these relationships.
This can be understood easily, as follows. If the zone resistances are computed without
applying corrections for the Onsager relationship, there will be deviations from the real
electrical resistances actually present. The zone resistances calculated will be smaller
than the actual resistances because relaxation and electrophoretic effects, which decrease
TABLE 4.6
SOME EXPERIMENTAL AND CALCULATED VALUES FOR CATIONS IN THE OPERATIONAL
SYSTEM AT pH 5.4 (SEE TABLE 11.3)
A values are given in C’ cm-I.
Cation
K+
&+
Na+
BaZ+
Ca ’+
Mg"
Fe 2+
l/h. los
without
correc-
tions
0.874
1.029
1.272
1.013
1.077
1.210
1.188
i/h 10'
with
correc-
tions
0.8930
1.0440
1.2825
1.1152
1.1818
1.3215
1.2969
-
Calculated
concentration of
the ionized part
(mole/ 1)
0.0100
0.0094
0.0086
0.0048
0.0046
0.0044
0.0045
PH Step height
(mm)
5.39 220
5.36 260
5.32 302
5.36 264
5.35 284
5.33 3 14
5.33 3 12

78 MATHEMATICAL MODEL FOR ISOTACHOPHORZSIS
TABLE4.7
SOME EXPERIMENTAL AND CALCULATED VALUES FOR ANIONS IN THE OPERATIONAL
SYSTEM AT pH 6 (SEE TABLE 12.1)
Ionic species l/h. lo3 l/h. lo3 Calculated PH Step height
without with concentration of (mm)
correc- correc- the ionized part
tions tions (molell)
Acetic acid
Benzoic acid
rn-Nitrobenzoic acid
pNitrobenzoic acid
Capric acid
Caprylic acid
Chloric acid
Crotonic acid
Formic acid
Glycolic acid
Hydrofluoric acid
Iodic acid
Lactic acid
Nicotinic acid
Nitric acid
Nitrous acid
Methacrylic acid
Pelargonic acid
Picric acid
0-Chloropropionic acid
Salicylic acid
Sulphamic acid
Sulphanilic acid
Isovaleric acid
Adipic acid
Maleic acid
dl-Malic acid
Malonic acid
Oxalic acid
Pimelic acid
Succinic acid
Sulphuric acid
Tartaric acid
Tartronic acid
2.036
2.504
2.561
2.560
3.075
3.064
1.230
2.414
1.474
1.996
1.468
1.977
2.268
2.526
1.120
1.115
2.295
3.100
2.648
2.283
2.334
1.623
2.483
2.660
1.543
1.689
1.402
1.257
1.098
1.626
1.511
0.996
1.257
1.203
*Concentration of the monovalent anions.
**Concentration of the divalent anions.
2.195
2.689
2.749
2.764
3.246
3.235
1.349
2.591
1.608
2.160
1.600
2.142
2.450
2.697
1.225
1.219
2.469
3.286
2.832
2.461
2.512
1.766
2.672
2.831
1.869
1.900
1.655
1.520
1.331
1.972
1.759
1.204
1.521
1.458
0.0082*
0.0078
0.0078
0.0078
0.0070
0.0070
0.0097
0.0078
0.0092
0.0084
0.0092
0.0085
0.0081
0.0076
0.0099
0.0099
0.0080
0.0070
0.0077
0.0081
0.0080
0.0090
0.0078
0.0075
0.0045**
0.0030 0.0027*
0.0042
0.0047
0.0049
0.0044
0.0013 0.0038*
0.0050
0.0047
0.0048
6.12
6.13
6.13
6.13
6.19
6.19
6.04
6.14
6.06
6.10
6.06
6.09
6.11
6.16
6.03
6.03
6.12
6.20
6.14
6.12
6.13
6.07
6.13
6.16
6.06
6.1 1
6.07
6.04
6.03
6.07
6.09
6.02
6.04
6.04
-
366
430
440
44 2
51 1
510
243
416
276
360
277
358
39 1
436
220
21 7
404
494
446
399
408
304
420
460
334
312
286
280
236
345
304
224
280
256

CHECK OF THE ISOTACHOPHORETIC MODEL
TABLE 4.8
SOME EXPERIMENTAL AND CALCULATED VALUES FOR ANIONS IN THE OPERATIONAL
SYSTEM AT pH 7 (SEE TABLE 12.2)
Ionic species l/h- lo3 l/h- lo3 Calculated PH Step height
without with concentration of (mm)
correc- correc- the ionized part
tions tions (mole/l)
Acetic acid
Benzoic acid
rn-Nitrobenzoic acid
p-Nitrobenzoic acid
Capric acid
Caprylic acid
Chloric acid
Crotonic acid
Formic acid
Glycolic acid
Hydrofluoric acid
Iodic acid
Lactic acid
Nicotinic acid
Nitric acid
Nitrous acid
Methacrylic acid
Pelargonic acid
Picric acid
p-Chloropropionic acid
Salicylic acid
Sulphamic acid
Sulphanilic acid
Isovaleric acid
Adipic acid
Maleic acid
dl-Malic acid
Malonic acid
Oxalic acid
Pimelic acid
Succinic acid
Sulphuric acid
Tartaric acid
Tartronic acid
1.5049
1.9019
1.9610
1.9608
2.2575
2.2567
0.9482
1.7952
1.1259
1.5239
1.1236
1.5171
1.7388
1.8520
0.8594
0.8536
1.7407
2.2594
1.7893
1.7398
1.7894
1.2454
1.8972
1.9680
1.1783
1.0679
1.0014
-
0.8422
1.2458
1.0411
0.7681
0.9611
0.91 82
*Concentration of the monovalent anions.
**
Concentration of the divalent anions.
1.5821
1.9711
2.0351
2.0299
2.3104
2.3016
1.0156
1.8668
1.2013
1.6009
1.1985
1.5920
1.8144
1.9182
0.9232
0.9137
1.8715
2.3100
1.8493
1.8140
1.8632
1.3194
1.9654
2.0322
1.3499
1.2207
1.1510
1.0831
0.9634
1.4228
1.1946
0.8931
1.1083
1.0642
0.0075*
0.0065
0.0064
0.0064
0,0059
0,0059
0.0093
0.0068
0.0087
0.0074
0.0087
0.0074
0.0069
0.0066
0.0097
0.0098
0.0068
0.0059
0.0068
0.0069
0.0068
0.0082
0.0066
0.0064
0.0042**
0.0042
0.0045
0.0046
0.0049
0.0041
0.0044
0,0051
0.0046
0.0047
7.1 3
7.18
7.19
7.19
7.23
7.23
7.03
7.17
7.06
7.13
7.06
7.13
7.16
7.18
7.01
7.01
7.16
7.23
7.17
7.16
7.17
7.08
7.18
7.19
7.07
7.06
7.04
7.18
7.01
7.08
7.05
6.99
7.03
7.02
281
340
340
345
4 00
400
190
326
216
286
218
290
314
342
174
170
312
393
350
316
323
244
344
360
252
216
222
204
180
264
224
169
216
195
79

80 MATHEMATICAL MODEL FOR ISOTACHOPHORESIS
Fig.4.15. Relationship between the measured step heights, as found in the linear trace of the thermo-
metric signal, and the calculated zone resistance for some cations in the operational system at
pH 5.4 (see Table 11.3), without corrections (left) and with corrections (right).
h I
40(
3M
Fig.4.16. Relationship between the measured step heights, as found in the linear trace of the
thermometric signal, and the calculated zone resistance for some anions in the operational system
at pH 6 (see Table 12.1), without corrections (left) and with corrections (right).

REFERENCES 81
Fig.4.17. Relationship between the measured step heights, as found in the linear trace of the
thermometric signal, and the calculated zone resistance for some anions in the operational system
at pH 7 (see Table 12.2), without corrections (left) and with corrections (right).
the mobility, have been neglected. Consequently, the resistances of the zones increase.
As these effects are stronger for divalent ionic species, two different curves can be
expected, as illustrated. The influence of the different temperatures on the pK values and
ionic mobilities and the influence of the activity coefficients do not differ very much
for mono- and divalent ionic species.
After corrections have been made for the effects of temperature, activity coefficients
and the Onsager relationship, only one curve is obtained for both mono- and divalent ionic
species (Figs.4.15b, 4.16b and 4.17b, in accordance with the theory. In all instances the
calculated pH values of the zones before and after applying the corrections do not differ
appreciably (not more than 0.01 pH unit for most ionic species). Therefore, no pH measure-
ments were used as a check on the theory. Reasonable values were obtained, however, by
Everaerts and Routs [ 111 .
REFERENCES
1 R. A. Alberty, J. Amer. Chem. Soc. 72, 2361, 1950.
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4 J.L. Beckers and F.M. Everaerts, J. Chromatog., 68 (1972) 207.
5 M. Bier, Electrophoresis, Vols. I and 11, Academic Press, New York, 1959 and 1967.
6 G. Brouwer and G.A. Postema, J. Electrochem. SOC., 117 (1970) 7 and 874.

82 MATHEMATICAL MODEL FOR ISOTACHOPHORESIS
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8 E.B. Disrnukes and R.A. Alberty,J. Amer. Chem. SOC., 76 (1954) 191.
9 V.P. Dole,J. Amer. Chem Soc., 67 (1945) 119.
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12 F. Kohlrausch, Ann. Phys. [Leipzig), 62 (1897) 208.
13 L.G. Longsworthand D.A. McInnes, Chem. Rev., 11 (1932) 171.
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15 R.J. Routs, Thesis, University of Technology, Eindhoven, 1971.
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17 D. Tondeur and J.A. Dodds,J. Chim. Phys., 3 (1972) 441.
18 L. Omstein, Ann. N Y. Acad Sci., 121 (1964) 321.
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Landolt-Bornstein-Zahlen werte und Funktionen aus Physik, Chemie, Astronomie, Geophysik
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20 B.P. Konstantinov and O.V. Oshurkova, Sov. Phys.-Tech. Phys., 11 (1966) 693.
21 S. Hjerth, Thesis, University of Uppsala, Uppsala, 1967.
22 J.L. Beckers, Graduation Rep., University of Technology, Eindhoven, 1970.
23 W.W. Edward, J.W. Clarence, F.R. Bichowsky, N.E. Dorsey and A. Klemenc, International
Critical Tables of Numerical Data, Physics, Chemistry and Technology, McGraw-Hill, New York,
London, 1933.
24 G. Kortiirn, W. Vogel and K. Andrussow, Dissoziations-konstanten Organischer Sauren in
Wussriger Losiing, Butterworths, London, 1961.

Chapter 5
Choice of electrolyte systems
SUMMARY
This chapter describes the method of choosing the most appropriate analysis for
separations according to pK values or mobilities and according to differences in solvents.
Several examples of problems are discussed and the solution of these problems by the use
of isotachopherograms and practical data is illustrated.
5.1. INTRODUCTION
In this chapter, we shall consider the choice of electrolyte systems for isotachophoretic
separations.
In isotachophoresis, ionic species can be separated if their effective mobilities differ
sufficiently. The effective mobility is defined as
meK, = Z ai yimi
i
The degree of dissociation, ai, depends mainly on the pK values, temperature and pH in
the zones. The value of yi, a correction factor for the decreasing effects on the mobility
of the relaxation and electrophoretic effects as described by Onsager, depends mainly
on the ionic concentrations. The value of mi depends on several factors such as solvation,
the radius and charge of the ions and the dielectric constant and viscosity of the solvents.
All of these parameters influence the effective mobility and a well considered choice of
the electrolyte system makes a good separation possible. The use of different solvents
(influence of the dielectric constant and solvation) or different buffers (change in pH
and the influence of complex formation) allows numerous possibilities.
The separation of ionic species can be carried out in different ways, as follows.
(1) The differences in absolute ionic mobilities can be used for the separation of the
ionic species. A particular pH of the buffered system is chosen such that all ionic species
are almost completely dissociated. We shall call these separations ‘separations according
to mobilities’.
(2) The differences in the pK values of the ionic species can be used for the separation.
A particular pH is chosen such that most ionic species are not completely dissociated,
especially when many ionic species have about the same ionic mobility. A pH is chosen
in such a way that maximal differences in effective mobilities are obtained. These
separations will be called ‘separations according to pK values’.
(3) Other solvents can be applied in order to obtain a complete separation. This
technique can be used if the ionic species have about the same ionic mobilities and pK
values, and/or are not or only slightly soluble in a certain solvent.
83

84 CHOICE OF ELECTROLYTE SYSTEMS
(4) Further, other factors such as complex formation, precipitation [ 1 ] and other
specific interactions can be used. We shall not look into these possibilities too deeply,
although they automatically affect the effective mobility. In the Section Applications,
where practical information is given, this subject is discussed in more detail.
A general method of findmg a suitable electrolyte system cannot be given, but in this
chapter we shall discuss several important factors that play a role in the choice of
electrolyte systems (operational systems) and must therefore be taken into account.
At the end of the chapter, we present examples of some problems and separations
that have been solved in order to illustrate the principles of choosing electrolyte systems.
5.1.1. General remarks
In choosing a suitable electrolyte system, several factors play an important role. In
general, the most important requirement is that the system must be chosen in such a
way that the ionic species to be separated have maximal differences in effective mobilities,
in order to achieve a rapid and complete separation. Sometimes, however, it is not
possible to follow this general rule as particular conditions may be necessary for the
sample and there may be limitations to the apparatus available. The choice of a suitable
electrolyte system wil often be a matter of experience by which, sometimes intuitively,
the advantages and disadvantages of the different possibilities must be weighed against
each other. In some instances, a suitable electrolyte system can only be determined
experimentally (Section Applications). In this chapter a number of factors that can
play a role in choosing the electrolyte system are discussed, and some practical examples
are given of electrolyte systems for which data and separations are considered elsewhere
in this book.
The most important factors in choosing electrolyte systems that are discussed are:
the choice of the solvent;
the choice of the buffering counter ionic species;
the choice of the pH of the leading electrolyte;
the choice of the leading ionic species;
the choice of the terminating ionic species;
additions such as spacers (and carriers), stabilizers, surfaceactive compounds and
reference compounds for identification and concentration calibration (internal
standards).
5.2. CHOICE OF THE SOLVENT
The first problem in choosing an electrolyte system is to decide which solvent can be used.
In most instances, water is used as a solvent in electrophoresis, not only because of its
price, availability, etc. but particularly because of its superior solubility properties and its
ionization power. However, other solvents can be used that are more suitable for particular
applications, such as for the separations of substances that are insoluble or only slightly
soluble in water (fatty acids, amino acids, proteins and complexes). When a non-aqueous
solvent has to be used, the choice depends mainly on the properties of the sample.

CHOICE OF THE SOLVENT 85
In general, a solvent suitable for isotachophoresis in capillary tubes must meet the
following requirements:
(1) It must have as small a self-conductance as possible, as a large conductivity results
in undesirable elution phenomena.
(2) The sample must dissolve in the solvent chosen. With regard to the process of
dissolution, two aspects of solvent behaviour are important. The first aspect is the
tendency of the solvent molecules to interact with or to solvate the sample molecules.
For example, water molecules can be considered as dipoles that can arrange themselves
around either positive or negative ions. For this reason, the dipole moment of the solvent
has to be taken into account. The second important role of the solvent is to decrease the
electrostatic interactions between the oppositely charged particles of the ionic substances
as a result of its dielectric constant, according to Coulomb’s law:
The dielectric constant alone is not an adequate measure of the suitability of a solvent
and plays only a minor role in the solubility of ionic substances. Of particular importance
is the specific solvation of anionic and cationic species.
(3) The sample must form charged particles in the solvent.
The sample substances can form charged particles by accepting or losing a proton and by
dissociation, whereby the ions formed will be solvated. In a strongly acidic solvent, the
dissolved substance will accept a proton. For example, an amino acid in 98% formic acid
will give
RNH2 + HCOOH RNg, + HCOO- (5.2)
In a basic solvent, e.g., liquid ammonia, the dissolved amino acid will lose a proton:
RCOOH + NH3 RCOO- f NP4 (5.3)
The dissociation of salts can be expressed by an equilibrium:
M+X-zW+X- (5 -4)
In solvents with high dielectric constants and in very dilute solutions, only free ions are
present, while at lower dielectric constants, the equilibrium lies towards the left-hand side.
According to Brdnsted’s theory (see ref. 2), solvents can be divided into eight classes,
with respect to three properties of water, viz., the basicity, the acidity and the dielectric
constant. The dielectric constant is assigned as positive for a value higher than 30 and as
negative for a value lower than 30. The acidity and basicity can also be expressed
numerically.
Analogous to the expression of Peters (see ref. 2) for a redox system:
RT ox
E,, = E& +- In -
F red
*The terms ‘ox’ and ‘red’ indicate the activities of the oxidant and reductant.
(5.5)

86 CHOICE OF ELECTROLYTE SYSTEMS
Schwarzenbach [3] expressed the acidity as the so-called normal acidity potential,
which is the potential on a platinum electrode, saturated with hydrogen gas at 1 atm,
when dipped into a conjugated acid-base system.
This gives
where 'acid' and 'base' represent the activities of these substances. If E: for water is
taken as zero, the values listed in Table 5.1 are obtained.
TABLE 5.1
NORMAL ACIDITY POTENTIALS ACCORDING TO SCHWARZENBACH 131
Solvent E; -EQ
H,O
NH, -1.00
N,H, -0.92
H2O 0.00
CH,CN 0.24
HCOOH 0.72
The eight classes of solvents according to Brbnsted are given in Table 5.2.
For the equivalent conductance, Onsager gave the following expression (see ref. 4):
where A = equivalent conductance, A, = equivalent conductance at infinite dilution,
D = dielectric constant, T= absolute temperature, z = valency, c = concentration and
1) = dynamic viscosity.
Assuming that for the dissociation constant we can write
it will be clear that the effective mobility decreases rapidly as the dielectric constant
decreases, so that solvents with low dielectric constants are not suitable for isotachophoresis
in general as too high potentials would be required and the heat production would be too
high and could cause boiling of the solvent*. After the division of the solvents into eight
classes and considering the above remarks, it is clear that solvents suitable for isotacho-
phoresis should be chosen from the classes with high dielectric constants. For polar
substances, amphprotic solvents are useful, whereas for the analyses of very weak acids
and bases, basic and acidic solvents, respectively, are. suitable. When considering these
last classes, it must be borne in mind that not all types of apparatus can be used because
of the aggressive properties of some solvents (e.g., formic acid on acrylic ware). A reason-
*It is possible to I;se very thin narrow-bore tubes, and moreover, very effective cooling systems are
now available. This procedure obviates the problems mentioned.
(5.7)

CHOICE OF THE SOLVENT
TABLE 5.2
THE CLASSES OF SOLVENTS ACCORDING TO BRONSTED’S THEORY
For further explanation, see text.
Class Members Examples D Acidity Basicity
I
I1
I11
IV
V
VI
VII
VIII
Amphiprotic media
Acidic media
Basic media
Aprotic media
with high D
Amphiprotic media
with low D
Acidic media
Basic media
Aprotic media
with low D
Water, methanol
Hydrocyanic, formic, hydrofluoric and
Sulphuric acids
Formamide, ethanolamide
N-methy lpropionamide
Dimethyl sulphoxide, dimethylformamide
ace tonitrile
Ethanol, cyclohexanol
Acetic and propionic acids
Pyridine, diosan, diethyl ether
ethylenediamine
Acetone, benzene, carbon tetrachloride,
cyclohexene
+ +
+ +
+ -
+ -
able solvent for most types of substances is methanol. The apparatus developed in our
laboratory meet all of the demands made by the use of methanol as a solvent, and it is
discussed in more detail in the Section lnstrumentation.
5.2.1. Methanol as a solvent
5.2.1.1. Comparative behaviour with water
Methanol, like water, belongs to the group of amphiprotic solvents with relatively high
dielectric constants. Its dielectric constant (31) is lower than that of water (81), however,
and hence the interionic forces in methanol are larger than those in water, according to
Coulomb’s law. In order to compare the acid-base behaviour of water and methanol, we
can study the following reactions:
(a) NW4 +HzO =NH3 + H3@
(b) NV4 + ROH=NH3 + ROP2
(c) CH3 COO- + H2 0 =CH3COOH + OH
(d) CH3 COO- + ROH CH3 COOH + OR-
In these reactions, the number of ions is the same on each side of the equations, so that
the influence of differences in dielectric constants is mainly eliminated. Measurements
on those equations showed that the acid-base characters of water and methanol do not
differ much.
If the number of ions is not the same on each side of the equation, the differences
in the dielectric constants can cause differences in the acid and base constants. For
-
+
+
-
+
t
-
+
87

88 CHOICE OF ELECTROLYTE SYSTEMS
example, for the reaction of an acid HA with the solvent S, we can have the following
reaction :
HA + S=A- + SH'
During the reaction, the number of ions increases. If the interionic forces between A-
and SH+ are small (high dielectric constants), the equilibrium lies on the right-hand side.
With lower dielectric constants, the equilibrium lies towards the left-hand side. On a purely
electrostatic basis, it can be derived that [2]
where K = equilibrium constant; zA = zB + 1 ; zA and zB are the charges of the acid-base
pair; e = standard electrical unit of charge; r = radius of the ions; k = Boltzmann constant;
T= absolute temperature. This equation gives the relationship between the acid and
base constants in the different solvents, and some examples are discussed below.
The acid constant of NH: in water and methanol
Reaction:
NP4 + S=SW + NH3
In this instance, zA =1 andzB = 0. Substituting the constants in eqn. 5.8 for water and
methanol we obtain
log (k)
KCH, OH
= - 1.24 (22,- 1)= - 1.24
Thus the acid constant of NH; in methanol is about 17 greater than that in water.
The acid constant of acetic acid in water and methanol
Reaction:
CH3 COOH + S ECH3 COO- + SH+
Here zA = 0 and zB = -1. Substitution in eqn. 5.8 shows that the acid constant for acetic
acid in methanol is about 17 smaller than that in water.
It can be seen that the change in the acid constants depends on the type of reaction.
In practice, the differences are much larger because, for example, the dimensions of the
molecules are not known exactly and the electrostatic model is not valid as the influence
of the acid-base behaviour of the solvents is neglected.
Another point in comparison with water is the self-dissociation of methanol:
2 CH3 OH
CH3 0- + CH3 OK2
The dissociation constant of methanol is about (for water it is about 10-14). As
with water, we can define a pH and a pOCH3 value for methanolic solutions. Especially
when choosing the pH of the electrolyte systems and when choosing the counter ions,
the pK values of the substances must be known for the solvent chosen. The way in which

CHOICE OF THE SOLVENT
pH and pKvalues can be determined in methanolic solutions is dealt with in the next
section.
5.2.1.2. Determination of pK values in methanolic solutions*
Determination of pff in methanolic solutions
The operational definition of the pH determined electrometrically in water is based
on E measurements of cells of the type
Aqueous solution
?t:ti: 1 I of standard; pH,
KC1, reference electrode
Aqueous solution [ KCI, reference electrode
In general, the indicator electrodes are glass electrodes and the reference electrodes are
calomel electrodes. Es and Ex can be expressed as (25°C)
E s = E ref -Eo md -0.05916l0ga~,~+l$~ (5.9)
Ex = Emf- Eqmd - 0.05916 log^^,^ +E;:,
Combination of eqns. 5.9 and 5.10 gives
Ex-Es -E),x-Ej,s
- log aH,x = - log aH,s + ____
0.0591 6 0.0591 6
The operational definition of the pH is
EX -4
pHx = pHs + ___
0.0591 6
Comparison of eqns. 5.11 and 5.12 shows that
PH, = -logaH,x
if pH, = -log I Z ~ and , ~
89
(5.10)
(5.1 1)
(5.12)
(5.13)
Ej,x = Ej,s (5.14)
In general, this is not exactly true. Bates [5] of the National Bureau of Standards
determined the pH, values of some standard buffer solutions for which
PHs = -log aH,s
If the solution x has about the same ionic strength in the solvent used for the standxd
solution, Ej,S can be considered to be equal to Ej,x and then pH, can be interpreted as
-log aH,x'
For pH measurements in methanolic solvents, the same procedure can be followed.
Because of the different liquid junction potentials for aqueous buffer solutions and
*For other solvents or combinations of soIvents, a simiIar procedure can be followed.,.

90 CHOICE OF ELECTROLYTE SYSTEMS
unknown methanolic solutcons, one must look for standard buffer solutions in the same
kind of methanolic solution as that used for the unknown solution. Using this standard
solution, the two liquid junction potentials will cancel each other again and the measured
pH can be interpreted in terms of hydrogen ion activity.
De Ligny et ul. [6,7] determined the pH (- log c,yk) for some standard solutions in
methanolic solvents according to the method of the National Bureau of Standards for
aqueous solutions.
In the determination of the pH* of standard solutions ( the asterisk here and on other
symbols indicates that the quantities refer to the solutions considered and not to aqueous
solutions) for methanolic solvents, corrections have to be made for the slight association
of ions to give ion pairs. Fuoss and Onsager [8,9] developed a method for the calculation
of the closest approach b and the dissociation constant K of an incompletely dissociated
electrolyte in water, but because for methanolic solvents no accurate values of the
conductivity of electrolytes were available, De Ligny et al. did not correct for the ion
pair association.
For the estimation of log y*, De Lgny et el. used the Gronwdl-LaMer-Sandved
equation:
(5.15)
The pH* values for some buffers as determined by De Ligny et al. are given in Table 5.3.
In the experiments, the reference electrode (calomel) was placed in a potassium chloride
solution of the solvent that was used to prepare the buffers. Using the values mentioned,
TABLE 5.3
pH* VALUES FOR THE OXALATE AND SUCCINATE BUFFER IN METHANOLIC SOLUTIONS
AS DETERMINED BY DE LIGNY [ 11 ]
Reproduced by permission of Dr. C.L. de Ligny.
0.01 MH,Ox + 0.01 MNH,HOx 0.01 MH, Succ + 0.01 MLiHSucc
Methanol PH* Methanol PH*
(%I (%I
0 2.15
10 2.19
20 2.25
30 2.30
40 2.38
50 2.47
60 2.58
70 2.76
80 3.13
90 3.73
100 5.19
0
10
20
30
40
50
60
70
80
90
100
4.12
4.30
4.48
4.67
4.87
5.07
5.30
5.57
6.01
6.73
8.75

CHOICE OF THE SOLVENT 91
TABLE 5.4
LIQUID JUNCTION POTENTIALS BETWEEN STANDARD SOLUTIONS IN AQUEOUS
MIXTURES 11 ]
Methanol and a saturated solution of potassium chloride in water for oxalate and succinate buffers.
Reproduced by permission of Dr. C.L. de Ligny.
Oxalate buffer Succinate buffer
Methanol
(%I
0
39.13
70
84.2
84.21
94.2
100
100
0.0046
0.0091
0.01 14
-0.009
-0.0082
-0.0435
-0.1338
-0.1347
Methanol E;
(%)
0 0.0041
43.31 0.0083
64.2 0.0132
84.1 -0.0091
84.2 -0.0086
94.19 -0.0485
100 -0.1329
De Ligny et al. determined the liquid junction potential between the buffer solutions in
methanol and a saturated solution of potassium chloride in water.
The liquid junction potentials between standard solutions in methanol-water mixtures
and a saturated solution of potassium chloride in water are given in Table 5.4. When the
pH of a solution of methanol-water mixtures is measured by means of a pH meter, standardised
by a solution of potassium chloride in water, the error due to the liquid junction
potential can be calculated by means of the equation
E:* (methanol-water) - E:,...-&-
(5.16)
For higher percentages of methanol, this dpH* value can be very high. In order to
calibrate the pH meter for measurements in methanolic solutions, a standard buffer
solution in water can also be used [lo, 111 and then the correct pH* can be calculated
from the observed pH by subtracting a correction factor (6) [12]. The 6 values are given
in Table 5.5.
The liquid junction potentials at the standard solution (alcohol-water mixtures)/
saturated aqueous potassium chloride boundaries are independent of the nature of the
buffering solution.
TABLE 5.5
6 CORRECTION TERMS FOR SOME METHAhOL-WATER MIXTURES
Methanol
(%I
6 (pH* units)
0 -
43.3 0.11
64 0.22
94.29 -0.86

92 CHOICE OF ELECTROLYTE SYSTEMS
In the pH* measurements carried out in the work described in this chapter, the same
procedure was used as described by De Ligny et al. Standard buffer solutions and pH*
values used were as determined De Ligny et al.
Determination of pK values in methanolic solutions
The determinations of pK values [13] can be carried out in several ways, the most
important of which are the conductivity, electrometric, spectrometric and colorimetric
methods. In this section, the electrometric method is discussed,
Rorabacher et al. [14] gave some definitions relating to the pK. The activity
equilibrium constant is defined as
The equilibrium constant based on concentrations is
K,* = [HI [A1 /[HA1
and the mixed-mode equilibrium constant is defined as
K$ = a$ [A] /[HA] or K z = K:rfi
Hence
In the electrometric method, the concept of the half neutralization point (HNP) is
used. The HNF’ is the point in the acid-base titration at which half of the amount of
acid (or base) present has been neutralized. According to eqn. 5.1 1 , this means that the
pK; is determined. The thermodynamic equilibrium constant can be calculated from
the mixed-mode constant by correcting for the activity coefficients (eqn. 5.20).
(5.17)
(5.18)
(5.19)
Ex penmen tal values
In order to choose an optimum electrolyte system in isotachophoresis, the pK values
of the buffers and the pK values of the sample ionic species must be known. Some
pKk values for anionic species and bases have been determined in 95% methanol by
the electrometric method and the results are given in Table 5.6.
5.3. CHOICE OF THE BUFFERING COUNTER IONIC SPECIES
(5.20)
With regard to the choice of the buffering counter ionic species, three important
points can be distinguished. Firstly, the buffering counter ions act as a counter ion by
which the principle of electroneutrality is obeyed. At the same time, the counter ionic
species can be used to form complexes with the sample ionic species in order to affect
the effective mobility of these ionic species in a favourable manner, particularly if the
ionic species to be separated have similar mobilities. Even buffering counter ionic species
with which particular sample ionic species do not migrate or are precipitated can be
chosen; by this they do not disturb the separation procedure. Deman [ 11 used this

CHOICE OF THE pH OF THE LEADING ELECTROLYTE
TABLE 5.6
EXPERIMENTALLY DETERMINED pK& VALUES FOR SOME ANIONIC SPECIES IN 95%
METHANOL
Ionic species PKh Ionic species PKL
Acetic acid
Adipic acid
Azelaic acid
Benzoic acid
Bu tyric acid
Caproic acid
Caprylic acid
Die thanolamine
Formic acid
Glutaric acid
Hippuric acid
Histidine
Irnidazole
Lauric acid
Linoleic acid
7.9
7.55-9.1
7.65-9.0
7.5
8.0
8.0
8.0
9.6
6.45
7.5-9.2
6.95
6.0- 10.15
6.55
7.9
7.9
Maleic acid
Malonic acid
Monoethanolamine
Myristic acid
Orotic acid
Oxalic acid
Palmitic acid
F’imelic acid
Pyruvic acid
Salicylic acid
Suberic acid
Succinic acid
Trie thanolamine
Tris
Isovaleric acid
4.6-?
5.9-9.7
9.6
8.1
8.8
4.5-8.3
8.0
7.6-8.95
5.9
6.2
7.6-8.95
93
7.25-9.4
7.9
9.05
8.05
principle for his ‘precipitation electrophoresis’. In isotachophoresis in capillary tubes,
precipitation is undesirable as it may produce stoppages in the capillary tube.
Asecond point to be considered is the use of buffering counter ions with a W
absorption power, if a W detector is used. The concenqations of the counter ions and
the pH are different in the various zones and, if we use a buffer with a molar extinction
coefficient that is influenced by the pN (i.e., if only a particular ionic form of the buffer
shows a UV absorbance), the different zones can be detected by the differences in the
UV absorption of the buffer ions in these zones. This method of detection can be
applied particularly if the sample ions show no or only a slight UV absorption.
The third point and the most important function of the buffering counter ionic
species is its buffering capacity for the stabilization and regulation of the pH in the
different zones, by which effective mobilities of the ionic species are fixed and by which
a ‘steady state’ can be maintained. The selection of a suitable pH, in combination with
the type of buffer, is considered in the next section.
5.4. CHOICE OF THE pH OF THE LEADING ELECTROLYTE
In this section, we shall consider separations according to pK valves because these
separations are closely related to the choice of the pH of the leading electrolyte. For
separations according to mobilities, the pH is of less importance. Two points are very
important here: firstly, the choice of the pH itself, and secondly, the choice of the type
of buffering counter ionic species, which defines the pH at which the analysis is to be
performed. In general, the pH is chosen in such a way that maximal differences in
effective mobilities can be obtained according to eqn. 3.18, but a limitation is that if the
pH in a zone differs more than 2 pH units from the pK values of the ionic species in that

94 CHOICE OF ELECTROLYTE SYSTEMS
zone, such low effective mobilities are obtained that the potentials required rise above
the maximal potential of the stabilized d.c. power supply (this factor is of minor
importance for the buffer.)
Another limitation is due to the buffering capacity of the counter ions. A maximd
buffering capacity is obtained if pK + 1 pK - 1. Hence the buffer will have a
low buffering capacity if its pK value differs more than about 2 pH units from the pH
in a zone. The pH lies between the pK values of buffer and sample ionic species, so
that we cannot use a buffer when its pK value differs more than about 3 pK units from
that of the sample ionic species (this is valid when the pK is lower than the pK values
of the sample ions). In order to demonstrate this important effect, the relationship
between the pK values of ionic species in the sample and the pH values of their zones
is shown in Fig.5.1 for a pKB of 6 and a pH of the leading electrolyte, pH, of 5.75.
Fig.5.1 shows clearly that the buffer has a low buffering capacity if its pKB differs
more than about 3 pH units from the pK of the ionic species. In general, a buffer should
be chosen with a pKB less than 3 pH units lower than the pK value of the anionic species
to be separated.
After considering the limitations concerning demands for the buffering ions at a
chosen pH, the next problem is to choose the pH, value, and two approaches are
possible.
If no data such as pK values and mobilities are available, the experimental method must
be used. All step heights of the substances concerned have to be measured at different
pH, values and for several electrolyte systems, after which a pH, value can be chosen
for the optimal separation (maximal differences in step heights). A combination of
systems can be applied.
If all data are known, theoretically all effective mobilities can be calculated and from
the results obtained the pH, that shows maximal differences in effective mobilities can
be decided.
In order to demonstrate those two approaches to separations according to pK values,
eleven anionic species that cannot be separated according to mobilities in the system
histidine/histidine hydrochloride at a pH, of 6.02 (see Table 12.1) were selected. The
effective mobilities were computed with a computer program for five electrolyte systems
at different pH, values and all step heights were measured for the systems. In Table 5.7,
the conditions are given for the five electrolyte systems and Table 5.8 gives the calculated
effective mobilities (theoretical method) and measured step heights (experimental
method). In Fig.5.2 the step heights are plotted for the different systems. The results
indicate that the differences in effective mobilities (and step heights) are much greater
at lower pH values of the leading electrolyte, which permits better separations to be
achieved.
Some separations have been carried out. Fig.5.3A shows the electropherogram for
the separation of a mixture of trichloroacetate, P-chloropropionate, benzoate, crotonate,
paminobenzoate and trimethyl acetate at a pH, of 6.02. The terminator was glutamate,
and the leading ion was chloride. No complete separation could be achieved. In Fig.5.3B,
the separation is shown for the same mixture in system E (see Table 5.7) at a pH, of4.
Trimethyl acetate was used as the terminator and a complete separation could be easily
achieved. A thermometric detector (copper-constantan thermocouple) was used.

CHOICE OF THE pH OF THE LEADING ELECTROLYTE
9
8
7
6
0
z-
a
t,
- 0 6
PKion- apeciea
/
/
I
/
/
/
I
/
Fig.5.1. Relafonship between the pH of the zone and the pK value of an anionic species for pHL =
5.75 and pK (counter ion) = 6.
10
/
95

96 CHOICE OF ELECTROLYTE SYSTEMS
TABLE 5.7
ELECTROLYTE SYSTEMS FOR SEPARATIONS ACCORDING TO pK VALUES
System Leading electrolyte PHL
A 0.01 NHCl+ pyridine 5.5 I0
B 0.01 NHQ+ pyridine 5.0 70
C 0.01 NHCl + aniline 5.0 70
D 0.01 NHQ +aniline 4.5 70
E 0.01 NHQ +aniline 4.0 70
TABLE 5.8
EXPERIMENTAL AND CALCULATED VALUES FOR ANIONS IN SYSTEMS A-E (SEE TABLE
5.7)
The mobilities (meK) are given in 10 cm2 /V.sec.
NO.* System A System B System C System D System E
1
2
3
4
5
6
7
8
9
10
11
meff H(mm) meff H(mm) meff H(mm) meff H(mm) meff H(mm)
26.33 428
28.13 424
29.59 403
31.20 380
32.21 317
34.58 338
31.16 377
31.97 372
30.24 380
34.26 361
36.60 344
22.39
24.36
25.94
27.81
30.45
33.26
30.80
31.16
30.24
34.10
36.60
-
455
432
41 3
382
363
377
360
313
347
343
21.42 590
23.41 533
25.08 518
27.08 478
30.24 428
33.15 394
30.19 429
31.12 398
30.24 418
34.09 379
36.60 370
16.76 73.2
18.62 629
20.20 618
22.19 547
26.78 477
30.25 435
29.80 426
29.15 404
30.24 408
33.60 382
36.60 372
13.81 810
15.44 722
16.85 688
18.65 624
23.31 522
26.86 449
27.99 433
26.41 409
30.24 408
32.49 392
36.60 370
*1= Trimethylacetic acid; 2 = p-aminobenzoic acid; 3 = butyric acid; 4 = crotonic acid; 5 = benzoic
acid; 6 = 0-chloropropionic acid; 7 = p-nitrobenzoic acid; 8 = sulphanilic acid; 9 = picric acid;
10 = salicylic acid; 11 = trichloroacetic acid.
5.5. CHOICE OF THE TERMINATING AND LEADING IONIC SPECIES
As already described earlier (section 2.4), the sample is introduced into the apparatus
between a leading and a terminating electrolyte. The choice of the buffering counter ions
for these electrolytes depends on the choice of the pH. The terminating and leading ions
are generally chosen such that the leading ion has a higher and the terminating ion a lower
effective mobility than those of all sample ionic species. Another requirement is that all
substances must be very pure. Small amounts of impurities in the terminating solution,
having higher mobilities than that of the terminating ions, will be pushed forwards by the
large potential gradient on the spot, will migrate through all preceding sample zones and
will create zones of impurities at the separation boundaries according to their effective
mobilities. These impurity zones become elongated with time, depending on the effective

CHOICE OF THE TERMINATING AND LEADING IONIC SPECIES 97
SOD-
100-
300 -
-
System
E D C 8 A
Fig.5.2. Graphical representation of the step heights (mm) as measured from the linear signal of a
thermometric detector. The step heights were obtained in five electrolyte systems: see Table 5.7 for
the definition of the electrolyte systems A-E and Table 5.8 for the key to the numbers 1-11.

98
c
t
CHOICE OF ELECTROLYTE SYSTEMS
Fig.5.3. Difference in the separation according to mobilities and the separation according to pK
values. The electropherograms were obtained with a thermometric detector. The electric current was
stabilized at 70 PA. T = Increasing temperature and t = time. Operational system: A, pH = 6
(Table 12.1); B, system E (Tabie 5.8). 1 = Chloride; 2 = trichloroacetate; 3 = p-chioropropionate;
4 = benzoate; 5 = crotonate; 6 = p-aminobenzoate; 7 = trimethylacetate; 8 = glutamate.
mobilities and concentrations of the impurities and the time required for the analyses.
In a similar way, impurities in the leading electrolyte will remain behind if their effective
mobilities are lower than that of the leading ions. Impurities in the terminating electrolyte
with mobilities lower than that of the terminating ion and impurities in the leading
electrolyte with mobilities higher than that of the leading ion do not affect the separation
procedure.
In experiments with thermocouples as the detector, electrolyte solutions were used
that were later found to be too impure when using detectors with a higher resolving
power such as W and conductivity detectors. Especially in these instances and if very
dilute samples are analyzed, the purity of the electrolyte solutions is very important. The
purity of the electrolyte solutions chosen can be checked by running a blank experiment
without a sample. If a counter flow of leading electrolyte is used in order to extend the
time of analysis, small amounts of impurities can be detected. It is not remarkable that
chemical substances that are chromatographically pure often appear to be very impure in
isotachophoretic analyses.
Sometimes, the rule mL >n~,,,,,~, >m, need not be followed. If one is interested

ADDITIONS TO THE ELECTROLYTE SOLUTIONS 99
only in a particular component of the sample solution, a leading andlor terminating ion
can be chosen with a mobility such that only some particular sample ionic species will have
a mobility between those of the leading and terminating ions. Only these components of
the sample will form consecutive zones between the terminating and leading zones.
Sample ionic species with higher or lower mobilities run in front of the leading zone or
remain behnd the terminating zone, respectively*. Such a choice of the electrolyte
system can facilitate i;ie interpretation of complicated isotachopherograms. It is
particularly useful when ionic species at very large concentrations are present in the
sample, e.g., Na', K’ and Cl- ions in biological fluids. These ionic species can be removed
from the sample by using a leading ion with a lower effective mobility. Long analysis
times, however, can still be expected.
5.6. ADDITIONS TO THE ELECTROLYTE SOLUTIONS
5.6.1. Stabilizers
In several chromatographic and electrophoretic techniques, stabilizing media are used.
Stabilization can be effected by choosing suitable supporting media (e.g , paper,
cellulose acetate, glass walls) or by additions to the solvents used (e.g. , starch gels,
Sephadex, sucrose gradients). In many instances, these systems can be used only once,
because of adsorption phenomena.
For isotachophoresis in capillary tubes, no stabilizing media are needed for the
separations of particles with an average molecule weight of less than about 3000. As the
methods of stabilization are treated in detail in the literature, only those stabilization
media used in our own experiments are considered.
5.6.2. Surface-active chemicals
Sometimes, additions are needed in isotachophoretic experiments in which high-
resolution detection systems are used. In Chapter 6, additions when W and conducto-
metric detectors are used are described. In the Section Applications (Chapters 8-17),
additions are given for the electrolyte systems used. These components are added in
order to suppress the undesirable electroendosmotic flow.
5.6.3. Reference materials for identification and calibration of concentrations
Reference materials for the identification and calibration of concentrations can
sometimes be used. These additions are described in the Section Applications.
5.6.4. Spacers and carriers
Spacers can sometimes be used in electrophoretic separation methods. In isotacho-
phoresis, the use of spacers can never improve separations according to differences in
effective mobilities, for the following reasons. A spacer is generally an ionic species with
an effective mobility between those of the ionic species to be separated. Hence, if the
*These ions do not influence the separation process.

1.00 CHOICE OF ELECTROLYTE SYSTEMS
differences between the effective mobilities of the ionic species to be separated are too
small for them to be separated, the addition of a spacer will further reduce the differences
in mobilities so that a separation cannot be achieved. Sometimes, however, it can be
advantageous to use a spacer, for instance if ionic species can be separated easily, but if
their zones are small and close together so that there is a risk that the detector cannot
distinguish the separated zones. In such a case we can use, for example, a non-UV-
absorbing spacer (if a UV absorption detector is being used) that separates these zones so
that they can be detected separately. Similarly, we can utilize a UV-absorbing spacer in
order to make the detection of consecutive zones of non-UV-absorbing ionic species
possible.
Another application (see Chapter 13) of spacers is in the separation of molecules
with a high molecular weight, e.g., proteins. For the separation and detection, a series of
compounds can be used with a large range of effective mobilities in the operational
system chosen and, almost always related to this, a large pH gradient. Of course, these
additives are a combination of spacers and carriers (see Chapter 13), but proteins normally
require stabilization with electrolytes. If a protein is introduced in such a gradient, it will
migrate together with the carrier, by which it is diluted, at such a position that its effec-
tive mobility corresponds to the related pH. This subject is dealt with in greater detail in
the Section Applications, where practical information on protein separations is presented.
5.7. DISCUSSION
Prescriptions for the selection of electrolyte systems that are always valid cannot be
given, because the choice of an electrolyte system depends to a great extent on the sample
being analyzed. In order to demonstrate the method of choosing a suitable electrolyte
system, some examples of electrolyte systems are considered in the remainder of this
chapter. Further information and experimental data are given in the Section Applications.
A scheme that can be of help in choosing an electrolyte system is shown in Fig.5.4.
5.8. EXAMPLES
Example A. Suppose we wish to separate two anionic species A and B with ionic
mobilities* of 30 and 50 and with pK values of 1 and 3, respectively. We must decide the
preferred type of separation (according to pK values or according to mobilities), the
pH, and the limits for the effective mobilities of the leading and terminating ionic species.
The effective mobility of the leading ionic species must be greater than 50 and that
of the terminating ionic species must be less than 30. (One must consider the possibility
that at certain pH values the effective mobilities of the sample ionic species can be
much lower than 30 or 50). In this instance we would prefer a separation according to
mobilities. Here the differences between the ionic species are sufficiently large and the
pH, should be about 2 (because the pK values are 1 and 3) for a separation to be carried
out according to pK values. At this pH, the concentration of the hydrogen ions is so high
that the current carried by the counter ions and hydrogen ions is the largest part of the
*.10-5 - cm*/V sec.

EXAMPLES
Choice of solvent.
Can water be used?
NO -
YES - Look for another
solvent **
I
in chosen solvent
Choose several electrolyte
systems that could be
possible*
c
YES t
with computer program an
optimal electrolyte system
can be calculated
I -
NO
-
Follow experimental method
in order to find an
optimal system
Separations can be
carried out?
If no separations can be
carried out because the
differences in effective
mobilities are too small,
look for another solvent
+
t
Electrolyte system
can be chosen
*This step is very important. Here one must take into account all possibilities
dependent on the sample given. Keep in your mind factors such BS complex
formation, precipitations, pH, the choice of leading and terminating ionic
species, concentrations etc.
**Consider the choice of basic and acidic media. Consider also the possibility
that the substances must take a charge.
Fig.5.4. Scheme for the choice of an electrolyte system.
total current. Hence the effective current carried by the anions is very small, so that long
analysis times can be expected (small step heights may be the result). We can choose a
pH, of about 5 or above, at which level both anionic species are nearly completely
ionized. If the pK values are 5 and 7 and the mobilities remain 30 and 50, for instance,
we could apply a combination of separations according to pK values and mobilities. At
a pH of about 6, the effective mobility of anion A would be nearly 50, but that of anion
B would be about 3 because its degree of dissociation is about 0.1. In that event, a
terminating anionic species with an effective mobility of less than 3 should be used.
101

102 CHOICE OF ELECTROLYTE SYSTEMS
Example B. Let us consider the type of buffer solution that would be preferred for
the separation of Al*, Ba2+ and Na': (a) hydrochloric acid; (b) potassium acetate-acetic
acid; or (c) potassium hydrogen sulphate.
The best system to choose is system (b), for the following reasons. Alw is a so-called
cationic acid; it undergoes partial protolysis according to the reactions
Al(H20): Al(0H) (HZ0)y + H’
Al(0H) (HzO)?
Al(0Wz (HzO); + W
etc. As a result of this property, a solution of aluminium chloride has a rather low pH,
which means that A13+ loses H’ according to the above reactions. During the analysis,
the H’ migrate away from the Al zone because its effective mobility is much greater than
that of N3'; hence the equilibrium in the zone is disturbed and the reaction tends
towards the right-hand side. By this procedure, we shall not have an isotachophoretic
system but a moving-boundary system if we do not use a buffering counter ionic species.
For this reason, system (a) is not suitable, and system (c) is not suitable because barium
sulphate is not soluble and it also does not have a buffering effect.
ExampZe C The determination of the alkali metals is one of the more difficult
problems in analysis. Complicated treatments are necessary in order to determine them,
and a mixture of all of them is particularly difficult to analyse.
Let us consider the type of electrolyte system that could be used for the isotacho-
phoretic separation of mixtures of alkali metals. In the first instance, we could choose
water as the solvent. The ionic mobilities of the alkali metals in water are known (see
Table 5.9). The pK values of these metals are not important because all ionic species are
completely ionized.
On considering the mobilities, it will be clear that in water Cs+, Rb' and probably K’
cannot be separated as the differences in their effective mobilities are too small. The use
of different counter ions and different pH, values will also not be successful as the alkali
metals show a similar behaviour. For this reason, the next step is to look for another
TABLE 5.9
MOBILITIES AND STEP HEIGHTS OF SOME CATIONS MEASURED AS THE LINEAR SIGNAL
OF A THERMOMETRIC DETECTOR
The step heights are measured on top of the leading ion H+, in the system MHC1.
Ion m. lo5 (cm'/V*sec) H(mm)
Water Methanol
H+ 362.2 149.7 -
a+ 81.3 62.5 17
Rb+ 80.3 58.4 25
K+ 76.7 54.4 31
Na+ 52.8 46.8 46
Li+ 40.2 40.4 61
a" -
- 125

EXAMPLES 103
solvent, hoping that a different solvation will affect the ionic mobilities in a favourable
way. As the ions concerned do not show any proton interaction, we must seek a
suitable solvent in the class of amphiprotic solvents, preferably with a high dielectric
constant. In that case, we can try methanol. The ionic mobilities of the alkali metals in
methanol are given in Table 5.4. It can be seen that the differences in the ionic mobilities
are much more favourable in methanol, so that we can use methanol for the separation of
the alkali metals.
The second problem is the use of a leadtng ion. In nearly all solvents, the alkali metals
have higher ionic mobilities than other positive ions except H’. Therefore, we should
choose H’ as the leading ion (in methanol there are some positive ions with a higher
effective mobility than CS', e.g., the tetramethylammonium ion). As the terminating ionic
species many positive ions can be used; in this example we tried Cu2+ ions.
In general, it is preferable to use a buffering counter ion, but for the separation af the
alkali metals the pH is not important and, because no disturbances can be expected, we
chose a non-buffering ion (chloride) as the counter ionic species.
We carried out some experiments in order to check this system, The step heights
measured with a thermocouple are given in Table 5.9. As the leading electrolyte we used a
solution of 0.OlNhydrochloride in methanol (95%) (MHCl) and as the terminator a solution
of 0.1N coppel(I1) chloride in methanol. Firstly, all step heights of the alkali metals
were measured and then a separation of a mixture of the alkali metals was carried out.
The isotachopherogram for this separation is shown in Fig.5.5. A rapid and complete
separation was easily obtained.
In Chapter 16, more data about cationic separations with both water and methanol as
solvents are given.
Example D. In Chapter 14 the separation of some nucleotides is considered; in this
example, we shall discuss how to choose a suitable electrolyte system for the separation
of nucleotide diphosphates.
As the solvent we use water as it dissolves all ionic species easily. As not all pK values
and mobilities of the diphosphates are known, we have to use the experimental method
in order to find a suitable electrolyte system. We have chosen some electrolyte systems
with pH, values of 3.4,3.7,4.2,4.6, 6.0 and 7.0 (these values were chosen because
several nucleotides have pK values between 2 and 5).
As the diphosphates will be separated as negative ions at the chosen pH values, we use
chloride as the leading ion and positively charged ionic species as the buffering counter
ionic species. It can be seen that the pH, values of the systems do not differ much from
the pK values. The conditions for the chosen electrolyte systems are given in Table 5.10
and the measured step heights are given in Table 5.1 1 and are shown graphically in
Fig.5.6. It can be seen from Fig.5.6 that maximal differences in effective mobilities are
obtained at lower pH values. For the separation of a mixture of diphosphates, we chose a
pH, of 3.7. The isotachopherogram of this separation is shown in Fig.5.7. The detection
was performed with a thermometric detector (copper-constantan thermocouple).
Example E. Fatty acids, especially the higher fatty acids, are slightly soluble in water,
so that water cannot be used as the solvent for their separation. Methanol is a better

104
7>
I:
CHOICE OF ELECTROLYTE SYSTEMS
Fig.5.5. Isotachopherogram for the separation of alkali metals in methanol (for operational system,
see Table 16.4). The detection was performed with a thermometric detector. T= increasing
temperature; t = time: The electric current was stabilized at 70 PA. 1 = H+; 2 = Cs+; 3 = Rb'; 4 = K+;
5 = Na+; 6 = LT; 7 = Cu2+.
TABLE 5.10
DIFFERENT ELECTROLYTE SYSTEMS FOR THE SEPARATION OF NUCLEOTIDES
For operational systems, see section 14.2
No. System pH Leading electrolyte Electric current Terminator
o( A)
I WAdQ 3.4 0.01 NHCl + adenosine 70 Caproic acid
I1 WaNCl 3.7 0.01 NHCl + a-naphthylamine 70 Caproic acid
111 WAnCI(1) 4.2 0.01 N HCl + aniline 70 Pivalic acid
IV WAnCl(I1) 4.6 0.01 NHCl +aniline 70 F'ivalic acid
v WHiScl 6.0 0.01 NHCl + histidine 70 Cacodylic acid
VI WImCl 7.0 0.01 NHCl+ imidazole 70 Benz yl-dl-asparigine

EXAMPLES
TABLE 5.11
STEP HEIGHTS OF THE DIPHOSPHATES OF NUCLEOTIDES MEASURED AS THE LINEAR
SIGNAL OF A THERMOMETRIC DETECTOR
The step heights are given in millimetres from the level of the leading electrolyte zone,
Ionic
System
species 1 I1 111 IV V VI
ADP 318 268 224 170 186 108
GDP 230 21 0 176 152 192 112
CDP 356 312 276 192 184 100
UDP 172 164 168 136 178 100
solvent for the higher fatty acids and in this example we shall consider the separation of
some fatty acids in methanol.
The mobilities of the fatty acids and their pK values in methanol are not known
exactly, and we therefore have to use the experimental method in order to find a suitable
4
300
200
100
""*\
0 I I1 111 .,.,
IV v VI -
I
S
Fig.5.6. Graphical representation of step heights of nucleotide diphosphates, (iz mm) measured as the
linear trace of a thermometric detector. The step heights were measured in different operational
systems (s), as given in Table 5.10.
105

106
67
/
6
i-
CHOICE OF ELECTROLYTE SYSTEMS
Fig.5.7. Isotachopherogram for the separation of a mixture of ADP, GDP, CDP and UDP in the
operational system at pH = 3.7 (Table 14.2). The detection was performed with a thermometric
detector. T = increasing temperature; t = time. The time in this experiment was short (15 min)
because there is a great difference in the effective mobilities. The electric current was stabilized
at 70pk 1 = Chloride; 2 = UDP; 3 = GDP; 4 = ADP; 5 = CDP; 6 = caproate.
electrolyte system for their separation. In Table 5.6, some pK values of fatty acids in
methanol are given; most of them are about 8, and we therefore have to choose a pH,
of 8-9 in order to separate them according to pK values. As a buffering counter ionic
species, Tris or Triethanolamine can then be used.
We chose as the counter ionic species Tris in combination with chloride as the leading
ion. The step heights of some fatty acids were measured for some concentration ratios
of hydrochloric acid and Tris, and the results are given in Table 5.12. With the chosen
electrolyte, the fatty acids could easily be separated and in Fig.5.8 the isotachopherogram
for the separation of some fatty acids is given for system A. The detection was performed
with a thermometric detector (copper-constantan thermocouple).
Example E If sample ionic species do not show any W absorbance, a UV detector
can still be applied by using a W-absorbing counter ionic species. The sample zones can

EXAMPLES
TABLE 5.12
STEP HEIGHTS OF SOME FATTY ACIDS IN METHANOL MEASURED AS THE LINEAR
SIGNAL OF A THERMOMETRIC DETECTOR
The step heights are given in millimetres from the level of the leading electrolyte zone.
Ionic species Leading electrolyte
Formic acid
Acetic. acid
Butyric acid
Isovaleric acid
Caproic acid
Caprylic acid
Pelargonic acid
Capric acid
Lauric acid
Myristic acid
Palmitic acid
Stearic acid
Terminator solutions
Litocholic acid
Cacodvlic acid
0.02 N Tris- 0.0085 N Tris- 0.01 N Tris-
0.01 N HCI 0.01 N HC1 0.018 N HCl
(system A) (system B) (system C)
3’
88
128
137
148
168
180
190
204
220
240
252
-
400
21
64
91
-
104
114
1 24
126
136
146
156
166
be detected, because the counter ions have different concentrations and have different
pH values in those zones, and hence show differences in UV absorbance (see section 5.3).
In order to demonstrate this effect, we carried out separations with the sample ionic
species chlorate, formate, acetate and glutamate, which do not show UV absorbance.
Firstly, a separation was carried out with a non-UV-absorbing counter ionic species and
then with a UV-absorbing counter ionic species, for which the molar extinction
coefficient is a function of the pH.
216
18
78
110
122
132
144
148
156
172
184
204
222
For the first separation we chose as the leading electrolyte a solution of 0.01 N
hydrochloric acid and e-aminocaproic acid at a pH of 4.5. The terminator was a solution
of 0.0 1 Nmorpholinoethanesulphonic acid (MES). In the second separation, the leading
electrolyte was a solution of 0.01 N hydrochloric acid and creatinine at a pH of 4.5 and
the terminator was a solution of 0.01 N MES. As detectors we used a conductivity detector
(a.c. method; see Chapter 6 and Fig.6.18) and a UV detector (256 nm; see Chapter. 6). The
isotachopherograms are shown in Figs.5.9a and 5.9b. It can be seen that the influence of
the buffering counter ions is sufficiently large for the sample zones to be detected with a
UV detector. The presence of several impurities that form zones between the sample zones
has already been mentioned in section 5.5. The conductimetrically measured signals are
nearly identical in both figures although differences in step heights occur owing to the
differences in the effective mobilities of the counter ions.
256
-
107

108 CHOICE OF ELECTROLYTE SYSTEMS
Fig.5.8. Isotachopherogram for the separation of some fatty acids in a methanolic system (for
operational system see Table 16.1). The detection was performed with a thermometric detector.
T= Increasing temperature; t = time. The electric current was stabilized at 70 PA. 1 = Chloride;
2 = formate (C, 1; 3 = acetate (C2); 4 = butyrate (C4); 5 = n-caproate (C6); 6 = ncaprylate (C,);
7 = n-caprate (&); 8 = n-laurate (C12); 9 = n-myristate (C14); 10 = n-palmitate (C16); 11 = n-stearate
(CIB); 12 = cacodylate.
Example G. Sometimes, different sample ionic species have identical effective mobilities
at a certain pH, so that they form stable mixed zones. By using different electrolyte
systems with different pH, values, they can generally be separated according to their
pK values. However, if we do not know the composition of the ionic species present in the
sample, difficulties can arise. The use of both a conductivity and a UV detector can
then be advantageous.
In Fig.5.10, the isotachopherograms are shown for phosphate, salicylate and a mixture
of them. The conductimetric signals are identical and the mixed zone cannot be recognized.
In this experiment, the leading electrolyte was a solution of 0.01 N hydrochloric acid
and 0-alanine at a pH of 4 and the terminator was 0.01 N glutamic acid. Because the pK
values of orthophosphoric acid are 2.12,7.21 and 12.67 and the pK value of salicylic acid
fT

Fig.5.9. Isotachopherograms of the separation of 1 pl of a mixture of chlorate (0.01 M), formate (0.0 1 M),
acetate (0.01 M) and glutamate (0.01 M). In both instances morpholinoethanesulphonic acid was
used as the terminator, which is the reason why the electric current was stabilized at 30 fiA. Detection
was performed with a linear conductivity detector (a.c. method) and a UV absorption detector (256
nm), both of which are described in Chapter 6. R = Increasing resistance; t = time; A =increasing UV
absorbance. The time of analysis was 15 min. For a comparison with the isotachopherograms
obtained with a thermometric detector (e.g. Fig.5.8), it should be noted that the speed of the
recorder paper was five times higher in the isotachopherograms shown here. In the experiment shown
on the right, the buffering counter ion E-aminocaproic acid, which shows no UV absorption, was
used as the buffer, added to 0.01 N hydrochloric acid (pro analysigrade) to a pH of 4.5. On the
left, an isotachopherogram is shown for the operational system consisting of creatine (as the buffering
counter ion), which has a molar extinction coefficient that is influenced by the pH. The creatine was
also added to 0.01 N hydrochloric acid to a pH of 4.5. Special attention should be paid to the
impurities, revealed by both the detectors, and of course the shift in the pH, as normally obtained in
an isotachophoretic separation but now shown in the linear W trace in the left-hand isotachophero-
gram [examine the pH of the zone of glutamate (S)]. The difference in step height, as obtained in
the linear trace of the conductivity detector, must be ascribed mainly to the difference in the counter
ion taken, rneff. and pK. 1 = Chloride; 2 = chlorate; 3 = formate; 4 = acetate; 5 = glutamate;
6 = morpholinoethanesulphonate.
109

110
1
CHOICE OF ELECTROLYTE SYSTEMS
Fig.5.10. Isotachopherograms for the separation of phospate and salicylate. The leading electrolyte
was 0.01 N hydrochloric acid (pro analysi grade), adjusted to a pH 4 by the addition of recrystallized
p-alanine; the terminating electrolyte was glutamic acid (0.01 N), adjusted at pH 4 by the addition of
Tris. The electric current was stabilized at 70 PA. The detection was performed with a linear
conductivity detector (a.c. method) and a UV absorption detector (256 nm), both of which are
described in Chapter 6. In the left-hand experiment 10 nmole of phosphate, in the centre experiment
10 nmole of phosphate plus 10 nmole of salicylate and in the right-hand experiment 10 nmole of
salicylate was introduced. Attention should be paid to the difference in step heights, as obtained
in the linear traces of the W absorption detector. t = Time; R = increasing resistance; A =
increasing UV absorption. I = Chloride; 2 = salicylate; 3 = glutamate; 4 = phosphate.
is 3.1, we can separate them according to their pK values. In Fig.5.11 this separation is
shown for three different pH values. The left-hand isotachopherogram shows the
separation with a leading electrolyte consisting of 0.01 N hydrochloric acid plus 0-alanine
at a pH of 3.2 and glutamate as terminator. In the centre as shown the mixed zone as in
Fig.5.10 and on the right is shown a separation at a pH of 7 with a leading electrolyte
consisting of 0.01 N hydrochloric acid plus imidazole and glutamate as terminator. It can
be seen in Fig.5.11 that the problem is not whether the ionic species can be separated,
but how to recognize a mixed zone. The UV detector can provide the solution. The UV
signals for zones with only one ionic species of the sample show sharp step heights and
each sample zone has its own characteristic step height in a particular electrolyte system.
However, when mixed zones are present, a typical form is often shown as can be seen in
Fig.5.10 (centre), while its average step height lies between those of the pure sample zones,
depending on the concentrations of the sample ionic species present in the mixed zones.
Example H Suppose we wish to separate anionic species with ionic mobilities of about
30, but with pK values of 2, 3, 5,7 and 8.
In this instance, of course, we have to choose a separation according to pK values.
Thus we have to choose a leading electrolyte with a pH lying between the pK values of
*
1
A

EXAMPLES
II
4 3
Fig.5.11. Isotachopherograms for the separation of phosphate and salicylate in various operational
systems, to show the difference in separation according to pK values and according to mobilities.
The centre isotachopherogram is as described in Fig.5.10 (centre). AU other conditions are as in
Fig.5.10, except for the operational systems. Left-hand isotachopherogram: leading electrolyte,
0.01 N hydrochloric acid (pro analysi grade) adjusted to pH 3.2 by the addition of p-alanine.
Right-hand isotachopherogram: leading electrolyte, 0.01 N hydrochloric acid (pro analysi grade)
adjusted to pH 7 by the addition of imidazole. In both instances, a complete separation could be
achieved. t = Time; R = increasing resistance; A = increasing W absorption. 1 = Chloride; 2 = phosphate;
3 = salicylate; 4 = glutamate.
the ionic species to be separated. We could use a leading electrolyte with a pH, of about
5, but there is then a problem. When the pH in the zones is about 5, the ionic species
with pK values of 2 and 3 have nearly identical effective mobilities and they cannot be
separated. For the ionic species with pK values of 7 and 8, the effective mobility is rather
low and it is then preferable to carry out the separation in two runs.
In the first run, we use a leading electrolyte with a pH of, e.g. ,3.5, so that we can
easily separate the anionic species with pK values of 2,3 and 5. Further, a terminating
ionic species is chosen such that its effective mobility is higher than those of the ionic
species with pK values of 7 and 8.
In the second run, we take a leading electrolyte with a pH of about 6.5 and we can
easily separate the anionic species with pK values of 5,7 and 8 (other ionic species
will generally form a mixed zone). With the two runs, we can thud separate the whole
mixture. In such a separation, we can speak of a ‘combination of systems’. As with
differences in pK values, we also can combine electrolyte systems with the aim of using
different properties such as complex formation for the separation of metal ions and
amino acids (see section 13.1.4).
111

112
00-
t,
/ I
/
/ I
WHCl WKAC MHCl MKAC
Pb
Y.
K
CHOICE OF ELECTROLYTE SYSTEMS
Fig.S.12. Step-height differences (differences in effective mobility) of some cations for different
operational systems. For more information, see Chapters 11 and 16.
mo f

REFERENCES 113
Example I. As example C shows, pronounced differences in effective mobilities can be
expected if instead of water methanol is used as solvent. Example G shows some differences
in effective mobilities, due to the change in pH. The differences in the effective
mobilities, as found in the various systems, always must be interpreted carefully. The
influence of the counter ion, the solvent and the pH always results in changes in the
effective mobilities of the various ionic species considered. Therefore another example of
these various effects can be given.
Fig.5.12 shows clearly the influence of the various systems on the effective mobilities
(step heights) of the cations. The systems WHCl, WAC, MHCl and MKAc are listed in
Tables 11.1, 11.3, 16.4 and 16.5, respectively.
The behaviour of the cations K, Na and Li is similar, while highly charged cations such
as Ce, Al and Pb show shifts due to effects of pH and complex formation. A large shift is
shown for the tetraethylammonium ion in the aqueous and methanolic systems due to the
effect of solvation and change of dielectric constant of the solvent.
Much more information about this subject can be found in the literature considering
“structure breaking” and “structure making” properties of ionic species.
Moreover, in methanolic systems the influence of a change in pH on the divalent
cations is remarkable.
REFERENCES
1 J. Deman, Anal. Chem, 43 (1970) 321.
2 R.P. Bell, Acids and Bases, Methuen, London, 2nd ed., 1969.
3 G. Schwarzenbach, Helv. Chim Acta, 13 (1930) 870.
4 J. Dingemans, Electrochemie, Waltman, Delft, 5th ed., 1964.
5 R.G. Bates, Electrometric pH Determinations, Wiley, New York, 1964.
6 C.L. de Ligny, P.F.M. Luykx, M. Rehbach and A.A. Wieneke, Rec. Trav. Chim. Pays-Bas, 79
(1960) 699.
7 C.L. de Ligny, P.F.M. Luykx, M. Rehbach and A.A. Wieneke, Rec. Trav. Chim Pays-Bas, 79
(1960) 713.
8 R.M. Fuoss,J. Amer. Chem. SOC., 79 (1957) 3301.
9 R.M. Fuoss and L. Onsager, J. Phys. Chem., 61 (1957) 668.
10 W.J. Gelsema, Thesis, University of Utrecht, Utrecht, 1964.
11 C.L. de Ligny, Thesis, University of Utrecht, Utrecht, 1959.
12 C.L. de Ligny and M. Rehbach, Rec. Trav. Chim Pays-Bas, I9 (1960) 727.
13 J. Kucharsk; and L. Safarik, nitrations in Non-Aqueous Solutions, Elsevier, Amsterdam, 1965.
14 D.B. Rorabacher, W.J. MacKellar, F.R. Shu and M. Bonavita, Anal. Chem. 43 (1971) 561.

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INSTRUMENTATION

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Chapter 6
Detection systems
SUMMARY
In the previous chapters, various theoretical and practical aspects were considered,
and it was shown that the choice of the electrolytic system in which an analysis is to be
performed determines whether an electrophoretic experiment will be carried out according
to isotachophoretic principles.
This chapter is devoted to detection systems that can be used with electrophoretic
equipment specially developed for isotachophoretic analyses, including the thermometric
detector, the conductivity detector (a.c. method), the potential gradient detector (d.c.
method) and the UV absorption meter. Some recent developments such as the highfrequency
detector with micro-sensing electrodes in indirect contact with the electrolyte
inside the narrow-bore tube and the polarimetric detector are mentioned only briefly, as
these types of detector need to be developed more thoroughly.
Some effects caused by the additives that must be added to the electrolytes in order
to suppress electroendosmosis or electrode reactions, and the coating of the micro-
sensing electrodes of the conductivity meter or potential gradient detector are briefly
discussed. It is shown that these effects which may occur if high-resolution detectors are
applied, are obtained only if the necessary precautions are not taken. Attention is paid
to these effects so that once they occur they can quickly be recognized and appropriate
measures can be taken.
6.1. INTRODUCTION
Because the principle of isotachophoresis can be applied to separations on both
analytical and preparative scales, the detection systems that can be used are numerous and
of different construction for the two types of instrumentation involved. In this book,
most attention is paid to analytical applications, although the operational systems
considered may be applied in both types of application. Even in analytical isotachophore-
sis, various types of instrumentation may be chosen. With these instruments, a choice
can be made as to whether or not stabilizing agents should be used. Again, we shall focus
attention on the equipment in which narrow-bore tubes are used. In such equipment, no
stabilizing agent needs to be used and also volatile and aggressive solvents can be used in
addition to water. Because the volume of the separating chamber and the concentrations
of the electrolytes in it are small, this type of equipment is particularly applicable to
analyses of samples that consist of different components present in low concentrations or
of samples of which only small amounts are available.
Basically, the isotachophoretic equipment consists of a narrow-bore tube [l-31 made
of an insulating material (glass or PTFE) with an inside diameter of cu. 0.4-0.6 mrn and
an outside diameter of cu. 0.7-1.0 mm. The inside diameter must not vary by more than
117

118 DETECTION SYSTEMS
ca. 2%. The choice of the material of which the equipment is constructed influences the
electroendosmosis considerably. The length of tube needed for a complete separation
depends on the difference in the effective mobilities of the ions in the sample that are
most difficult to separate, the concentrations of the constituents of the sample and the
availability of a counterflow of electrolyte (see Chapter 7).
Because the choice of detection system determines the type of construction of the
equipment, the detection systems that are currently available for analytical isotacho-
phoresis are summarized first. Some equipment is considered in detail in Chapter 7.
The detectors that are available can be divided into three main classes: universal
detectors, specific detectors and combinations of both, and these types are considered
in the following sections.
6.1.1. Universal detectors
When a universal detector is used, the information obtained is directly proportional
to the effective mobilities of the ionic constituents [4], and the information derived
therefore has a continuous stepwise character. From the height of a step, qualitative
information can be deduced, while the length of a step provides quantitative
information.
Universal detectors may be divided into two classes:
(1) Detectors of which the sensing element is not in direct contact with the electrolytes
inside the narrow-bore tube [S] . This class can be divided into two sub-classes:
(la) Detectors with a low resolving power, e.g., temperature-recording detectors [l].
(lb) Detectors with a high resolving power, e.g., high-frequency conductivity detectors.
(2) Detectors in which the sensing element is in direct contact with the electrolytes
inside the narrow-bore tube [6]. This class can also be divided into two sub-classes:
(2a) Detectors that involve a.c. recording of the conductivity between two micro-sensing
electrodes mounted equiplanar or axially [7].
(2b) Detectors that record the potential gradient directly, making use of the direct
driving current between two axially mounted micro-sensing electrodes [8] .
6.1.2. Specific detectors
When a specific detector is used, the information obtained is not directly proportional
to the effective mobilities of the ionic constituents. A series of components, separated
isotachophoretically, may have different, non-continuous, responses on the detector, so
that the absolute value of the measuring signal may be different from zone to zone. One
can use the principle of absorption of light measured during the analysis, or polarimetric
detection can be used [9]. The succesful use of radiochemistry has so far been applied
only in analyses on strips [ 101 .

THERMOMETRIC RECORDING
6.1.3. Combinations of universal and specific detectors
There are two main possibilities for combining universal and specific detectors:
(1) Specific detectors and universal detectors both mounted in a similar piece of
equipment. This will be discussed in Chapter 7, where the construction of the equipment
is dealt with.
(2a) A universal detector may serve simultaneously as a specific detector. Particularly
if micro-sensing electrodes, which are in direct contact with the electrolyte inside the
narrow-bore tube, are coated with a suitable polymer [7], these electrodes give specific
infor mation.
(2b) A specific detector may serve simultaneously as a universal detector. If, for
instance, a W-absorbing component, for which the electrophoretic migration in the
operational system applied is almost zero and the UV absorption is a function of the
pH, is added to the leading electrolyte, the W detector gains ‘universal characteristics’
if non-UV-absorbing species are present. This effect is due to the different pH values
characteristic of each zone [l 11 .
Before the detectors are discussed in detad in the following sections, a survey is given
of the phenomena that occur during isotachophoretic analyses (Table 6.1).
A survey of the detectors used in analytical isotachophoresis is presented in Table 6.2.
6.2. THERMOMETRIC RECORDING
6.2.1. Introduction
The electric field strength (V/cm) varies from one zone to another and is inversely
proportional to the effective mobilities of the ionic species, giving all zones the same
speed. If a stabilized electric current is applied, the heat production increases from the
front side towards the rear in a well defined way. Consequently, the zone boundaries
are characterized by sharp changes in temperature [l, 121. This effect can serve to
indicate the positions of the zone boundaries or to characterize the zones themselves if
the actual temperature is measured simultaneously. While the zone length provides
quantitative informtion, the actual temperature characterizes the ionic species in a
particular zone. Hence thermometric recording in an isotachophoretic analysis provides
all the qualitative and quantitative information required.
6.2.2. Construction
The temperature of the individual zones can be measured with micro-thermocouples
[13, 141 or micro-beat thermistors [15]. Both types of detectors can be mounted around
the narrow-bore tube with a suitable adhesive (elastic type). This adhesive, in addition to
fixing the detector, also improves the thermal contact of the detector with the narrow-bore
tube.
119

120 DETECTION SYSTEMS
TABLE 6.1
SURVEY OF SPECIFIC PROPERTIES THAT CAN BE RECOGNIZED IN ISOTACHOPHORETIC ANALYS:
Type of electrolyte Initial conditions Steady state
Leading electrolyte (L.E.)
Sample zone(s)
Conductivity
Determined by the choice of the
operational system in which
the analysis is carried out.
Unknown, but generally adjusted
to the conditions of the L.E.
with respect to pH and the
concentration of the ionic
species (roughly).
Terminating electrolyte (T.E.) Chosen to be of approximately
the same order of magnitude
as the leading anion (anion
separation) or cation (cation
separation), to minimize the
effect of fast-moving
impurities.
(pH is adjusted approximately to
the pH of the leading elec-
trolyte to prevent sample
ions being 'missed'.)
Conductivity
Determined by the initial choice of
the operational system.
AU zones adjusted to the concen-
tration of the leading anion
(anion separation) or cation
(cation separation).
The concentration is adjusted to the
concentration of the leading
anion (anion separation) or
cation (cation separation).
The conductivity decreases from the
L.E. towards the T.E. (except
for 'enforced' isotachophoretic
systems).
An easy method of making these thin thermocouples is as follows. Copper (30 pm) and
constantan (25 pm) wires are twisted symmetrically together, after the ends of each have
been fixed in a small piece of shellac placed on a rod [ 1 ] .
The difference in the diameters of the wires is necessary because otherwise, owing to
their very different flexibilities, the twisting of the two wires of identical diameter results
in an asymmetrically wound thermocouple, the copper wire being wound around the
constantan wire. The procedure for mounting a thermocouple that is not symmetrically
wound around the narrow-bore tube is very difficult and the thermocouple easily breaks
on the copper side, especially if both diameters are 30 pm. Copper wires of 25 pm diameter
easily breaks during the twisting procedure, especially if the wires are corroded.
The tightly twisted part is cut so that a length of about 1 mm remains twisted. This
twisted end is silver soldered by inserting it into molten silver solder, protected with a
small amount of flux. This silver solder is kept molten by utilizing the heat capacity of
a thick-walled Pyrex glass tube (Fig. 6.1). Other methods of making thin thermocouples
are given in ref. 1.

THERMOMETRIC RECORDING
Potential gradient Temperature PH
Determined by the direct driving
current chosen and the conductivity
of the L.E.
Determined by the direct driving
current chosen and the concen-
tration of the anion of the L.E.
(anion separation) or the cation of
the L.E. (cation separation) and the
effective mobility of the ionic
constituent present.
Determined by the direct driving
current chosen and the concentration
of the L.E. initially chosen
and the effective mobility of the
terminating ion.
Determined by the direct driving Determined by the ratio of the
current and the conductivity
of the L.E.
121
concentration of the ionic
species of the leading electro-
lyte and their pKa values.
Determined by the direct driving Determined by the ratio of the
current and the concentration concentration of the ionic
of the anion of the L.E. (anion species present in each zone
separation) or cation of the and their pKa values.
L.E. (cation separation).
Determined by the direct driving Determined by the ratio of the
current and the concen-
concentration of the ionic
tration of the L.E. initially species present in this zone
chosen. and their pKa values.
The potential gradient is a constant The temperature is constant for The pH tends to increase in
for each zone and increases
each zone and increases from anion separation and to
from the L.E. towards the T.E. the L.E. towards the T.E. decrease in cation separation
(except for ‘enforced’ isotacho- (except for ‘enforced’ iso- from the L.E. towards the
phoretic systems). tachophoretic systems). T.E.
If this thermocouple is mounted around the narrow-bore tube, a linear stepwise
recording of the isotachophoretic zones is obtained as soon as they have passed this fixed
thermocouple. In order to make the zone boundaries more pronounced electronically,
the differential of this signal can be obtained. An example of a possible circuit is given in
Fig.6.2.
Another possibility is to make a differential thermocouple with two copper-constantan
junctions, about 2 mm apart. This thermocouple can be mounted around the narrow-bore
tube in a similar way by means of a suitable elastic adhesive. The two twisted (and silver
soldered) ends must be bent until they are approximately at right-angles to the surface
of the narrow-bore tube. While the values from the passage of a zone boundary recorded
by a normal thermocouple can be expected to be of the order of millivolts, a differential
thermocouple indicates the passage of a zone boundary with a signal of the order of
microvolts, and amplification is therefore necessary.
The differential thermocouple has to be balanced. Normally, there is a difference in
temperature between the two junctions of the differential thermocouple, because both

TABLE 6.2
SURVEY OF THE DETECTORS USED IN ANALYTICAL ISOTACHOPHORESIS
Lmin. = minimum detectable zone length; Qmin, = minimum detectable amount of ionic component in gramequivalents; t,, = average time for
analysis; I = direct driving current; Scan = possrbdity of scanning.
Type Performance ‘min.(mm) Qmin. t,,(min) Scan Dependence on 1 General information
Thermal Thermocouple, 25 fim 5
(Cu-constantan)
thermistor
(Philips micro-beat)
Conductivity Micro-sensing electrodes 2
(high frequency) not in contact with
electrolyte
Conductivity Micro-sensing electrodes
in contact with
electrolyte
0.05
Potential gradient Micro-sensing electrodes 0.05
in contact with
electrolyte
uv Micro-cell wavelengths: 0.05
205,256,280 and
340 nrn
0.5*109 30 No I=
2.10 0 20 Yes(?) No
0.5 * lo-” 10 No No
0.5 * lo-’ 10 No I
Qualitative
Quantitative
General detector
Qualitative
Quantitative
General detector
Qualitative
Quantitative
General detector*
Qualitative
Quantitative
General detector
0.5 lo-” 10 Yes No Quantitative
Specific detector**
*A coating on the sensing electrodes gives the detector ‘specific’ characteristics.
**The use of strong Wabsorbing counter ions of which the W absorption is a function of the pH changes the specific characteristics of this detector
into ‘general’ characteristics. 8
i3
2:
cn
- N

THERMOMETRIC RECORDING
/2
Fig.6.1. Preparation of micro-thermocouples by silver soldering twisted copper and constantan wires
by means of a thick-walled Pyrex glass tube. 1 = Silver solder; 2 = flux.
the contact with the wall of the narrow-bore tube and the heat loss at each junction are
different. This difference in temperature will not remain constant, but will be greater or
smaller after the passage of a zone boundary. The differential thermocouple can be
Fig.6.2. Electronic circuit for differentiating the stepwise linear signal derived from the thermocouple
during the passage of the zone boundaries. All resistances are given in aTunless stated otherwise.
123

124 'DETECTION SYSTEMS
balanced by cutting a piece from one of the junctions, by setting one of the junctions
at a different angle to the wall or by putting extra adhesive on one of the junctions (or
alternatively by removing some of the adhesive from the other junction with a suitable
solvent). In order to check if the differential thermocouple is well balanced, the electric
current is switched on and off, when the signal derived from the differential thermocouple
should not vary. However, an effect can still be obtained, even if the differential
thermocouple is well balanced, if difference in heat capacity between the two junctions
is large. The switching on and off of the electric current may then result in a peak or a
dip, the height of which and the time required to reach the balanced position (zero)
again may be such that they cannot be considered to be negligible. This may disturb the
electrophoretic pattern recorded by the differential thermocouple, especially if large
temperature differences need to be recorded. The impression can be given that a zone is
preceded by a small zone of lower temperature (enforced isotachophoretic system) or
that an extra component (impurity) is present. If the electronic differential is taken, of
course, no balancing procedure needs to be carried out.
The great advantage of a differential thermocouple, however, is that the direction of
movement of the temperature step is recorded. Mistakes can always be made in the
interpretation if a zone of high conductivity (e.g. , H'ions that originate from the pH
jump at the membrane [16] moving through the narrow-bore tube) migrate in a direction
opposite to that of the sample ions. This zone causes a lower temperature, which migrates
and is therefore detected by the differential thermocouple as a hot zone coming from the
opposite direction. Because the differential thermocouple never has a position on the
narrow-bore tube similar to that of the linear thermocouple, which has only a single
junction on the wall, the time of recording clearly shows this effect. This effect, however,
may cause severe problems, especially because in practice a lower temperature zone may
migrate in front of zone of higher temperature in exceptional cases (e.g., enforced isotachophoretic
systems). If electronic circuits are used in order to differentiate the signal derived
from the linear thermocouple, this problem can be solved if at least two thermocouples
are mounted axially on the wall of the narrow-bore tube. The simultaneous recording of
these thermocouples indicates both the rate of separation (are mixed zones still present?)
and the direction of movement of various zones. Although a thermometric detector is
very cheap, its resolving power is, compared with that of other detectors, not very high,
although for many applications it is sufficient. This will be shown in later chapters, where
some applications are discussed.
The fronts, as finally detected by the thermometric detector, lack sharpness with
respect to the concentration profiles inside the narrow-bore tube, because the heat
generated inside has to pass through the wall*. The longitudinal conduction of heat, both
in the liquid and the insulator, spreads the temperature change along the tube. Also,
considerable time is required to heat the part immediately behind the front in order to
obtain dynamic equilibrium of the temperature step. The wall must not be too thin,
however, because the thermocouple and the instrumentation connected to it must be
insulated from the high potential inside the narrowbore tube.
Even if another precaution is taken, e.g., by using an insulated amplifier, and the
thermocouple is mounted closer to the centre of the narrow-bore tube, the final recording
is not improved very much (Fig.6.3), because the time required for dynamic equilibrium
*See also Appendix B.

THERMOMETRIC RECORDING
T t l
Fig.6.3. Temperature profiles of zone boundaries in isotachophoretic analyses, as derived from
micro-thermocouples. In the direction X, the micro-thermocouple is mounted closer to the centre
of the narrow-bore tube. In the initial phase, the transient response of the thermocouple, mounted
closer to the centre, is more rapid, but the resolution is not improved much. T= Increasing
temperature; t = time.
of the temperature step to be attained is not changed very much; the total mass is
not changed or is changed in the wrong direction.
Calculations show that the sharpness of the temperature step approaches that of the
concentration step if the narrow-bore tube is made of ‘infinite’ thickness [ 171 .
Alist of data for thermometric detection is given in Tables 11.6 and 12.3.
The resolution and stability of thermistors are comparable with those of thermocouples,
but thermistors are not discussed further here because more complicated electronic
circuits are necessary. Optical means of detection have not been tested so far, because
the temperature differences are relatively small and the optics are very expensive,
compared with the thermocouples and thermistors [18] . The information derived from
liquid crystals [19] painted on the wall of the narrow-bore tube is poor and needs
expensive instruments for automatic recording.
6.2.3. Experimental
Thermometric detectors lack high resolution because the heat generated by the electric
current has to diffuse through the wall of the narrow-bore tube. The various heat transi-
tion coefficients influence the final recording of successively migrating narrow zones. As
a general rule, the zone length needed for a full qualitative and quantitative recording
must be about 5 mm, making use of a PTFE (or Pyrex glass) narrow-bore tube with an
outside diameter of 0.7 mm and an inside diameter of 0.45 mm. This value can vary,
depending on the heat production of the adjacent zones, the electric current applied, the
type of solvent used and some other minor factors (e.g., the addition of surfactants to
the electrolytes in order to depress the electroendosmotic flow).
In Fig.6.4, some graphs are shown of actual temperature measurements carried out
with a thin thermocouple mounted on the outside of a narrow-bore tube.
125

126 DETECTION SYSTEMS
30 4'0
T
Fig.6.4. Relationship between the temperature inside a narrow-bore tube and the output signal of a
thermocouple mounted on the outside of the tube. 1 = Theoretical relationship; 2 = practical
relationship, found when water at a known temperature flows through the narrow-bore tube; 2%
of the surfactant Mowiol (polyvinyl alcohol) was added to the water; 3 = as 2, with no surfactant;
4 = as 2, with methanol instead of water. The experiments with methanol were difficult to perform
because a suction pump was used, and the methanol began to boil.
These temperatures were compared with the actual temperatures inside the narrow-
bore tube as follows. Water was pumped through the narrow-bore tube until the signal
derived from the thermocouple reached a constant value. Simultaneously, the water
temperature before entering the narrow-bore tube was measured. The results indicated
that in the region of the temperature of the terminating electrolyte (35-45"C), mistakes
can be expected in the qualitative recording, especially if surfactants are present.
6.2.4. Resolution
If the concentration of an ionic species in the narrow-bore tube is about 0.01 g-equiv./l
and the cross-section is about 1.6 - cm2, the minimum amount of that ionic species
which can be detected is about g-equiv. If the volume of the sample injected is
3 pl, the minimum concentration in the sample that can be detected is thus 2.7
g-equiv./l [20].
To illustrate the effect of the introduction of samples of different sizes, some isotachophoretic
separations are shown in Fig.6.5 of the anions oxalate, formate, acetate, and
P-chloropropionate in the operational system at pH 6*. The conditions for this system are
listed in Table 12.1. The concentrations of the various anions were: oxalate, 0.005 N;
formate, 0.01 N, acetate, 0.01 N, and P-chloropropionate, 0.015 N. The volumes injected
were (a) 1 pl, (b) 2 1.11 and (c) 3 p1. The ameunts that can be detected are therefore
5 * 1 O+, 1 O* , 1 O-' and 1.5 - 1 0-8 moles for the anions in the order listed for the case
when 1 pl was injected. It can be stated that the isotachopherogram in Fig.6.5 for the
separation of 1 p1 of sample represents a complete separation of the anions in the mixture.
*For a more extensive description, see Chapter 10.

THERMOMETRIC RECORDING
7
127
It-- I
a b
Fig.6.5. Isotachopherogram showing the separation of some anionic species in the operational system
at pH 6. The signals were derived from thermometric recording of the passage of zone boundaries.
T= Increasing temperature; t = time. The injected volumes were (a) 1 pl, (b) 2 p1 and (c) 3 pl.
1 = Chloride (leading ion); 2 = oxalate; 3 = formate; 4 = acetate; 5 = p-chloropropionate; 6 = glutamate
(terminating ion).
The steady state during isotachophoretic separation is attained earlier if a smaller amount
of ionic material is injected. Thus, although this isotachopherogram simulates an incom-
plete separation, the steady state has been reached*. From this isotachopherogram,
however, only quantitative information can be deduced, so the actual sequence must be
known. It should be remembered that for quantitative analyses, only the transition of
*This is in contradiction to similar looking records in chromatography.
C

128 DETECTION SYSTEMS
the zone boundaries is required. The other two isotachopherograms contain both the
qualitative and the quantitative information.
In Table 6.3 the results of the separation are given. The time interval between two
peaks, measured with a stop-watch. is given in seconds. The use of electronic equipment
for measuring the time intervals more accurately decreases the detection limit. The
detection limit can be decreased further by using a leading electrolyte with a lower
concentration, because all other zones will adjust their concentrations in the zones
according to the concentration of the leading electrolyte. It must be emphasized, however,
that a decrease in the concentration of the leading electrolyte automatically implies that
the pH limits between which experiments according to the isotachophoretic principle can
be carried out safely are narrower. While at a concentration of the leading electrolyte of
lo-* N a pH of about 3.5 is the lower limit and a pH of about 10 is the upper limit, at
TABLE 6.3
QUANTITATIVE INFORMATION THAT CAN BE DERIVED FROM A THERMOMETRIC
DETECTOR
The figures given are zone lengths, expressed in seconds. The quantitative information that can be
derived from conductivity and W detectors is discussed in Chapter 10, where the quantitative aspects
of the thermometric detector are also discussed in more detail.
Compound Amount injected (nmoles)
Oxalate 20 43 64
21 43 64
21 43 63
43 64
64
Average 21 43 64
Formate
Acetate
Average
Average
5 10 15 20 30 45
20
20
19
20
22
22
21
22
39 62
41 61
41 61
40 61
63
40 61.5
47 72
47 72
47 71
47 71
72
47 71.5
0-Chloropropiona te 37 71 111
37 74 108
39 73 107
73 108
110
Average 38 73 109

THERMOMETRIC RECORDING
a concentration of the leading electrolyte of 10-3N these pH limits are narrowed to
4 and 9, respectively. The upper limit is determined mainly by the interference in the
analyses by carbon dioxide from the air.
The detection limit can also be decreased by the injection of a larger sample in a
suitably constructed sample tap. Some possibilities for this approach are considered in
Chapter 7. The sample tap may have a volume of 30 pl, which decreases the minimum
detectable concentration by a factor of 10 compared with sample introduction via a micro-
syringe. An important aspect of the use of a sample tap is that the sample is already
separated from the leading and terminating electrolytes and the separation takes place in
the tap. On injection with a syringe, the sample is always mixed with the highly mobile
chloride or with the terminating electrolyte, which may decreaFe the pH of the sample
generally in anion separations and increase it in cation separations. If the average concen-
tration of the ionic species in the sample is low, a sample tap is always recommended, if
no sample pre-treatment can be applied.
The detection limit can also be decreased by using a regulated counter flow of
electrolyte, again because larger sample volumes can be used., in spite of the short length
of the narrow-bore tube available for separation. The time of analysis, of course, will
increase considerably.
Because detectors are available with a higher resolving power than that of a thermo-
metric detector, the detection limit in an isotachophoretic analysis must not be estab-
lished by using a thermometric detector, and therefore the use of a sample tap and a
counter flow of electrolyte in combination with a thermometric detector will not be discussed,
It is important, however, to establish the limit of concentration that can be detected
with a thermometric detector. For this purpose, analyses with a leading ion concentration
of g-equiv./l were carried out. Again the operational system at pH 6 was chosen. The
basic information is listed in Table 12.1. The concentration of the hydrochloric acid was
decreased to 0.001 N. Because excessive heat production may render the analysis useless
and also the driving potential available is limited, the direct driving current was decreased
to 7 pA. Fig.6.6 shows the isotachopherogram of the separation of nitrate, chlorate,
formate, citrate and adipate. Acetic acid (0.001 N) was used as the terminating electrolyte.
A complete separation could easily be obtained, but the disadvantage is shown very
clearly. Because at these low concentrations only small temperature differences can be
expected, the signals derived from the thermocouples need to be amplified too much.
6.2.5. Conclusion
Thermometric recording acts contrary to the requirement that for a complete reproducible
analysis by electrophoretic techniques, optimal thermostating is necessary. If thermometric
detection is used, all of the equipment can be optimally thermostated, but at the
position where the detector is mounted good thermostating will destroy the temperature
differences necessary for measurement. Another disadvantage is that in thermometric
detection the direct driving current plays such an important role (I2) and the detection
is only proportional to the concentration adjustment (R). This disadvantage is partly
overcome by the construction of a good current-stabilized power supply, which can now
be obtained commercially.
129

130
DETECTION SYSTEMS
Fig.6.6. Isotachopherogram of a mixture of anions using the operational system at pH 6. The
concentration of the leading anion (acetate) was decreased to 0.001 N, and the direct driving current
co:sequently was stabilized at a lower value of 7 PA. A thermometric detector was applied. T =
Increasing temperature; t = time. 1 = Chloride; 2 = nitrate; 3 = chlorate; 4 = formate; 5 = citrate;
6 = adipate; 7 = acetate.
The influence of the direct driving current on both the leading and terminating
electrolytes is shown in Fig.6.7. A current of 250 yA increases the temperature inside the
narrow-bore tube to 62OC for the leading electrolyte, while the terminating electrolyte
attains this temperature if a driving current of 150 yA is applied. Fig.6.7 shows that a
current of 100 pA gives a temperature of 26°C for the leading electrolyte and 42OC for
the terminating electrolyte. These values are obtained if the narrow-bore tube of PTFE
(I.D. 0.45 mm, O.D. 0.7 mm) is hanging free in air; a cooling device will decrease these
values, of course.
6.3. HIGH-FREQUENCY CONDUCTIVITY DETECTION
6.3.1. Introduction
In order to give all zones an identical speed, the field strength per zone increases from
the leading electrolyte side towards the terminator side. The current density is the same
for all zones. Hence the electric resistance increases from the front side towards the rear.
This resistance can be measured with a high-frequency detector in which the measuring
electrodes are not in direct contact with the electrolyte inside the narrow-bore tube.
Polarization of the measuring electrodes, caused by the driving current, is impossible [7].
By this means, undesirable oxidation (or reduction) reactions, which occur on the micro-
sensing electrodes and which may influence both the separation and the detection are
prevented.

HIGH-FREQUENCY CONDUCTIVITY DETECTION
i- h
100
50-
0-
I, ,
20
1
40
1 -
I
60 80
T OC
Fig.6.7. Temperature differences expected when isotachophoretic experiments are carried out in
narrow-bore tubes hanging free in air. The temperatures were measured in the operational system at
pH 6 (Table 12. l), with chloride (0.01 N) as the leading ion (B) and glutamate as the terminating ion
(A). The values on the arrows indicate the direct driving current (B for the leading electrolyte and A
for the terminating electrolyte). T= Increasing temperature; h(mm) = step height as found in the
linear trace of the thermometric detector. The direct driving currents are given in @A.
The high-frequency conductivity detector needs good shielding [21] and zone lengths
of 2 mm can be detected. The reproducibility, however, is poor. The development of this
type of detector is in the initial phase, so that it would be premature to state that it is
possibly the detector of the future for isotachophoresis.
6.3.2. Construction
The conductivity changes that occur during an analysis inside the narrow-bore tube of
the isotachophoretic equipment can be measured with the circuitry shown schematically
in Fig.6.8.
The signal produced by the generator is led via the symmetry transformer TI (coil ratio
1 : 10) to the emitting electrodes El and E2. Two trimmers, the capacitors C3 and C4,
permit exact symmetry of the high-frequency signal on the emitting electrodes to earth.
131

132
DETECTION SYSTEMS
Fig.6.8. Schematic diagram of the high-frequency conductivity detector. The four electrodes
(E, , E,, E, and E4), which are not in direct contact with the electrolytes inside the narrow-bore
tube, are mounted equiplanar. By using screening, the resolution is improved, although a much higher
amplification is necessary, which decreases the signal to noise ratio. A = Amplifier; G = generator
(cu. 1 MHz).
The emitting electrodes El and E2 are in contact with the receiving electrodes E3 and
E4 via the capacity of the narrow-bore tube. The electrodes El, E2, E3 and E4 are
mounted equiplanar. In order to prevent a fan-shaped high-frequency signal between the
emitting and receiving electrodes, which of course would decrease the resolution,
shielding is necessary, as shown in Fig.6.8. The signals picked up by the electrodes E3
and E4 are fed to the second symmetry transformer (coil ratio 1 : 1) and, after arnplifica-
tion and rectification, are recorded with a potentiometric recorder. The signals derived
from the probe are directly proportional to the effective mobilities of the ionic material
at the position of the electrodes El, E2, E3, E4 and between the shielding.
For optimal operation of the conductivity probe, a good symmetry of the emitting
electrodes El and E2 to earth is necessary. If this symmetry is not correct, boundary
passages of various shapes are recorded, which, of course, do not exist in reality. It was
even found that some boundary passages were not recorded at all. Poor symmetry to
earth of the receiving electrodes E3 and E4 influences only the final amplification, and
does not influence the shape of the recorded transition.
In some isotachophoretic analyses using the high-frequency detector, the reproduc-
ibility was found to be poor. Particularly when a series of zones needed to be detected, the
resolution was far lower than that with a conductivity detector with the micro-sensing

CONDUCTIVITY DETECTION 133
Fig.6.9. Boundary passage of an isotachophoretically moving zone (cNoride/glutamate) recorded with
a high-frequency conductivity detector (solid line) and compared with the a.c. method of conductivity
determination (broken line), discussed in section 6.4. A small amount of impurity (sulphate) was
detected by the a.c. conductivity detector, which was 'missed' by the high-frequency conductivity
detector. The analysis was performed in the operational system at pH 6 (Table 12.1). The direct driving
current was stabilized at 40 pA. The speed of the recorder paper was 6 cmlmin. R = Increasing electric
resistance: t = time.
electrodes in direct contact with the electrolyte. The high-frequency detector, if it is
possible to make an operational type, still has advantages, however, especially if aggressive
solvents are chosen for electrophoretic analysis. A further small advantage is that the
means of detection does not interfere with the electrophoretic separation procedure.
There is the possibility of making a scanning detector, although in practice deviations in
the resistance of the wall will be greater than the variations in electric conductivity.
Although the detector is not yet operational, Fig.6.9 shows the boundary passage of the
zone chloride/glutamate, carried out in the operational system listed in Table 12.1. If
more ions were introduced, the resolution was found to be greater than that with the
thermometric detector, and the reproducibility was found to be smaller.
6.4. CONDUCTIVITY DETECTION
6.4.1. Introduction
In isotachophoretic analyses, the sample ions separate according to their effective

134 DETECTION SYSTEMS
mobilities and form discrete zones with concentrations that are constant with time,
homogeneous throughout each zone and directly related to the concentration of the
leading ion. If the current is stabilized, all of the velocities of the various zones, in the
steady state, are identical and constant with time. The boundary between two successively
moving zones is sharpened by the electric field, which increases stepwise, following
each zone in which it is a constant, to compensate for the less mobile ions. This stepwise
increase in the electric field causes a stepwise increase in the electric resistance according
to Ohm’s law. In addition, this stepwise increment is automatically directly proportional
to the effective mobilities of the ionic species actually present in each zone. If the
operational systems, and hence electrolyte systems in which the isotachophoretic analyses
can be carried out properly, are chosen well, the electric resistance of each zone will
characterize the ionic species in the zones. These conductivities are not defined by the
electric current, supplied by the current-stabilized power supply, if the contribution of
the temperature is left out of consideration. This is in contrast to thermometric detection,
where the zone characteristics are determined by p. As already discussed in section 6.2,
this is a disadvantage of thermometric detection.
Special attention should be paid to the fact that even if only a stabilized voltage power
supply is available and this source is used in isotachophoretic analyses (the electric current
is thus constantly decreasing), the conductivities of the various zones are still identical
with those in experiments with a stabilized-current power supply. All of the concentrations
of the various zones are determined by the concentration of the leading electrolyte. If a
voltage-stabilized power supply is used, the velocities of the various zones, which
characterize the amounts present, decrease but all remain identical with each other. Hence
the simple relationship between zone length and the amount of an ionic species injected
is lost.
The conductivity of the various zones can be determined in different ways: (A) with
the micro-sensing electrodes not in direct contact with the electrolytes inside the narrowbore
tube (see section 6.3), and (B) with the micro-sensing electrodes in direct contact
with the electrolytes inside the narrow-bore tube. No further attention will be paid to the
type of detector in class A, because they are classified not as conductivity detectors but
as detectors in which the sensing element is not in direct contact with the electrolyte
(universal type).
The method of detection mentioned under B needs further specification. This class can
be subdivided into sub-classes indicating the way in which the detector is constructed:
(1) with the micro-sensing electrodes mounted axially, and (2) with the micro-sensing
electrodes mounted equiplanar. For the micro-sensing electrodes, of course, various noble
metals can be used (Pt- 10-30% Ir was found to be the best). The electrodes can be coated
with a suitable polymer or plated with platinum black. The frequency of the measuring
current can also be varied, but this does not give a new class of detector.
Only two of many possibilities in subclass B will be considered. In one case the driving
current itself and in the other case an external current source for the measuring current
is applied. These two cases are considered briefly below, and are then considered in more
detail in sections 6.4.2-6.4.4.

CONDUCTIVITY DETECTION 135
The d.c. method of resistance determination or the potential gradient detector. This
type of measurement of the resistance of the various zones makes use of the potential
gradient that characterizes each zone. While in thermometric detection there is a quadratic
relationship with the driving current, in this method of detection there is a linear
relationship. While in thermometric detection low driving currents could not be applied,
because the heat has to pass through the wall of the narrow-bore tube and too small a
signal remains for detection, the d.c. method allows low driving currents to be used. The
current for measuring is about A; if this value increases to A, electrode
reactions (see Fig.6.43) will result.
The a.c. method of resistance determination. This type of measurement of the resistance
makes use of an external measuring current (1-lOpA). Th~s method is applied
especially in sub-class 2 (see above), where the micro-sensing electrodes are mounted
equiplanar. Special care must be taken in insulating the low-potential circuit from the
high-potential circuit, in order to minimize the leak current. This leak current (as hgh
as 10+ A) may disturb the detection because this method of detection is particularly
sensitive to all types of coatings formed by the electrode reactions.
The d.c. and a.c. methods of resistance determination are discussed in detail in the
following sections. Special attention is paid to the various types of electrode reactions
and their influence on the recording of the isotachopherograms finally obtained. The
construction of the various measuring cells is shown, and the coatings required and the
necessary additives to the electrolytes are discussed. These additives need to be added
both in order to prevent electrode reactions, which in many instances create unwanted
coatings on the micro-sensing electrodes, and in order to reduce the electroendosmotic
flow by increasing the viscosity in the vicinity of the wall of the narrow-bore tube.
6.4.2. The d.c. method of resistance determination
The construction of a conductivity cell for use in combination with the narrow-bore
PTFE tube is shown schematically in Fig.6.10 [22].
Pieces 1 and 2 are made of brass in order to prevent an unnecessary increase in the
temperature of the electrolyte in the detector. These pieces clamp the interrupted
narrow-bore PTFE tube liquid tight without the use of adhesive (even if 6 atm pressure is
applied) in the central hole drilled through them, the diameter of which is identical with
the outside diameter of the narrowbore tube. The narrow-bore tube is first drawn over
a certain length and then pulled through the hole until it fits tightly. The narrow-bore
tube is cut off after about half an hour in order to compensate for shrinkage of the PTFE
material of which it is made. The ends of pieces 1 and 2 are supplied with a piece of
Perspex (acrylic) for three reasons:
(1) Perspex is an extremely good material for clamping the PTFE narrow-bore tube;
a brass fitting without this piece of Perspex did not clamp the tube satisfactorily. Other
plastic materials were less effective.
(2) Electric leak currents towards this piece of brass must be avoided.
(3) The thin electrodes are finally mounted between these pieces. If no plastic material

136 DETECTION SYSTEMS
5 4 1
e
2 3 6
-a
0. ....
I 1
Pt Pt
Fig.6.10. Conductivity probe for use in combination with a narrow-bore tube. 1,2 = Pieces for
clamping the narrow-bore tube; 3 = brass housing, inside which is a cylinder of Kel-F for insulating
pieces 1 and 2; 4 = screw-cap; 5,6 = electric connections, A = Pt measuring electrodes, sputtered on a
disc of insulating material; B = Pt (or Pt-Ir) foil measuring electrodes, separated by a disc of
insulating material.
is present, damage to the micro-sensing electrodes can be expected during clamping.
Two types of measuring electrodes were tested, as shown scaled up in A and B in
Fig.6.10. A disc of insulating material (0.05 mm) with R sputtered on both sides, and a
configuration in which two discs of Pt-Ir, separated by a similar disc of insulating material
(0.05 mm), were used. The electric contacts were provided by a small copper pin and a
spring mounted in the brass pieces 1 and 2. The pieces 1 and 2 and the measuring electrodes
were all aligned by precise boring in the insulating material (Amite) inside the brass
housing 3.
The brass clamping piece 2, of course, must not make electrical contact with the brass
housing, as the clamping piece 1 does, which is why an insulating disc is fixed at the
bottom. The whole unit is clamped by the screw-cap 4. The final contact to the trans-
former, to give a good galvanic separation of the micro-sensing electrodes from the
measuring electronics, is made by cables fixed in the rings 5 and 6. In this construction,
the inner profile of the narrow-bore tube is n,ot perturbed locally.
Conductivity determinations in the various zones during isotachophoretic analysis by
the d.c. method can be performed in several different ways: with an insulated d.c.
volt meter; with a floating high-voltage power supply (HSP) source; and with an insulated
d.c. amplifier. The 272 J amplifier of Analog Devices (Norwood, Mass., U.S.A.) is such an
amplifier, although the measuring current is rather high.
A possible electronic circuit for measuring the resistance by the d.c. method is shown
in Fig.6.11.
Because the driving current is used for the determination of the conductivity between

CONDUCTIVITY DETECTION 137
Hi V
1kR
15~F
330pF 4.7 k n
Fig.6.11. Electronic circuit for the determination of the conductivity of various zones by the d.c.
method (potential gradient measurement). A much better device is shown in Fig.6.14.
the two discs, the resulting potential between them is a direct measure of the resistance,
and a correction must be made for variations in the electric driving current. The input
impedance of the 272 J amplifier was found to be high enough to prevent the formation
of disturbing gas bubbles, but other electrode reactions were not completely suppressed.
Because the maximum voltage on the input of the 272 J amplifier is limited, an attenuator
(20 and 5 Ma) has to be applied. If a 272 J operational amplifier is used, only those
HSP stabilized power supplies which are equipped with a polarity switch for the case
when both anions and cations need to be separated can be chosen. One can only select
the high voltage side to be as far away from the measuring cell as possible. This decreases
the chance of destruction of the operational amplifier, which has a limited voltage
(maximal value) of 5 kV. In addition to this advantage, the electrioleak currents via the
micro-sensing electrodes will be as small as possible because the electrodes are always at
relatively low potentials. As already mentioned, these leak currents may cause a build-up
of a layer on the electrodes that may obscure the detection, especially in the a.c. method
of resistance determination, which is often applied simultaneously with the d.c. method.
In order to measure the small difference in d.c. voltage that will be obtained after a zone
boundary on the micro-sensing electrodes, on the output of the 272 J amplifier, a certain
voltage has to be subtracted for zero adjustment. Experimentally, we found that the
Diff

138
DETECTION SYSTEMS
addition of an external source, in spite of its high stability, gave poor results with respect
to the drift. There appears to be no other explanation that no compensation can be
made on the input of the 272 J amplifier. The drift obtained if an external source is used
for zero adjustment originates mainly from the variations in the current of the currentstabilized
power supply. Experimentally, we found that for simple compensation, the
driving current itself could be taken, as shown in Fig.6.11.
The value of the compensation is chosen such that the resistance halfway between the
resistances of the leading and terminating electrolytes is optimally compensated. This
method of compensation works better if the relative change in conductivity of the various
zones is smaller or if the distance between the micro-sensing electrodes is reduced as
much as possible. The signals finally obtained were of such a value that an attenuator
had to be applied for recording on a 100-mV recorder. Because the driving current is
used for compensation, changes in the electric current have less influence on the detec-
tion, as mentioned before. Because the measuring current must be low, if the d.c. method
is chosen for resistance determination, the micro-sensing electrodes may be made
extremely thin. In order to demonstrate this, experiments were carried out in which
electrodes were made by sputtering Pt on both sides of a foil of an insulator (0.05 mm
thick). An isotachopherogram is shown later in Fig.6.50. If these thin electrodes are
mounted in the conductivity cell, no simultaneous a.c. measurements can be made
because this destroys the electrode surface.
The disadvantage (a.c. method) of the axial construction of the sensing electrodes is
that the measuring current will flow mainly directly along the wall between the sensing
electrodes. Wall effects, e.g., electroendosmosis, will influence the detection more than
when the measuring current can flow through the centre of the narrow-bore tube. The
addition of surface-active substances, which directly influence these wall effects, improved
the detection by the a.c. method more than that by the d.c. method, in which much
less current has to pass the electrode-electrolyte interface. Nevertheless, surface-active
substances need to be added in order to achieve high resolution (as was found to be neces-
sary, too, if W detection was applied).
In order to make a comparison of a detector with the sensing electrodes in direct
contact with the electrolytes inside the narrow-bore tube with a thermometric detector
possible, a thermocouple was mounted around the same narrow-bore tube. The micro-
sensing electrodes and the thermocouple were mounted as close to each other as possible.
The result is shown in Fig.6.12.
The measured isotachopherogram was compared with a theoretical curve calculated
from a rough model of the current distribution. Fig.6.13(2) shows how, in this model,
the current used for the detection is distributed over the electrolytes in the neighbourhood
of the micro-sensing electrodes. Two cross-sections of the narrow-bore tube are shown;
one is perpendicular to the axis (Q), located between the two electrodes, and the other
coincides with the axis (P). When the narrow-bore tube is homogeneously filled, the
detection current is perpendicular to the cross-section Q. The simplified current pattern
is represented by parallel currents through different resistances a and b in Fig.6.13(2).
These two resistances are assumed to be proportional to the length of the lines a and b
and to the resistivity of the electrolyte.
In this model, the passage of a zone boundary can be dealt with by dividing each

CONDUCTIVITY DETECTION
Fig.6.12. Comparison of proffles obtained from thermometric recording and detection with a
conductivity probe. The dotted curve is the profile of the boundary choride/glutamate determined
with the conductivity probe, and the solid curve is the thermometxjc profile. The current was
stabilized at 70 MA. The speed of the recorder paper was 6 cm/min in both instances. The analysis
was performed in the operational system at pH 6, and the recording was made simultaneously in a
PTFE narrow-bore tube (I.D. 0.45 mm, O.D. 0.75 mm). R = Increasing electric resistance; T=
increasing temperature; t = time.
resistance in sections with different values of resistance per unit length. On passage of a
boundary, this approximation procedure leads to a resistance versus time curve as shown
in Fig.6.13(1), curve A. The correspondence with the measured curve B is acceptable,
apart from the deviations at the top of the curves. These deviations probably have three
causes: (a) the model is highly simplified; (b) in the region directly behind a boundary,
both the composition and the temperature of a zone may not be completely homogeneous;
and (c) parts of the measuring equipment introduced a certain time delay.
The detectox discussed in this section has also been applied in experiments where
coatings and additives were studied, which are described later in this chapter. The
influence of a coating on the micro-sensing electrodes can be illustrated by comparing
results from both the d.c. and a.c. methods of resistance determination during isotacho-
phoretic analyses. A small coating on the micro-sensing electrodes only slightly influences
the signal derived from the detector if the d.c. method is applied.
139

140
--- I-,-
B
-a; - --
z E E
0
DETECTION SYSTEMS
Fig.6.13. (1) Comparison of a theoretical model (A) with a practical curve (B) for conductivity
determination with the micro-sensing electrodes. (2) Current distribution in the electrolyte near the
micro-sensing electrodes, from which the theoretical model is calculated. R = Zone boundary; re,
rb and rc satisfy the equations ri = r:l4 and r;l = ri14.
6.4.3. The d.c.-a.c. converter
Fig.6.14 shows an electronic circuit (d.c.-a.c. converter) that can be used in combination
with the conductimeter discussed later (Fig.6.18).
The conductimeter was developed for resistance determinations between 50 kS2 and

CONDUCTIVITY DETECTION
Fig.6.14. The d.c.-a.c. converter.
100 kR
10 Ma, although between 10 and 50 kR a fairly linear response can be obtained. With
the converter, as shown in Fig.6.14, a maximal potential difference of 10 V can be
measured between two points, which has a maximal common mode potential of 6 kV with
respect to earth. The impedance between the two points, at the input of the circuit
shown in Fig.6.14, is greater than 10l2 !d and the common mode impedance is greater
than 10’’ R.
The junction-field effect transistor (FET) is used as a source follower. This junction-
FET is supplied by a battery, which eventually can be replaced with an electronic circuit,
although it is used here in order to minimize both the leak current towards earth
(discussed in section 6.6.4) and the parasitic capacitance towards earth. An advantage is
that the supply current of the circuit given is very small (

142 DETECTION SYSTEMS
where Yis the voltage on the potential recorder, and Int and Zero are the positions of
the controls ‘Int’ and ‘Zero’, respectively. The pA meter indicates 50 pA if V, = 0 V and
100 pA if Vh = 10 V. The accuracy in measuring Vin is better than 50 mV. If the ambient
temperature changes from 10 to 35 C, the difference in the measured value of Yb,
which of course does not change during this procedure, is less than 20 mV. In Fig.6.15,
an isotachopherogram of a test mixture of anions in the operational system at pH 6
(Table 12.1) is given. In the conclusion of this chapter, more attention is paid to this
method of detection.
It can be seen that Fig.6.15 is comparable with the linearized isotachopherogram
obtained with the a.c. conductimetric circuit, shown in Fig.6.18; in fact, we did not find
any characteristic differences.
10
10
d.C.
6
-
rO
I
7T/J1 10
Fig.6.15. Isotachopherogram of a test mixture of anions in the operational system at pH 6 (Table 12.1),
as derived from the d.c.-a.c. converter in combination with the circuit shown in Fig.6.18. For
comparison, an isotachopherogram obtained with the circuit shown in Fig.6.18 is also given. This
test mixture was applied in almost all isotachopherograms, for pattern recognition, in order to show
the various effects that may occur if insufficient precautions are taken. The leading electrolyte
consists of 0.01 N hydrochloric acid (pro analysi grade) and histidine, adjusted to pH 6; 0.05% (w/w)
of Mowiol (polyvinylalcohol) was added to the electrolyte. Glutamic acid (0.005 N) was used as the
terminating electrolyte, adjusted to pH cn. 6 by addition of Tris. 1 = Chloride; 2 = sulphate; 3 =
chlorate; 4 = chromate; 5 = malonate; 6 = pyrazole-3,5-dicarboxylate; 7 = adipate; 8 = acetate; 9 =
P-chloropropionate; 10 = glutamate. R = Increasing electric resistance; V = increasing potential gradient;
t = time.
i
8
-
a.c.
1

CONDUCTIVITY DETECTION 143
6.4.4. The a.c. method of resistance determination
In order to measure the conductivity (resistance) of an electrolyte, in which a
potential of about 6 kV towards earth is present, good galvanic insulation between the
sensing electrodes and the electronic circuit (i.e., the conductimeter) at low potential is
necessary. This can be realized by measuring the conductivity with an a.c. current that
passes through a transformer with two separated coils, the construction of which will be
described in detail. All types of electric leak currents must be prevented; even a leak
current via the sensing electrodes of 1 0-9 A will have a considerable influence on the
measurement of the conductivity. This is shown particularly in this chapter, where the
coating of electrodes is dealt with. We found that the conductivity of isotachophoretic
zones could be determined optimally with a probe in which the micro-sensing electrodes
were constructed to be equiplanar. In order to prevent an electric current flowing from
one sensing electrode towards the other, if the electrodes are mounted badly so that a
potential difference exists between the electrodes by the driving current via the trans-
former, a capacitor is arranged in series with the transformer. It can be repeated (see
section 6.4.1) that in isotachophoretic analyses the sample ions separate according to their
effective mobilities and form discrete zones with concentrations that are constant with
time, homogeneous throughout each zone and directly related to the concentration of the
leading electrolyte. If the electric current is stabilized, all of the velocities of the zones,
in the steady state, are identical and constant with time. The boundary between two
successively moving zones is sharpened by the electric field, which increases stepwise,
following each zone in whch it is a constant, to compensate for the less mobile ions. This
stepwise increase in the electric field causes a stepwise increase in the electric resistance
according to Ohm’s law. In addition, this stepwise increment is automatically directly
proportional to the effective mobilities of the ionic species actually present in each zone.
If the operational system is chosen well (e.g., Table 12.1), the electric resistance of each
zone is determined by the electric resistance of the leading electrolyte zone. These
conductivities are not defined by the electric driving current, assumed temperatures can be
neglected. Because the conductivities of the zones are determined, the length of the zones
provides the quantitative information.
6.4.5. Conductivity probe with equiplanar-mounted sensing electrodes
The construction of the measuring cell is shown in Fig.6.16, which shows also the
principle of its construction. In a round piece of transparent insulating material, e.g.,
Perspex (acrylic), a hole of diameter 0.4 mm is drilled along part of its length (a). A
metallic foil (e.g., Pt- 10-30% Ir of thickness 0.01 mm) is glued to this piece of trans-
parent insulating material at the opposite end to that where the 0.4-mm hole has been
drilled. Cyanolite@ proved to be a good adhesive when Perspex was used as the insulating
material. If different transparent material (e.g., TPX) is used, a different procedure has to
be followed. A small core of TPX is prepared, which is surrounded with a cylinder of
acrylic that fits exactly. The remainder of the procedure can then be followed as for
Perspex. With aid of a template (shown in Fig.6.16a), the centre is located with aid of a
0.2-mm drill. Special care has to be taken to ensure that this 0.2-mm hole does not

144 DETECTION SYSTEMS
Fig.6.16. Conductivity probe with the measuring electrodes mounted equiplanar. All dimensions are
given in millimetres. For explanation, see text.
C
d

CONDUCTIVITY DETECTION 145
contact the 0.4-mm hole opposite to it, because Cyanolite is applied again in a subsequent
step and if it penetrates into the 0.4-mm hole, this hole will be rough.
With alancet under a microscope, excess of the Pt- 10-30% Ir foil is cut away such that
the profile shown in Fig.6.16b remains. A new piece of insulating material is now glued
to the first one with Cyanolite, applying a high pressure for at least 5 min. The pieces
are glued well if the entire piece is completely clear again, which can easily be checked
by immersing the acrylic in kerosene; if TPX is used, immersion in glycerol is necessary
because TPX dissolves in kerosene.
If the two pieces are glued satisfactonly (Fig.6.16c), they are placed in alathe and,
after turning the piece, the 0.4-mm hole can now be drilled through the entire piece. On
both sides, collars are made for mounting the brass pieces with a screw-thread (Fig.6.16d).
These pieces are also glued to the Perspex with aid of Cyanolite. Finally, the brass pieces
are futed stably with a lock pin, which penetrates the brass cylinder that covers the whole
piece (Fig.6.16e).
The cables that provide the electrical contact are supplied with extra insulation;
generally the PTFE narrowbore tube can be used so as to minimize the chance of
contact of the brass housing with the cables. The cables are fixed to the micro-sensing
electrodes with a suitable metal paint, which is covered with a small amount of Cyanolite
for optimal solidity. The connection with the interrupted narrow-bore tube is made in a
simple manner. A piece of Perspex is provided with a hole with an inside diameter equal
to the outside diameter of the PTFE narrowbore tube. The narrow-bore tube is first
drawn out over a certain distance and is then pulled through the piece of Perspex. After
a few minutes, the narrow-bore tube is cut off straight with a lancet. These clamping pieces
are pressed in the detector with a clamping screw. To prevent any leakage, on the top of
the clamping piece of perspex, where the narrowbore tube is cut, a small O-ring of soft
rubber is applied. A film of electrolyte between the clamping piece of Perspex and the
Perspex of the detector may cause a leak current to pass through towards the brass
detector housing, which may render the analysis useless*. The basic principle of the
electronic circuit is shown in Fig.6.17.
This conductimeter is the result of the latest research and gives a linear response as a
function of the resistance to be measured. Some of the isotachopherograms, however,
were obtained with conductimetric equipment in which this linear relationship did not
hold. This will be discussed separately because the isotachopherograms obtained by the
older electronic measuring circuits differ. Later, the linearities of various conductimeters
are compared.
If the electric resistance of both coils of the transformer can be neglected and the
coupling of both coils is assumed to be unity, the transformer can be considered to be an
ideal transformer with a resistance R, and a coil L in parallel (Fig.6.17). The losses in
iron and copper are responsible for this resistance R,. If the material of the core is not
saturated, then R, can be considered to be a constant. The capacitance C and the coil
L together form a resonance circuit with a resonance frequency given by
*According to this principle, also a probe for potential gradient measurements (d.c. method) has been
made with the electrodes mounted axially.

146 DETECTION SYSTEMS
Fig.6.17. Electronic circuit suitable for the determination of the conductivity of the various zones
by the a.c. method.
1
or=- m
If the ratio of the number of turns is unity, the impedance of the circuit between the
inverting input and output of the operational amplifier of those signals which have a
frequency or can be written as
Impedance = - R"R
R, +R
The resistance R is the unknown resistance, for example between the micro-sensing
electrodes of the conductivity cell.
If vc = V, cos art (v = a.c. voltage, V= d.c. voltage) and we assume that the amplification
of the operational amplifier is infinity and dl input currents are zero, we can write
v1 =v2 (6.4)
and
If
RZ =R1
R3 R,
then

CONDUCTIVITY DETECTION 147
We can now write
and
or
Then
Thus
(6-9)
(6.10)
(6.1 1)
(6.12)
R2 R1
We can conclude that vc is a constant if - = - and the amplitude of vu is proportional
R3 Rv
to R.
After rectification and smoothing of vu, the resulting potential is also proportional to
R. In order to keep the frequency of vc equal to the resonance frequency a,., a
comparator is used, which generates vc. This comparator is controlled by vu, a squarewave
voltage; in all of the above equations we have considered only the first harmonic of
this square-wave voltage.
The higher harmonics are suppressed by the circuitry applied, assuming that R is not
too small. These higher harmonics can be neglected in this case. The circuit as finally
applied is shown in Fig.6.18.
IC2 is the operational amplifier as already discussed in Fig.6.17. lC3 rectifies the
voltage vu in a single-phase d.c. mode. The pA meter measures the average value of this
rectified signal. By means of IC5, a pre-adjusted voltage can be added to this signal, whch
is then smoothed and amplified by IC4. The smoothmg capacitcr is chosen such that the
maximal time constant involved in the amplification of IC4 is 0.1 sec. The potential
recorder used in our laboratory in combination with the conductimeter also has a time
constant of ca. 0.1 sec.
Both the d.c. voltage and the amplification can be adjusted by two potentiometers
(P3 and P4), which are supplied with a set of Multi-dials (Spectro Electronics, Plainview,

120kR Lin
fc,-IC5: $7 +15V rnetul film I*/.
1q: pA 709
1C2-IC5: pA741
Dq-Dg: i N 4148
p1 # P2 :
P3 :
P4 :
P5 :
@4 -15v
udjusting potentiometer, 100kfi (10 tui’ns)
potentiometer, 100 k n (10 turns)
adjusting potentiometer, 10 kSL
potentiometer, 50 k h
AH resistances : i/e w
TR:
Potcore :
2 x 1000 turns, 6 0.1 mrn
P 36122, 387 or 3H1 , pe= 2030
Fig.6.18. Circuit suitable for the recording of isotachophoretic zones present in consecutive zones inside the narrow-bore tube.
out
Lin
U
m
4
rn
a

CONDUCTIVITY DETECTION 149
N.Y., U.S.A.). The lowest position of both of these dials corresponds with an output
voltage of ICs and amplification of IC4 of zero. The circuit with IC1 represents the
comparator. The 1-pF capacitor and the 1000-kQ resistor are included so as to ensure that
the oscillation of the oscillator formed by ICI , IC2 and IC3 is always guaranteed. The
47-kS2 resistor in series with the 47-pF capacitor prevents undesirable oscillation of ICI
during triggering.
Th~s circuit for resistance determination was developed for use with micro-sensing
electrodes. The volume of the conductivity cell is approximately 16 nl. The output
voltage IC1 is attenuated 11 or 110 times, depending on the ‘Range’ switch, if the ‘Range’
switch is in the open position, resistances can be measured between SO kS2 and 1 MQ,
while if it is closed, resistances can be measured between 1 and 10 MQ, less accurately
than in the open position. The proportions and construction of the transformer are
mainly determined by the measuring range chosen. If the inductance of the primary coil
is too high, the quality factor of the resonance circuit is too small, which is particularly
inconvenient if small resistances have to be measured. The measurement of these resistances
are no longer accurate because the square-wave voltage from IC, is fdtered badly.
If the inductance of the primary coil is too low, the transformer is already saturated if
the resistance between the micro-sensing electrodes is high. Consequently, the voltage
over the primary coil is low, if the resistance between the micro-sensing electrodes is low.
A high capacitance of the capacitor results in rapid saturation of the primary coil, but
a low capacitance makes the influence of parasitic capacitances too great.
Of many possibilities, a capacitor of 2.2 nF, a self-induction of the primary coil of
about 800 mL and a ratio of number of turns of unity were chosen. The resonance
frequency is then CQ. 4000 Hz and the quality factor is about 1, if the resistance to be
measured is of the order of 20 kQ. Thls value is acceptable for a sufficiently accurate
measurement of the resistance. The quality factor is, moreover, dependent on the quality
of the capacitor applied, and a mica capacitor is therefore recommended as the dielectric
losses are small.
It should be noted that the circuit does not work efficiently with resistances below
1 kQ, as the coupling of the coils can no longer be assumed to be unity. If one measures
a resistance that is, for example, a factor A smaller, the number of turns of the secondary
coil must decrease with a factor of t/x; the number of turns of the primary coil remains
unchanged.
The core material is P 36/22. 3B7 or 3H1, p, (permeability) = 2030. This potcore
is provided with a gap by applying a foil of insulating material between the two parts in
order to limit the temperature drift of the inductance. A potcore P 33/22, pe = 220,
can also be used; this is already provided with an air gap.
The micro-sensing electrodes can have a maximum potential of approximately 6 kV
towards earth, otherwise the leak current towards earth is intolerable. In order to attain
this value, the secondary coil must be well insulated, eg., by constructing both the
primary and secondary coils in a PTFE housing. A possible construction is shown in
Fig.6.19.
The extra PTFE ring is mounted around the secondary coil. The wires leaving the
PTFE via a small hole made in the PTFE are insulated with an extra PTFE narrow-bore
tube. This transformer must be mounted as near as possible to the micro-sensing
electrodes so as to diminish losses of electricity towards earth.

150 DETECTION SYSTEMS
/
primary coil
Fig.6.19. Construction of the transformer for a good galvanic separation of the high potential at the
micro-sensing electrodes from the low potential of the conductivity measuring circuit. A good
insulation is necessary because a leak current towards earth often causes the formation of a coating
that may obscure the recording. All dimensions are given in millimetres.
In our equipment, this transformer, which separates galvanically the sensing electrodes
from the circuitry at low potential was mounted on the electrophoretic equipment itself
about 3 cm away from the conductivity probe. Even if a well insulated cable is used, a
length of about 1 m is enough to influence the recording by the parasitic capacitance
resulting from it.
This, as already mentioned, results in the formation of a coating deposited on the
micro-sensing electrodes, built up by the electrolytes present inside the narrow-bore tube
during the analysis. This effect is particularly large if pyridine is used as a buffering
counter-ion, but even with moderate buffers such as histidine the effect is far from
negligible in the long term. More attention is paid to these undesirable effects in this
chapter, where the coating of electrodes is dealt with. Uncontrolled coating of the micro-
sensing electrodes decreases the resolution of the detection of the isotachophoretically
moving zones and, in particular, the passage of a zone boundary is recorded more slowly.
The detector must be cleaned either with aqua regia while it remains mounted in the
electrophoretic equipment or with a suitable metal polish while it is dismounted. For
optimal detection, the electrodes, made of Pt or Pt-Ir, must be passivated, which can be
30

CONDUCTIVITY DETECTION 151
achieved by passing a 5-PA current for 5 min. After the passivation, a new equilibrium
must be attained, which takes about 1.5 h. Passivated electrodes were found to be less
sensitive for all types of electrode reactions under the conditions used in our electrophoretic
equipment. It should be particularly noted that the difference in resistance
determined for an arbitrarily chosen electrolyte, after a procedure during which
hydrogen or oxygen is evolved on the micro-sensing electrodes, may be as great as the
difference in resistance between the leading and terminating electrolytes. If nonpassivated
electrodes are applied, automatic passivation can occur during the analysis
by the driving current, especially if reactive ions are present, e.g., chromate. It is clear
that the qualitative information will be obscure if the micro-sensing electrodes are
partially passivated during the recording of an isotachophoretic experiment*.
Several noble metals and their alloys were tested, and some electropherograms
obtained with these electrode materials can be found in Section 6.6. Experimentally, we
found that hard electrode materials were especially suitable for our purpose.
The circuitry for the determination of the conductivity by the a.c. method (Fig.6.18)
can be tested as follows. Make the offset voltage of IC3, IC4 and lC5 minimal. Connect
the output ‘Lin’ with a voltmeter with a measuring range of 100 mV and an accuracy
better than 0.1%. Short-circuit the diode D4. Turn the dial ‘Zero’ to its minimum
position, the dial ‘Int’ to position 1 and the switch ‘Range’ in the l-Ma position. The
adjusting potentiometer ‘Lin’ is arranged in a position such that on the output a value of
approximately 90 mV is obtained if the conductivity probe, with no liquid in it, is connected
to the secondary coil. The resistance is now defined as ‘infinity’. The capacity of
the conductivity probe, which is part of the measuring circuit, is compensated in this way.
The short-circuit of the diode D4 must now be removed and a 1 -Ma resistor connected in
parallel with the conductivity probe. The output voltage is adjusted to 100 mV by means
of the adjusting potentiometer ‘Gain’. The correct shunt resistance for the pA meter to
give it a full-scale deflection is then sought. The potentiometer ‘Zero’ is turned to its
maximum position and the output voltage is corrected for zero by means of the adjusting
potentiometer ‘Zero adjust’. The circuit is now ready for use in the determination of
resistances up to 1 Ma.
If higher resistances need to be measured, e.g., if the concentration of the leading
electrolyte is decreased to
or even N, the following procedure should be
followed. Turn the potentiometer ‘Zero’ to its minimum value. Select the range 10 Ma
and mount a 9-Ma resistor in parallel with the conductivity probe. Adjust the output
voltage to 90 mV by means of the adjusting potentiometer ‘Lin’. Replace the 9-MQ
resistor with a 1-Ma resistor. Set the ‘Range’ switch in the’ 1-MS2 position and adjust the
output voltage to 100 mV by means of the adjusting potentiometer ‘Gain’. Repeat these
last procedures until no further corrections need to be made.
For continuous recording of the resistance, the output ‘En’ is connected with a
potential recorder with a sensitivity of 100 mV.
If the ‘Range’ switch is in the 1-MSZ position, the resistances to be measured can be
determined from the equation
-.-
+-
-- R
-
V 0.1 Zero
1 MC! 100mV Int 1
*This was found especially when the electrodes were made of Pt instead of Pt-Ir.
(6.13)

152
DETECTION SYSTEMS
where R is the resistance to be measured, Pis the output voltage of ‘En’, and Int and
Zero indicate the ratio between the real and the maximum values of the multi-dial unit
connected to the potentiometers ‘Int’ and ‘Zero’, respectively.
For the determination of resistances higher than the 1 Ma, the ‘Range’ switch is set
in the second position. In a similar way, the resistance can be determined from the
equation
Fig.6.20. Results from the circyit shown in Fig.6.18 with two measuring ranges: 50 kR-1 Mn and
1-10 MR. Between these limits, a linear response as a function of the resistance is obtained. The
linearity (a) is compared with that of an electronic measuring circuit (b), which was used amongst
others during the study of the effect of additives and coatings on the microsensing electrodes
discussed in this chapter. R = Increasing resistance.

UV ABSORPTION METER 153
-_-.-
+-
R - V 0.1 Zero
10MR l00mV Int 1
(6.14)
The potentiometers ‘Zero’ and ‘En’ must be arranged in a position such that during the
analysis the output voltage on ‘Lin’ always remains between 0 and 100 mV. An example
of the linearity of the circuit shown in Fig.6.18 is given in Fig.6.20, in comparison with
that of the older circuits. Most of the isotachopherograms considered in this chapter were
obtained with non-linear circuitry, but the data collected for the various operational
systems that are given in Chapters 11-17 were obtained with linear circuitry.
At a resistance of 1 MR, the accuracy of the resistance determination with linear
circuitry is better than 2 kf2, whde at 10 MR the accuracy is better than 30 kR. If the
ambient temperature increases by 10°C, the difference in resistance determination at a
level of 1 Mi2 is less than 1 kR and at 10 Mi2 it is less than 20 kR.
The stepwise trace obtained during an isotachophoretic analysis when the zones pass
the conductimeter can easily be interpreted for qualitative and quantitative information,
because the steps are sufficiently sharp. This is in contrast with thermometric recording.
Two main reasons can be given why a differentiator was constructed. (1) If small
zones are present, stacked between the others, these zones can be detected much easier
and better with an electronic differentiator. (2) If electronic devices are available and
applied for measuring the time interval between two successive peaks, a pulse is needed
for the printer. A possible circuit for such a differentiator is given in Fig.6.21.
The circuit with IC6 is actually the differentiator. Its amplification can be chosen by
the potentiometer ‘Diff. The time constant is 24 msec, which is small enough to differentiate
the signal on the output ‘En’. The circuit IC7 and lCs is a double-phase rectifier.
A potential recorder with a sensitivity of 100 mV can be applied in combination with
ths differentiator. IC9 forms, together with the two resistors on the non-inverting input,
a Schmitt trigger with a very small hysteresis. The 10-pF capacitor ensures that the
input signal is equal to the first differential of the output signal of the rectifier. If this
value is zero, the Schmitt trigger is triggered. This procedure is shown schematically in
Fig.6.22.
The printed values of the time intervals between the fast moving (about 1 m/h) zone
boundaries increase the accuracy of time recording and hence the quantitative data
handling and, moreover, simplifies the latter. The accuracy in time recording is better
than 10 msec, which represents an accuracy of 5 - g-equiv./l, assuming an electric
driving current of 70 /.LA and a concentration of the leading electrolyte of lo-* g-equiv./l.
6.5. UV ABSORPTION METER
6.5.1. Introduction
Because in the steady state of an isotachophoretic separation all components move in
individual zones with identical speeds and only the counter ion is mixed with these
components, specific detectors such as the W absorption meter can offer much additional

Fig.6.21. Differentiator for the a.c. conductivity detector and the circuit that produces signals suitable for the printing of the various zone transitions
needed for the quantitative measurements. This circuit can be used in combination with the circuit given in Fig.6.18.
154
DETECTION SYSTEMS

UV ABSORPTION METER
Fig.6.22. Control of the printer by signals derived from the ax. conductivity detector.
information about the ions of interest. The main disadvantage of these types of detectors
is the small cell volume for which specific information can be obtained, because narrow-
bore tubes are used. The enlargement of this cell volume by measuring the absorption in
an axial direction, as is commonly done in equipment for liquid chromatography, is
impossible because one expects small zone lengths in isotachophoresis. For this reason, a
new type of detector that is not yet commercially available has been developed. Another
disadvantage is that some of the non-UV-absorbing ions, especially cations, which are
present in the sample can easily be ‘missed’ if they form zones that combine with each
other. However, some possibilities will be mentioned for removing this last disadvantage
in some specific instances.
6.5.2. Construction of the UV source
Microwave mercury electrodeless lamps can be used successfully for the UV absorption
meter, because nowadays stable transistors are available that operate at a frequency of
100 MHz with a power of at least 5 W.
The lamps can consist of thin-walled quartz tubes with a diameter of about 1 cm. The
lamps that we tested had a volume of about 8 ml and were filled with about 5 mg of
mercury [23] . A clean gold-plated carrier is used to facilitate the weighing and transportation
of the mercury during the preparation of the lamp. The preparation is carried out
as follows (Fig.6.23). Some quartz tubes are melted on to a central tube, and the system
is then evacuated to a pressure of about
torr. Under this vaccum, these tubes are
baked at about 1000°C for several hours, while in the side-tube the mercury on its gold-
plated carrier is kept at the temperature of liquid nitrogen. In order to permit the escape
of water and other gases collected during the baking procedure, the liquid nitrogen must
be removed temporarily, but the temperature must not be allowed to rise too much.
After this procedure, the entire system is filled with dry argon to a pressure of 3 torr.
A discharge is then run in the quartz tubes for a few minutes. This discharge is not run
155

156 DETECTION SYSTEMS
Fig.6.23. Schematic diagram of device for the preparation of the mercury electrodeless microwave-
powered lamps. The mercury is easily handled by using a gold-plated carrier.
in the side-tube, where the mercury is kept on its gold-plated carrier, because the
temperature will rise too much and the mercury will be released. Also, this tube will not
be used as a lamp and substantial losses of the mercury must be prevented. The argon is
then pumped off and the liquid nitrogen is removed from the side-tube. Now the entire
system, Le., the quartz tubes and the side-tube, is separated from the pump by means of
a valve in order to prevent too much of the mercury from being lost during the next
step. The mercury is distilled in the quartz tubes, all of which are cooled in turn by
gently heating the side-tube. After the distillation has been completed, the liquid nitrogen
is placed around the side-tube again. The valve is opened and dry argon is admitted to a
pressure of about 2 torr. Again a discharge is run while the argon is pumped off very
quickly. If ths procedure is repeated about three times, all lamps light up brightly.
Because each time the pumping time is very short, only a small amount of mercury is lost.
Before the lamps are sealed off at the constrictions A, the mercury condensed at the
constrictions A and B is redistilled into the lamps with the aid of a burner, while the
lamps remain cool at the bottom. The brightness of each lamp is slightly different,
however even if stringent precautions are taken during the preparation to make all lamps
as reproducible as possible.
A circuit suitable for exciting the mercury electrodeless lamp is shown in Fig.6.24.
The microwave-powered mercury electrodeless lamp is placed between the plates of
the capacitor, clearly shown in Fig.6.25. The lamp itself is fixed in the housing, that
surrounds the electronic circuit. For optimal stability, this surrounding must be made of
metal. It must not be made too small in order to prevent the jump-over of the high-
frequency signal applied. The housing must be earthed. If modulation is not necessary, the
resistance R1 must be increased to 56 Q, and points B and C must be short-circuited. By
ths means, the capacitor C7 and the radio-frequency choke are disconnected.
The circuit shown can be modulated 100% via the modulation input 'Mod'. If the
voltage on this input is approximately - 15 V, the W source produces the maximum
amount of W light. When the electric current through R1 is zero, the UV source is
switched off.
Because the mercury lamp must ignite each time when the source has been switched

W ABSORPTION METER
r
+15V -15V Mod
Ti : 2N3553 C1,C2’: 4.7pF
D~ - D : ~ i ~414a c3 : 120 pF
R1: 33n,1/4w C4- Ge: 4.7 nF
u2: 10kh,1/aW c7- c9: ca. 5 n ~
Lsm : radio -frequency choke
L: 3 + 12.5 windings, @ 2mm
Fig.6.24. Electronic circuit suitable for continuous excitation of the mercury electrodeless lamps. L
is a coil made of copper wire (2 mm diameter) of 3 + 12.5 turns; the outside diameter is 15 mm.
off, the modulation frequency must not be lower than 100 Hz (safe limit). Sometimes, if
no modulation is required, low-pressure lamps are difficult to ignite. This can often be
remedied simply by rubbing with a wool cloth. The modulation input ‘Mod’ is connected
with the point ‘to Mod’ shown in Fig.6.29. The supply current has an average value of
80 mA, whether or not modulation is chosen. This means that in both instances the
average amounts of W light are comparable (Table 6.4).
It is very important not to decrease the frequency at which the lamp is excited too
much. Tests on mercury lamps operating at a variety of frequencies showed that these
lamps become discernibly darkened in a few days when operated at a frequency below
50 MHz. Lamps operated continuously at 80 MHz for at least 1000 h showed only slight
discoloration, which could easily be corrected with the circuitry used.
The transistor oscillator circuit needs careful construction. The coil must be fixed
stably to the printed circuit plate because vibration may alter the capacitance between the
various parts and influence the coupling of the coil and the lamp. The light produced by
the lamp would change in intensity and noise would result. The solder points must be
made as short as possible and the wires that have to be used must be made as flat as
possible, because both solder points and wires may influence substantially the coupling
and hence the brightness of the lamp. In order to make a UV source that operates well, it
157

158
DETECTION SYSTEMS
Mod +15V -15V
Fig.6.25. Mechanical construction of the UV source. The printed circuit plate is surrounded by a
metal box to give optimal stabilization of the source and to decrease the influence of the source
itself on the final recording by the W detector. For optimal results, all sources must be made as
similar as possible. All dimensions are given in millimetres.
is essential to build the circuit shown in Fig.6.25 so as to conform as precisely as possible
to the values given.
In order to prevent the electromagnetic field causing a disturbance, for instance by
radiation, the circuit is surrounded with a metal box. Even the cables for the power
supply and the connection of the modulation input ‘Mod’ towards the modulation output
‘Mod out’ are screened. If lamps are used under different laboratory conditions, the coil

W ABSORPTION METER 159
TABLE 6.4
PROPERTIES OF THE TWO TYPES OF UV SOURCES
Type of UV Noise voltage Microphonic Sensitivity Drift (10-50 C) Influence of external
source (mV) noise (V/N (mV) light source
Modulated

-+
+-@
m
> - In c
Fig.6.26. The UV detector to be used in combination With the non-modulated UV source. B is the Type R 330 UV-sensitive photodiode (Hamamatsu).
160 DETECTION SYSTEMS

W ABSORPTION METER
230 240 2% m zm 280 EC GO 30 m 330
wovelength (nm)
Fig.6.27. Optical filters used for the UV absorption measurements on the various zones. A combina-
tion of an interference filter and an end filter (coloured glass filter) was always used. From this figure,
an estimation of the band width can be made for the experiments included in this book.
impedance, the circuit must be surrounded with a metal frame in order to prevent
disturbances. Special care must be taken in selecting the 104-Ma resistor, which is
basically responsible for a bad signal-to-noise ratio and drift.
In the PTFE narrow-bore tube (I.D. 0.45 mm, O.D. 0.75 mm), in combination with
a slit width of 0.3 mm*, the difference in signal (uv) between dark and light is approxi-
mately 100 mV, which is the normal value if a non-absorbing component is inside the
narrow-bore tube with water as the solvent and the combination of interference filter
and coloured glass filter (end filter) is chosen, as shown in Fig.6.27. This value of 100 mV
is chosen arbitrarily and depends mainly on the sensitivity of the potential recorders
available.
6.5.4. UV detector in combination with a modulated UV source
In the UV detector, the alternating current, due to the alternating influx of W
quanta, in the phototube is changed to an alternating voltage, which is then detected
synchronously.
*Later experiments learn that with a slit of 0.1 mm and a narrow-bore tube (I.D. 0.2 mm, O.D.
0.4 mm), sharper boundaries could be detected between the various zones.
161

162
DETECTION SYSTEMS
The modulation frequency must always be chosen to be greater than 100 Hz. A
preferable modulation frequency in our laboratory was found to be 125 Hz, at which
value the influence of possible electric currents in the phototube with a frequency of
50 Hz (mains frequency) or higher harmonics of ths frequency is minimal. These
electric currents may originate in the 100-Hz modulation in the electric lighting and in
disturbances in the mains. The circuit is shown in Fig.6.28.
The circuit with IClo is comparable with the part shown in Fig.6.26. The resistor R,
may not have a value greater than 1 G!2 because otherwise the band width of the circuit
is adversely affected. If this value is not exceeded, a source follower on the input of IClo
can be applied. This source follower can be a normal junction-FET, which is less noisy
than an MOS-FET. By means of ICll , the alternating voltage on the output of IClo is
amplified by a factor of 100. The circuit is operated such that the noise voltage on the
output of the synchronous detector is minimal. If the sensitivity is too great, the value of
R, and/or the amplification of ICll can be decreased. The output voltage of IClo should
not exceed * 1OV. In order to minimize the influence of the UVsource on the W detector,
the circuitry is surrounded with a metal frame and all connection cables between them are
screened. The output of ICI is connected with the input ‘Dem in’, as shown in Fig.6.29.
A synchronous detector is shown, combined with the oscillator that controls both the
detector and the W source. The oscillator is a symmetrical emitter coupled with a multivibrator,
which controls the transistor T4 such that it is periodically saturated and blocked,
The UV source, of which the modulation input ‘Mod’is connected with the collector of
T4, is thus periodically switched off. With the l-kn potentiometer, the modulation
frequency can be adjusted to 125 Hz.
All resistances : l/8 W
IClo, IC1, : PA 741
Tg : 2 N 5245
Fig.6.28. UV detector that can be used in combination with the modulated W source. This circuit is
applied in combination with the synchronous detector shown in Fig.6.29.

to
Mod
(Fig 6.24)
oscillator
I synchronous
I
detector
AH resistances : 1/8 w Tq : 40361
U metal film 1 %
T5 : 2 N4124
2N4126
IC12 : 7796 Tg-Tg:
IC13 : pA 741 Dg,D10: 1N4148
100 mV
Fig.6.29. Synchronous detector that handles the signals derived from the electronics of Fig.6.28. The electronic circuit converts the current variations in
the R 330 phototube (Hamamatsu) into voltage variations.
c
m
W

164 DETECTION SYSTEMS
IClz is a double-balanced modulator/demodulator. The input 'Dem in' is connected
with the output of the circuit shown in Fig.6.28.
The potential difference between the connection points 7 and 8 of the ICll is a square
wave that is in phase with the modulation of the W source. Synchronous detection is
achieved by multiplying the voltage on the input by this square-wave voltage. If UV light
from the UV source penetrates the W-sensitive R 330 phototube on the input, a squarewave
voltage also results. On the output of the differential amplifier I&, a d.c. voltage
is the result, which is proportional to the amplitude of the square-wave current in the
phototube. Alternating voltages on the input with frequencies that are not identical with
the modulation frequency or the odd harmonics cause alternating voltages on the output.
These voltages are suppressed by the 6.8- and 68-pF capacitors. The time constants that
result from these capacitors in the amplification of the differential amplifier are of the
same magnitude as that of the potential recorder. The output voltage 1 ~ ~ lo9 x 3 i’,
where Pis the peak-to-peak value of the square-wave current in the phototube; vu can
never exceed 100 mV. The peak-to-peak value of the noise voltage on the output is less
than 0.5 mV.
6.5.5. UV cell
A schematic diagram of the UV detector cell is given in Fig.6.30.
The PTFE narrow-bore tube (1) is not interrupted and is pulled through a slit (3),
shown scaled up in order to demonstrate its construction. The slit is made of brass with
an axial hole of 0.7 mm so as to enable the narrow-bore tube to pass through. Perpen-
Fig.6.30. Schematic diagram of the W detector. A = direction towards the injection system
(terminator compartment). B = direction towards the counter electrode compartment. 1 = PTFE
narrow-bore tube; 2 =position free for mounting the conductivity detector; 3 = W slit; 4 = quartz rod
(optical quality); 5 = holder for the quartz rod; 6 = microwave-powered mercury electrodeless lamp;
7 = photodiode (R 330, Hamamatsu); 8 = UV detector; 9 = holder for end fiter and interference
filter; 10 = construction for assembling 11 and 9; 11 = holder for the quartz rod; 12 = construction
for fixing 1, 3, 5 and 11; 13 = construction for mounting 5,14 and a slide; 14 = UV source.

UV ABSORPTION METER 165
dicular to this hole, a hole is drilled of diameter 0.3 mm with a conical shape at the
outside on both ends, so that the quartz rod (4) can approach the central hole as close
as possible. The diameter of the quartz rods (optical quality) is 3 mm. The distance
between the quartz rods and the narrowbore tube must be made as small as possible,
otherwise some of the UV light is lost by absorption by oxygen. The quartz rods are
fixed by brass holders (5,ll). Before mounting, the outside of the optical quartz rods
and the inside of the brass holders must be cleaned carefully with cyclohexane in order
to remove all UV-absorbing material. After ths procedure, the quartz rods must be
handled with a pair of clean tweezers in order to prevent dirt from sticking on the surface.
An adhesive must not be used to fix the quartz rods in the holders. Any resin on the
surface of the rods absorbs large amounts of W light; when the rods were fixed in the
holders with Cyanolite, the signal was reduced to 1% of its original value. The two brass
holders, with the quartz rods fixed simply with an O-ring, clamp the slit holder (12) by
means of a set of O-rings. The flanges fit the central housing (not shown in Fig.6.30)
and fixes the slit holder at a pre-determined position.
The edges on the ends of the holders of the quartz rods act as an important lock for
daylight, together with components 10 and 12. This lock is not as important at the side
of the W source because the combined interference filter and end fdter is mounted at
the side of the UV detector (8). If this lock is not positioned in front of the photodiode
(R 330) at least a noisy signal may be expected and too much daylight may even destroy
the phototube.
The holder 9 contains the combined interference filter and end filter. In the W source
(14), the UV light is generated by the microwave-powered low-pressure mercury
electrodeless lamp (6). A slide (13) makes it possible for the narrowbore tube not to be
exposed constantly to the UV light, which may damage the PTFE. After a long exposure
time, the quality of PTFE coloured by UV light is poor. For optimal stability of the UV
lamp, it is preferable to have the lamp constantly in the bright mode. After switching on,
it requires at least 1.5 h to attain full optimal stability. The slit holder (12) contains an
extra hole that permits the conductivity detector to be mounted near the W detector
cell. It has proved to be unimportant if the sequence of either the UV detector and the
conductivity detector is altered. Of course, exceptional cases can occur.
6.5.6. Experimental
It does not need a long explanation about the way in which the UV detector will
produce supplementary information about the isotachophoretic separation, because many
ions lack W absorbance and so many successively moving zones that contain ions without
any UV absorbance will not be detected. In some instances, minor UV-absorbing
components (see Fig.6.15) are present in the electrolytes (the leading electrolyte, the
sample and the terminating electrolyte). These impurities can ‘mark’ a zone boundary,
because these components are also concentrated in zones, sandwiched between the
sample zones.
If a mixture of mainly non-UV-absorbing components is available, one can add to
the sample an extra amount of UV-absorbing markers in such a concentration that, if
these markers cannot be separated from the sample ions and form stable mixed zones, they

166 DETECTION SYSTEMS
do not really contribute to the increase in zone length (quantitative information).
These markers must not interfere with the sample ions or electrolytes of the operational
system, and they must be present in such concentrations that they can be detected (the
zone length is not important) by the W detector, if separated isotachophoretically.
In many instances, one can use another approach*, as follows. A concentration always
changes at a zone boundary, of the order of 2-20% for the ions to be separated. A change
in the concentration of the counter ion is also likely, although this component is selected
on the basis of its pK value and determines the pH at which the analysis is performed. If
all conditions are fulfilled satisfactorily, the counter ion always remains in its region of
dissociation. For this reason, the concentration step for the counter ion is only a few
per cent; it may be positive, negative or zero, because it is related to the change in
dissociation and pH across the zone boundary. As we are working in the buffer region of
the counter ion and therefore the acidic and basic forms of the ion can be compared, we
can select this counter ion on the basis of its pK value and its large difference in absorp-
tivity between the acidic and basic forms, especially in the W region of the detector
[ 111 . The pH difference across a zone boundary will give rise to an absorbance difference
that is sufficiently large to be detectable. This procedure is shown in the isotachophero-
gram in Fig.6.31.P-Alanine (of the highest commercial grade) and re-crystallized from
water-ethanol (1 : 1) was used as the counter ion in (b). Detection was carried out with
both a conductivity and a W detector. In (a), creatinine (also of the highest commercial
grade) was chosen as the counter ion. Both the leading electrolytes had chloride as the
mobile ion (0.01 N) and the pH in both instances was chosen as 4.1. Creatinine is well
known for the large difference in the molar absorptivities of the acidic and basic forms.
For this reason, the absorption must vary visibly if the pH of a zone varies, even if a
non-W-absorbing ion is present, and a ‘stepwise’ function of the W signal is generated.
The final signal is not proportional to the effective mobilities, especially if other UV-
absorbing ions are present and somewhat obscure.
It should be noted that the pH does not always increase from one zone to another in
the direction of the terminating zone in anion analyses and does not always decrease in
cation analyses. As indicated in the Section Theory, the pH of a zone is a function of
various parameters.
This method of indirect W detection can, of course, also be applied in operational
systems that are suitable for the separation of cations, many of which lack W absorbance.
The following are some of the counter ions that have been tested so far that can be used in
operational systems: sulphanilic acid, 4.23 > pH > 3,256 nm; fumaric acid, 4.94 > pH
>3,256 nm; and ascorbic acid, 4.6> pH>3.6,280 nm. The principle of indirect W
detection can also be applied in operational systems in which methanol is used as the
solvent. Attention has so far been paid to the separation of cations in methanol because
the mobilities of cations in water do not differ so much and separation according to
pK values is difficult because many of the cations have similar pK values in water. This
subject is discussed more extensively in the Section Applications and the data can be
found in Chapters 11-17.
Sulphanilic acid was found to work well in methanolic systems at 256 nm. Sulphanilic
*The ‘indirect UV method’.

Fig.6.31. Isotachopherograms for the separation of some anions that lack W absorbance. (a)
Creatinine, for which the molaf absorptivity of creatinine is function of the pH. Leading electrolyte
= HCl(O.01 M, pro analysi grade) t creatinine (purified); pH = 4.5. Terminating electrolyte =
morpholinoethanesulphonic acid (MES) (re-crystallized three times). (b) Counter ion for which the
change in pH of a zone does not influence the molar absorptivity (p-alanine). Leading electrolyte
= HCl (0.01 M, pro analysi grade) t e-aminocaproic acid (purified); pH = 4.5. Terminating
electrolyte = MES (re-crystallized three times). Non-W-absorbing ions can thus be detected by the
‘indirect UV method‘. A = Increasing UV absorption; R = increasing electric resistance; t = time. The
current was stabilised in both instances at 30pA. The chart paper speed was 2 cm/min. An injection
was made of 1 pl of the sample 0.01 Mchlorate t 0.01 Macetate + 0.01 M formate + 0.01 M
glutamate. Peaks: 1 = chloride; 2 = chlorate; 3 = formate; 4 = acetate; 5 = glutamate; 6 = MES. In (a)
the lower pH of the glutamate zone with respect to the zone preceding it and following it is clearly
visible. From this isotachopherogram, the pH can easily be checked as it can be calculated with the
computer program discussed in Chapter 4. The difference in the step height as found in the linear
conductivity trace, due to the difference in the counter ion, should be noted. The conductimeter
used is discussed elsewhere (Fig.6.18); the measuring electrodes were mounted equiplanar. Various
impurities commonly present in the chemicals can be observed in the traces from both the UV and
conductivity detectors.
167

168 DETECTION SYSTEMS
acid has a low electrophoretic mobility in methanol, although it dissolved sufficiently
in it. A solution of methanol saturated with sulphanilic acid can be used to indicate the
pH differences in the succesive zones in cation separations if acetate is used simultaneously
as the electrically conducting counter ion with buffering capacity. As already mentioned,
the contribution of sulphanilic acid to the buffering capacity and conductivity is
negligible.
The W detector can also be used for the determination of trace amounts of W-
absorbing material. An arbitrarily chosen component, salicylic acid, is used because it
shows moderate UV absorption. For the determination of trace amounts of, e.g., ATP
or ADP, the following method works more satisfactorily because of the higher molar
absorptivity of these ions.
Suppose the concentration of the salicylic acid is so small that even with the concen-
tration effect of isotachophoresis the zones are too small for complete qualitative and
quantitative determinations to be effected by the W detector or any detector with equal
resolution. One can decrease the concentration of the leading ion, because the subsequent
zones will be more dilute than the leading ion and longer zone lengths can be expected.
The contribution to the conductivity from the solvent itself will be greater the more
dilute are the solutions, because the mobility of, e.g., H’ and O H, is great if water is used
as the solvent. While at a concentration of the leading ion of 0.01 N a pH of 3 is a critical
value, at a concentration of the leading ion of 0.001 N a pH of 4 is critical. The use of a
I
Y 7
f
7
3 2 d
Fig.6.32. Isotachophoretic separation of phosphate, salicylic acid and a mixture of them in an
operational system at pH 4.2. HCI (0.01 M pro analysi grade) was taken and p-alanine (re-crystallized)
added until the pH reached 4.2; 0.05% of Mowiol was added to this electrolyte. The terminating ion
was glutamate. Separations: (a) phosphate; (b) 1:l mixture of phosphate and salicylic acid; (c)
salicylic acid. The shift in the step height of the UV traces should be noted. An enrichment of
salicylic acid was revealed by the W detector in (b), but not by the conductivity detector. 1 =
Chloride; 2 = salicylate; 3 = glutamate; 4 = phosphate. R = Increasing electric resistance;A = increasing
UV absorption; t = time.

UV ABSORPTION METER 169
50
Fig.6.33. Plot of step heights (h mm) in the UV traces for the mixed zone (Fig.6.32b) against the
concentration ratio of phosphate to salicylate (r). BY this ‘dilution’ technique, the W detector
sensitivity is improved for the UV-absorbing ion. For salicylic acid, the sensitivity is improved at
least 50-fold.
counter flow of electrolyte is feasible, but longer times of analysis are involved and very
pure electrolytes and even more complicated equipment are necessary. Alternatively to
these two procedures, a decrease in the concentration of the leading electrolyte and
counter flow of electrolyte may be applied for both W-absorbiag and non-UV-absorbing
ions.
Because salicylic acid shows UV absorption, another approach is possible. An opera-
tional system is chosen such that a stable mixed zone can be made of salicylic acid and a
non-UV-absorbing acid. At pH 4, phosphate has been found to be satisfactory.
In Fig.6.32, the isotachophoretic separation of phosphate, salicylic acid and a mixture
of phosphate and salicylic acid is shown. The leading electrolyte was 0.01 N hydrochloric
acid (pro analysi grade) plus 0-alanine, re-crystallized from water+thanol (1 : l), adjusted

170 DETECTION SYSTEMS
to pH 4.2. The electric current was stabilised at 70 PA and W detection was carried out
at a wavelength of 256 nm. The drop in the height of the UV trace is clearly visible. The
trace from the linear conductivity detector does not resolve these two acids in the mixture.
The step height in the UV trace for salicylic acid can be plotted against the concentra-
tion ratio of phosphate to salicylic acid, as shown in Fig.6.33’. A 50-fold dilution of
salicylic acid could easily be determined, which means that the resolution improves by a
factor of 50. The step height in the W trace may be influenced by the following factors:
the pH of the ‘mixed zone’ may vary as a function of the composition, which may
influence the step height if there is a large difference between the acidic and basic forms
of the molar absorptivity; and the molar extinction coefficient of the W-absorbing
component. It does not need to be explained that a larger improvement in the resolution
may be expected if the molar absorptivity increases. Moreover, the use of a En-Log
converter will further improve this method.
Anticipating on analyses discussed later, Fig.6.34 demonstrates that salicylic acid and
phosphate can be separated at another pH, the so-called separation according to pK values.
Thee isotachopherograms are shown, demonstrating the separation of phosphate and
salicylic acid (1 : 1) at pH values of 3.2,4.0 and 7.0. It can clearly be seen that these two
acids can be separated at pH values both below and above 4.0. At a high pH, the
influence of carbonate can be seen, because no precautions were taken.
w-
4 3 2
I-
7L
i l 1
I
~
a -
-7
2+3
L;@
, - 1
-L
Fig.6.34. Isotachopherograms of the separation of phosphate and salicylic acid, demonstrating that
the separation is possible at both a ‘high‘ pH (7) and a lower pH (3). Because no precautions were
taken during the preparation of the operational system at pH 7, the influence of carbonate can be
seen. This aspect is considered further in Chapter 12. (a) Separation at pH 3; (b) separation at pH 4.2:
(c) separation at pH 7. Glutamate was used as terminator. 1 = Chloride; 2 = phosphate; 3 = salicylate;
4 = glutamate. A = Increasing UV absorption; R = increasing electric resistance; t = time.
*The signal-to-noise ratio of the W detector is such that an amplification of at least 1000-fold is
possible.
R

ADDITIVES TO THE ELECTROLYTES 171
6.6. ADDITIVES TO THE ELECTROLYTES
6.6.1. Introduction
As soon as the high-resolution UV detector became avdlable and the results could be
compared with those of the conductivity detector, with comparable resolution, the
initially non-reproducible results of both the conductivity detector and the UV detector
could be studied more intensively. The UV detector mainly does not disturb the isotachophoretic
pattern by its presence, except for compounds that may be destroyed by the UV
light applied or if the material of whch the narrow-bore tube is constructed is eventually
affected by the UV light. The conductivity detector, however, may disturb the electrophoretic
pattern as a result of the polarization initiated by the driving current or due to
a leak current, or due to excessive heat produced by the measuring current. Because in
our systems the last mentioned current is small compared with the driving current, the
heat produced by the driving current can be neglected.
The aim of making additions to the electrolytes may vary. The addition of surfactants,
for instance, not only sharpens the zone boundaries by depressing electroendosmosis,
especially visible if the combination of a UV and a conductivity detector can be applied,
but also influences the overpotential against electrode reactions on the micro-sensing
electrodes of the conductivity detector.
Additives can be classified into three categories: additives that affect the electro-
endosmotic flow; additives that influence various electrode reactions; and additives that
show both of these effects.
A study was undertaken in order to elucidate these phenomena. Another purpose of
the study was to show the difficulties that might arise if the electrophoretic equipment
is not well constructed. Many of the problems that were initially present during the
development of the conductivity detector have been overcdme, but more useful informa-
tion can be gained from considering these problems than from presenting the final
solution only.
6.6.2. Effect of additives on the electroendosmotic flow
Electroendosmosis is the movement of a liquid with respect to a solid wall as the result
of an applied potential gradient. Although it is generally assumed that the electroendos-
motic flow can be neglected in a single narrow-bore tube, with high-resolution detectors
this is not so. In the beginning of isotachophoresis (displacement electrophoresis), the
viscosity of the electrolytes was increased in order to suppress the electroendosmotic
flow, to prevent hydrodynamic flow (semipermeable membranes were not used) and to
suppress convection. The viscosity was increased by the addition of hydroxyethylcellulose,
linear polyacrylamide, arrowroot, agar agar or methylcellulose. These viscous liquids were
purified by shaking them with a mixed-bed ion exchanger. One of the disadvantages was
that between analyses considerable time was needed for rinsing the narrow-bore tube.
In the early days, a precise classification could not be made. Most electrokinetic
phenomena have to be explained in terms of the interaction between a flow of liquid in
the double layer, but the exact structure of the double layer may generally be left out

172 DETECTION SYSTEMS
of consideration, especially if one is interested only in the suppression of the electro-
endosmotic flow.
In isotachophoretic analyses, the electroendosmotic flow is not constant in all zones,
but increases in the direction of the terminating zone. Ths effect increases the turbulence
of the liquid in each zone, but it is beyond the scope of this book to go into great detail.
Because hydrodynamic flow in the narrow-bore tube is blocked at one side by the semi-
permeable membrane, a profile as shown in Fig.6.35 may be postulated for both the
electroendosmosis and the temperature differences in the various zones.
There are still some differences of opinion concerning the boundary conditions for
the movement of liquids, especially if this is compared with the movement of the zone
boundaries. As in all types of calculation, the potential at the wall is taken determinative
for the electroendosmosis. This potential is often called the { (zeta) potential. When an
A B
Fig.6.35. Profile of a zone boundary in a narrow-bore tube that is blocked at one side by asemipermeable
membrane. The arrows indicate the movement of the zone boundary; Xindicates the direction in which
the sampk zones move. In both (A) and (B), the parabolic profile due to the difference in temperature
between the centre and the wall of the narrow-bore tube is in the same direction as the movement of the
zone boundary (-.-.-). A correction must be made for the difference in temperature on both sides of
the parabolic profile. In (A), the electroendosmotic flow is chosen to be in the same direction as the move-
ment of the zone boundary, while in (B) this flow is in the opposite direction. The dotted line indicates
this electroendosmotic proffle. A correction must also be made for the difference in electroendosmotic
flow on both sides of the boundary, because the potential gradients in the two zones are not equal.
The final profiie (hypothetical) is indicated in both (A) and (B) by a full line.

ADDITIVES TO THE ELECTROLYTES 173
electric field E is applied, a stationary state is reached after a short period of time. We can
divide the forces that are responsible for the electroendosmosis into two main classes: the
force exerted by the external fieldE on ihe ions in the double layer, the force being transferred
by these ions by liquid friction to the layer as a whole; and the force exerted by the
friction on the layer considered by the neighbouring layers, moving with a different
velocity. The force due to the difference in electroendosmosis in the consecutive zones
is not taken into consideration.
In order to gain an impression of the electroendosmotic velocity, vE, the following
equation can be given:
(6.16)
where E is the dielectric constant, { is the potential at the wall, E is the electric field
applied and q is the viscosity. The volume of liquid moving by electroendosmosis can be
measured for each zone, but the net result is zero, because one side is blocked. If this side
was not blocked, this volume transport would be
Q=vEO, (6.17)
where 0 is the cross-section of the narrow-bore tube. We can eliminate 0 by means of
Ohm’s law:
I
OE=-
x
where I is the current through the narrowbore tube and h is the specific conductivity of
the liquid.
For a rough estimation, the volume transport in a system in which no semipermeable
membrane is applied is
(6.18)
(6.19)
We have not considered the contribution of the surface conductance, because in the
systems applied by us they can be neglected.
Under normal operating conditions, e.g., if analyses are performed at pH 6 (Table 12.1),
an estimation can be made of the values of Q in the leading and terminating zones, the
values being about 40 and 80 pl/h, respectively. The final profde of a zone boundary is,
of course, influenced by these values.
By adding a suitable surfactant, the viscosity in the vicinity of the wall can be increased
at least 100-fold, which suppresses the electroendosmotic flow sufficiently. Finally, it
should be noted that the time of analysis is not influenced by the electroendosmosis,
again because one side is blocked by a semipcrrneable membrane.

114 DETECTION SYSTEMS
6.6.3. Effect of additives on the micro-sensirrg electrodes
The physical chemist usually distinguishes between two extreme types of ideal
electrodes [27, 281. The first type is the reversible electrode, on which ions from the
solution are actually charged and discharged, so that a steady current is possible. The
d.c. potential of the electrodes has a well defined value, which depends on the current
and the composition of the solution. The second type is the polarized electrode, in which
no transformation of ions take place, no steady current can pass and any current that
does pass represents the charging and discharging of a double layer made up of the
electrode and the ions very close to its surface. As is well known, the double layer is a
structure that acts as a capacitance, the value of which is dependent on the potential
across it. This second type of electrode has no well defined d.c. potential. It may vary
greatly under apparently identical circumstances and is greatly influenced by trace
amounts of substances or impurities, as will be shown.
Metallic electrodes are, in fact, always combinations of both types and their impedance
as a function of frequency shows the extent to which one or other mechanism dominates
their behaviour. For instance, a bright platinum electrode in a fluid that is rich in
adsorbable compounds has an impedance that is very nearly proportional to W' , over the
frequency range from 1 to 20 kHz, and it is therefore nearly an ideal polarized electrode.
For the same electrode in a saline solution, a more complicated behaviour is observed. By
the addition of, e.g., Triton X-100 to this saline solution the relationship mentioned above
is again obtained. Without the addition of an inhibitor for electrode reactions (redox
reactions), the electrode reaction is rapid and the current is limited by diffusion of the
reacting ions and the products between the surface of the electrode and the bulk of the
solution. The impedance will decrease proportional to w-f.
If the electrochemical reaction itself is slow, the impedance will be lower for small
w and higher for large w than in the case when diffusion predominates. In practical
cases, inhomogeneities in the electrode material will spread out the band of frequencies
for which the impedance decreases only slowly with frequency, as the rate of the reaction
is strongly dependent on the d.c. potential of the electrode, which varies over the surface.
In instances in which insoluble reaction products cover the electrode surface, the
multiplicity of diffusion paths may have a similar effect. This is the case with a silver-
silver chloride electrode in either a saline solution or a saline plus gelatin solution. It has
an impedance that decreases approximately proportional to w-t over a wide range of
frequencies. The d.c. potential of the electrode can make a considerable difference in
the impedance function by changing the rate or even the nature of the reaction carrying
current. Therefore, the balance between this potential and diffusion is responsible for
the impedance. The real and imaginary components of the impedance of a particular
electrode decrease as approximately the same function of w. A fl uid-filled micro-electrode
can be compared with a low-pass filter that is d.c.-stable. They must be used when signals
are large and for which d.c. and low potentials are of interest.
The metallic electrode is a high-pass filter, which is d.c. unstable. Its optimum use is
when rapidly varying signals are of interest and the amplitude of the signals may be close
to the noise level.
It should be remembered that the micro-sensing electrodes described in this book are

ADDITIVES TO THE ELECTROLYTES 175
also a combination of both types. Of course, those micro-sensing electrodes are meant
which have a direct contact with the electrolyte inside the narrow-bore tube. Owing to the
driving current, polarization of the micro-sensing electrodes occurs, Le., these electrodes
may act as charge-transfer electrodes. This depends on the potential gradient across the
electrodes caused by the driving current and the composition of the electrolyte. In
those cases when the driving current itself was used for measuring the conductivity (the
so-called d.c. method), difficulties similar to those found by several workers who used
metallic electrodes were observed. Partial polarization of the metallic electrode made
the recording of the boundaries obscure.
In order to study the interaction of the electrolyte. the driving current and the
micro-sensing electrodes, electrodes were constructed such that the electrolyte inside
the narrow-bore tube remained surrounded by an uninterrupted cylindrical wall (Fig.6.10).
In ths measuring probe, the distribution of the measuring current is much more linear
than in other constructions considered, although the measuring current flows mainly along
the wall of the narrow-bore tube. This is especially so if the results of current distribution
are compared with those for the probe in whch the micro-sensing electrodes are mounted
equiplanar (Fig.6.16). The capacitance of the measuring cell used was about 3 pF. In the
experiments described, the value of R,, varies from approximately 15 kf2 at the
beginning to 50 kS2 at the end*. While the driving current is kept constant, the potential
varies during the experiment from 4 to 12 kV between the anode and the cathode of
the electrophoretic equipment. The polarization of the micro-sensing electrode, which is
in direct contact with the electrolyte, due to the driving current is shown schematically
in Fig.6.36. Although the platinum has the same potential over all of its surface, it acts
as a bipolar electrode with the cathode directed towards the anode compartment of the
electrophoretic equipment and the anode towards the opposite side.
Depending on the potential gradient and the composition of the electrolyte, the
micro-sensing electrodes can be ‘ideally’ polarized or act as a charge-transfer electrode, as
discussed above. Normally the electrodes, under the conditions used, fall between the
two extremes.
The moment at which oxidation and/or reduction starts depends on various factors:
the roughness of the electrode surface, the composition of the electrolyte and the con-
figuration and material of the electrodes. Minor effects are the temperature, the pre-
treatment of the electrode surface, the pressure and the current density. All of these
values are determined in our equipment. Data from the literature show that under the
conditions used in our equipment, an overpotential of about 70 mV is adequate for thz
evolution of hydrogen, while the evolution of oxygen requires at least 700 mV (bright
platinum electrodes). In many instances the chloride ion (0.01 N ) is chosen as the mobile
ion, but for the evolution of chlorine a higher overpotential is required. For hard electrode
material, hgher values for the overpotential can be expected.
Of course, difficulties can sometimes be expected if the micro-sensing electrodes are
directly in contact with the electrolytes. If, for instance, ions are present that can be
oxidized more easily than the proton, e.g. , the equilibrium Fe3+ =+ Fe2+, particularly
*If the distance between the measuring electrodes (axially mounted) is decreased, or the micro-
sensing electrodes are very thin, these values increase.

176 DETECTION SYSTEMS
f V
t C
I
t
I I
Fig.6.36. Polarization of the micro-sensing electrode. The potential gradient inside the narrow-bore
tube at the position where the microsensing electrode is mounted decreases if these measuring
electrodes change from an 'ideal' polarized electrode to a charge-transfer electrode, owing to the
neghgible resistance of the platinum electrode. As a result of this effect, the concentration of the
electrolyte, again inside the narrow-bore tube at the position where the measuring electrodes are
mounted (l), will decrease if the electrode changes its character in order to fulfil the isotachophoretic
conditions (2). L = position in the narrow-bore tube; V = increasing potential gradient; c = increasing
ionic concentration; Z = centre of the electrode.
obscure results are obtained. Although under normal conditions some hydrogen may be
produced at the beginning of the experiment, the evolution stops because the difference
in potential between the anodic side of the bipolar sensing electrode and the electrolyte,
which is surrounded by this electrode, is insufficiently great to start the evolution of
oxygen (provided that no anion is present that can be oxidized more easily than the
hydroxyl ion). If a sensing electrode changes, for any reason, into a charge-transfer
electrode, the zone length, as actually measured, of the ionic species present in that zone
is longer than can normally be expected according to the concentration in the sample. If
the micro-sensing electrodes are made of Pt-Ir, Pt, Pd or Au considerable amounts of
hydrogen and/or oxygen can be bound in the first two metallic layers of the electrodes.
If hydrogen is bound, the impedance of the electrolyte increases, but with oxygen the
contact of the electrolyte and the metallic electrode improves, which causes an apparently
lower impedance of the same electrolyte. If the overpotential against the formation of
oxygen is exceeded, the bipolar electrode always starts to produce both hydrogen and

ADDITIVES TO THE ELECTROLYTES 177
oxygen. The zone boundary, which causes the electrodes to start the production of gas,
is mainly recorded with an overshoot (Fig.6.37).
Fig.6.37 shows a series of boundary passages. The leading electrolyte consisted of
hydrochloric acid (0.01 M), an unbuffered system. The cations themselves were used each
time as the terminating ions. The electric current was stabilized at 50 PA. For any ion
that is slower than Li' under these conditions, including also the construction of the
probe: the evolution of gas is such that it is produced continuously and cuts off the
electric current. This happens if, for example, Fe3+ is used as the terminating ion.
At the moment the electrodes start to conduct electricity, i.e., when the electrodes
change from polarized electrodes to charge-transfer electrodes, the potential gradient
over the electrolyte, surrounded by the charge-transfer electrode, decreases immediately.
Owing to the electroneutrality principle, the ions in this zone must follow the equally
charged ion of the same species in front of it. The only possible way in which the
condition can be fulfiled is for the concentration to decrease. The electrodes are slowly
coated with a layer of gas, and less of the driving current then passes through the sensing
electrodes. The potential gradient adjusts again and so does the concentration. If the
potential over the zone considered is not too great, the sensing electrodes change again
into polarized electrodes at the moment when the electrode surface is entirely covered
with gas. The evolution of gas in this case stops automatically, and the micro-sensing
electrodes become protected against the electrode reaction characteristic for this zone.
This can be seen in the lithium zone in Fig.6.37.
A second effect may result from the micro-sensing electrodes being first covered
with more hydrogen. As soon as enough oxygen has been produced, this influence on
the electrodes predominates. Electrodes on which oxygen is bound always records the
conductivity as a lower value than do electrodes on which hydrogen is bound.
Another possible explanation of the overshoot is the following. The more the zone is
situated towards the terminating zone, i.e., the smaller the net mobility is, the higher is
the temperature and consequently the Joule heat produced by the sum of the direct
driving current and the measuring current. The conductivity increases with temperature
and so an overshoot may be expected if a boundary passage is measured with a fairly
high difference in conductivity with respect to the leading zone, because time is required
for warming up the detector. Later experiments showed, however, that this effect is
negligible. Later experiments, in the period the conductivity detector has reached its final
construction, showed, however, that sometimes an overshoot can still be obtained. Such
overshoots were also seen with the W absorption detector if a buffering counter ion was
taken, for which the extinction coefficient was a function of pH. The overshoot appeared
in those instances when the buffering capacity of the counter ion was not sufficient.
Experiments in which no buffer was used showed the effect more clearly. Some overshoots
may therefore be ascribed also to the fact that two consecutive moving zones may
have a mutual effect on the pH of the zones involved. A lower pH of the first moving
zone may decrease the conductivity of the zone following at the front side if the buffer
capacity is not sufficient. If no buffer is used, even the higher pH of the zone moving in
the second position may cause a small region of higher conductivity in the zone of lower
pH moving ahead of it. Of course all of these effects may (partially) play a role.
The change of a polarized electrode to a charge-transfer electrode may also be due to a
*The measuring electrode used was rather thick (0.1 mm).

0
I
HCI : 0.01 M
Li
CS
Mg
Ca
Sr
Ba
K+NH4
Rb
Fig.6.37. Step responses of several zones after the leading electrolyte HCl(O.01 M). The electric
current was stabilized at 50 pA because rather thick measuring electrodes (0.1 mm) were used. Clearly
visible is the decrease in the concentration if a slow terminating ion (Li+) is applied, possibly owing to the
change in character of the measuring electrode. The potential gradient over the Li zone was not so
great that the analysis was disturbed by the production of too much gas. The overshoot may be also
explained by a pH jump, because a non-buffered system is applied (see Chapter 9). If, instead of
Li’, Few was chosen, the electric current was cut off.

ADDITIVES TO THE ELECTROLYTES 179
different cause. The potential on the electrodes may be so high that a leak of current to
earth results. This leak to earth often causes the micro-sensing electrodes to be coated
with a polymer derived from the components of the electrolytes, and this coating is sometimes
not easy to remove. Cationic buffers, which mainly bear reactive nitrogen-containing
groups, are especially liable to produce these coatings. These coatings are easily recognized
by the decay in resolution of the conductivity detector. This effect will be discussed later
in section 6.7.
If the surface of the bright platinum electrodes is covered with platinum black, the
contact with the electrolyte improves but the overpotential against oxidation and reduction
is decreased considerably. Hence low current densities must be applied for separation.
The adsorption of ions md uncharged substances can be distinguished in the treatment
of isotherms. Accurate measurements need to be carried out, and it appears that, at
present, the dependence of capacity-potential curves on the bulk activity of the adsorbate
provides the best criterion. The dependence of the standard free energy of adsorption on
the electrode potential (or charge) is different for charged and uncharged species. While
ionic adsorption (specific) leads to a linear relationship, uncharged particles give a
quadratic dependence. The addition of, e.g., Triton X-100 affects the d.c. measurement
of the conductivity, of course, more than the a.c. measurement, especially with respect
to the electrode reactions that may occur. In order to demonstrate this effect, two
isotachopherograms of the separation of oxalic, citric and acetic acids are shown in Fig.6.38.
The traces represent the conductivities of successive zones as measured by the a.c. method
(curve b) and by the d.c. method (curve a).
A mixture of histidine (0.01 M) and lustidine hydrochloride (0.01 M) was used as the
leading electrolyte. The terminating electrolyte was glutamic acid (0.01 M). The current
was stabilized at 40 PA. Because thicker electrodes are used than under normal
conditions*, a greater direct driving current would cause the prcduction of gas at the
beginning of the experiment. In general, the mobilities of the anions are low in com-
parison with those (absolute values) of the cations. It can be seen in Fig.6.38 that the
potential gradient from the oxalate zone is sufficiently great to start an electrode reaction.
Gas may be produced by this electrode reaction and a layer of a ‘histidine polymer’
coating may be deposited on the electrode surface by a combination of both a leak current
to earth and the affect of the bipolar electrode.
A close look at the two traces shows that the resistance, as measured by the d.c.
method, increases in each zone and the conductivity of each zone no longer seems to be
constant. The increment is greater in zones that are situated nearer the terminating zone,
which can be explained by the higher potentials that exist in these zones. The impedance
as measured by the a.c. method is initially not or only slightly influenced.
In a long run (30-50 experiments), the resolution of the as. method of conductivity
determination is smaller and effects characteristic of coatings are obtained (see section
6.7). Later experiments showed that a large proportion of the ‘histidine’ coating is rinsed
off by simply refilling the narrow-bore tube, contrary to the effect using other buffers
such as aniline and pyridine.
*In this experiment a thickness of 0.1 mm was used, while the thickness of the electrode material
used in other experiments was only 0.01 mm.

180 DETECTION SYSTEMS
R t
1 -
t
Fig.6.38. Detection of zone boundaries in isotachophoretic analyses performed by the a.c. method
(b) and the d.c. method (a) simultaneously. At the point marked with an asterisk, the micro-sensing
electrodes change their behaviour from polarized to charge-transfer electrodes. In this particular
instance, gas was produoed. ?he coating of the electrode is more visible in the stepcurves of the d.c.
method. The difference in inclination in the trace of the d.c. method in each zone should be noted.
1 = Chloride; 2 = oxalate; 3 = citrate; 4 = acetate; 5 = glutamate.
If stable coatings are obtained for any reason, the electrodes must be cleaned by
rinsing with aqua regia for about 10 sec.
The effect of the increment in the d.c. trace in Fig.6.38 must be ascribed largely to the
evolution of gas. The small layer of gas influences the d.c. method much more than the
a.c. method of conductivity determination.
6.6.4. Additives
Components that inhbit electrode reactions are characterized by strong adsorption on
the electrode surface. The electrode reactions may be inhibited in two ways: (a) the
transport of ions involved in the electrode reactions towards the electrode decreases as a
result of an increase in viscosity in the vicinity of these electrodes; (b) the inhibitor, as it
is adsorbed, inhibits the reaction by its presence. In general, adsorption on the electrode
surface is caused by the interaction of free-electron pairs (e.g., in oxygen, nitrogen and
sulphur compounds) or by n-electrons (e.g., in aromatic compounds). Again, two groups
can be distinguished; (1) surface-active compounds, including detergents; (2) organic
nitrogen or sulphur compounds, commonly used as corrosion inhibitors.

ADDITIVES TO THE ELECTROLYTES 181
TABLE 6.5
SURFACE-ACTIVE COMPOUNDS USED IN ISOTACHOPHORETIC ANALYSES
-
Compound Structure
Commercial source
Triton X-100
Ethomene T/20
Priminox 32
Serdox ZCA-10
Serdox NJADZO
Nonic 21 8
Mowi018-88
PVP
PEG 200
(C, H, 0)9- monoisooctylphenol
(C, H, O), -talc amine
(C, H, 01, -tertiary amine (chain length
unknown)
(C, H, O),,, -C,,-,, amine
(C,H, 01, -C,,-,, amine
(C, H,O),-,,-tert-dodecylmercaptan
(-CH, -CH-), (polyvinyl alcohol)
I OH
u- \
- CH- CH, -
Rohm & Haas, Philadelphia,
Pa., U.S.A.
Armour Industrial Chem. Co.,
Chicago, Ill., U.S.A.
Rohm & Haas
Servo, Delden, The
Netherlands
Servo
Pennsalt Chem. Corp.,
Philadelphia, Pa., U.S.A.
Hoechst, Frankfurt, G.F.R.
(polyvinylpyrrolidone) Fluka, Buchs, Switzerland
(-CH, -CH, -O-),, (polyethylene oxide, E. Merck, Darmstadt, G.F.R.
molecular weight 200)
Because the additives are to be used in electrophoretic analyses, compounds must be
selected that do not take part in the electrophoretic transport (non-ionogenic compounds).
Exceptions are those compounds which both inhibit the electrode reaction and can be
applied as the buffering counter ions, e.g. pyridine and related compounds. The surface-
active components studied were not only detergents, but also some soluble polymers.
Detergents consist of a polar and an apolar part. The non-ionic part consisted mainly of
8-20 units of ethylene oxide, condensed with units of 8-20 carbon atoms, with or
without functional groups. The polar part can be formed by alkylphenols, alkyl alcohols,
alkylamines, alkylmercaptans or alkanes. Some possibilities are shown in Table 6.5.
Of these additives, Triton X-100 and Mowiol 8-88 are especially useful. All of these
compounds were purified on a mixed-bed ion exchanger. The nitrogen and sulphur
compounds were expected to be particularly useful, because they tend to show surface-
active activity* and are used commercially as corrosion inhibitors. However, these
compounds adversely affect the analysis, possibly because they take part in the electro-
phoretic transport. These additives could not inhibit the interaction between the micro-
sensing electrodes and active components present in the electrolytes such as chromate or
malonate.
In order to give an impression of the effect of the addition of the different additives
on the recording and/or separation of the various ions, a test mixture was prepared as
*It should be borne in mind that not only the electrode reactions need to be suppressed, but also the
electroendosmosis.

182
DETECTION SYSTEMS
described in the legend to Fig.6.15 and all of the separations were carried out in the
operational system at pH 6 (operational system prepared with histidine, see Table 12.1).
The amount of additive differs enormously from compound to compound, and the
optimal amounts were therefore established in separate series of experiments. The
electric direct driving current was stabilized at 80 PA.
In Fig.6.39 and later in this chapter, unless otherwise stated, the isotachopherograms
were unfortunately obtained from an a.c. measuring circuit that was not as good as
those now available; the linearity is shown in Fig.6.20. The a.c. measuring circuit was
poor not only with respect to linearity, but also with respect to the insulation towards
earth and the RC time.
The measuring probe was constructed of 0.05-mm platinum foil (nowadays Pt-Ir of
0.01 mm thickness is used). Later experiments showed that the typical reaction during
the passage of chromate and malonate, components of the test mixture, could be
suppressed by addition of 0.05% of Mowiol8-88 (polyvinyl alcohol).
In order to check separately the influence of additives on electrode processes, current-
voltage characteristics were obtained. The equipment used is shown in Fig.6.40.
An improvement in the detection was found when either an a.c. or d.c. method was
used for the determination of the conductivity, although clearly a difference in behaviour
between these two modes was observed. In order to accentuate this difference, gold was
used as the electrode material, because it is known that it can easily be passivated and
the influence of possible electrode reactions is greater. Unusual effects were obtained
when gold was used. In order to give an impression of the recording of isotachophero-
grams with a conductivity detector with the sensing electrodes made of gold, the test
mixture of anions (Fig.6.1 5), in the operational system with histidine hydrochloride as
the leading electrolyte at pH 6 (Table 12.1), was again examined (Fig.6.41). The
isotachopherogams in Figs.6.39 and 6.41 show that additives need to be used.
In the experiments shown in Fig.6.41, the direct driving current was stabilized at
80 PA: It should be noted that in the trace shown in Fig.6.41 (l), where the impression
is given that the conductivity of the zones decreases towards the terminator zone, this
isotachopherogram was obtained by using the ax. method of conductivity determination.
The simultaneous detection of the conductivity with aid of the d.c. method, as in
Fig.6.41(2), shows the normal isotachophoretic tendency, viz., a stepwise increase in the
resistance. This difference must be ascribed to the dominating influence on the capacity
of a passivated oxide layer of the gold surface of the micro-sensing electrodes. These
effects are discussed in more detail in section 6.7. Many other coatings were also tested
with these gold electrodes because they easily adhered to gold. Sometimes the coatings
were formed automatically by the driving current during the electrophoretic process.
Some other unusual effects occurred when surface-active compounds were added. For
instance, the amount of a surface-active agent added does not influence the analyses
substantially; The concentration of many of them can be varied from 2 to 0.5% without
any recognizable difference in the recording of the zone boundaries. Of course, the
material added to the electrolytes must not contain ionic impurities. Another phenom-
enon that occurs is the ‘memory’ effect. When an analysis was carried out with Nonic 2 18
and the narrow-bore tube was then rinsed carefully, subsequent experiments without the
addition of Nonic 218 gave a low resolution. However, when experiments with Mowiol

ADDITIVES TO THE ELECTROLYTES
Fig.6.39. Influence of additives on the final recording of the isotachophoretic separation of a test
mixture of anions (see Fig.6.15), carried out in the operational system at pH 6 (Table 12.1). 1 = 0.05%
Ethomene T/20; 2 = 0.05% Serdox ZCA-10; 3 = 0.05% Serdox NJAD-20; 4 = 0.05% Priminox 32;
5 = 0.05% Triton X-100; 6 = 0.1% polyvinylpyrrolidone; 7 = 0.05% Nonic 218; 8 = 0.1% Mowiol
(polyvinyi alcohol). The isotachopherogam shown in the centre represents the analysis of the test
mixture of anions after the narrow-bore tube and the detector had been rinsed well with double-
distilled water after a series of experiments carried out with Mowiol, to show the ‘memory effect’ with
this surfactant. The resolution disappears again when about ten experiments have been carried out.
were performed, many subsequent analyses could be made without the addition of
Mowiol, as Mowiol is difficult to remove. It can therefore be concluded that the adsorp-
tion of surface-active components is really important. Other workers have also reported
183

184 DETECTION SYSTEMS
Fig.6.40. Equipment used to characterize the influence of additives and coatings on the micro-sensing
electrode via current-voltage curves. It includes a platinum double electrode, a calomel electrode (S.C.E.)
together with a Luggia capillary filled with agar agar (3%) and potassium chloride (30%) and a counter
electrode in a separate compartment filled with 0.1 Mpotassium chloride solution, provided with a
ceramic filter. AU experiments were carried out in 0.1 Mpotassium chloride solution.
that polyvinyl alcohol (Mowiol) shows little desorption if adsorbed. From our experiments
so far, Mowiol proved to be superior to the other additives, even Triton X-100, especially
if the effect is studied in long runs. Triton X-100 shows a type of saturation effect after
some time, which results in a poor resolution.
Some corrosion inhibitors were also tested in two groups of experiments: (1) small
amounts were added to the leading electrolyte, e.g. , thiourea and benzothiazole;
(2) compounds were used as the buffering counter ions, e.g., pyridine and 0-picoline.
These compounds also sharpened the pattern, but were not better than Mowiol. When
gold electrodes were used, coatings were formed during the analysis that were recognizable
in the electrophoretic recording because coated electrodes become sensitive to doubly
charged ions (Fig.6.47). As usual, the d.c. method, applied as before, gave a slightly
different behaviour. When gold was used as the electrode material, a layer was formed
more easily, possibly containing the sulphur component, as it is known that thiourea can
easily form such layers.
In the experiments in which pyridine and /3-picoline were used as the buffering counter

ADDITIVES TO THE ELECTROLYTES 185
Fig.6.41. Isotachopherograms of the test mixture of anions (Fig.6.15) obtained in the operational
system at pH 6 (Table 12.1). The isotachopherograms were derived from a conductimeter (a.c.
method) with the micro-sensing electrodes made of gold. 1 = kc. recording with passivated electrodes;
2 = simultaneous detection by the d.c. method; 3 = a.c. recording when no addition of surfactants
was made to the leading electrolyte; 4 = a.c. recording with the addition of 0.05% of Nonic 218;
5 = resolution of the a.c. recording increases if experiments lasting several hours were carried out
with the addition of 0.05% of Nonic 218; 6 = as. recording with the addition of 0.1% of Mowiol
(polyvinyl alcohol).

186 DETECTION SYSTEMS
ions, the pH of the leading electrolyte was about 6, containing 0.01 Nhydrochloric acid
(pro analysi grade). The pKa values for pyridine and P-picoline are 5.25 and 5.69,
respectively. Analyses of the test mixture of anions (Fig.6.15) were carried out and sharp
zones were observed. Unfortunately, W detection cannot be used, because the W
absorption of these counter ions is strong between 250 and 300 nm.
It must be remembered that the effective mobilities of pyridine and 0-picoline are
greater than that of hstidine. This results in the need for longer narrow-bore tubes for
the separation of similar mixtures, because mixed zones are formed much more easily as
components with a higher effective mobility transport a higher proportion of the elec-
tricity. Nevertheless, here also the disturbance in the detection of the chromate zone
(electrode reaction) was found to be a function of the driving current, which illustrates
further that an electrode reaction indeed occurs, as shown in Fig.6.42 (1-3). The
electrode reaction is shown to be a part of the profile finally recorded if chromate is
used as the terminating ion. The reaction time for the electrode is therefore of the order
of seconds. All analyses, carried out with the 0.05-mm platinum electrodes and the coil,
for galvanic separation of the high potential of the micro-sensing electrodes and the low
potential of the conductivity-measuring electronics, which are not well insulated, show
this typical reaction, but at low pH (e.g., 4) it is less pronounced.
If the chromate zone has passed the measuring electrodes, these electrodes are
(partially) passivated. All other zones following the chromate zone are measured correctly
if the chromate zone instead of the leading electrolyte is taken as a reference. Thus a
shift is obtained: all zones before chromate (of the test mixture of anions) are of correct
height and all zones after chromate are of correct height. An extra impedance, reversible
and stable, arises during the analysis, but if the narrow-bore tube is rinsed after the
experiment has been completed, this impedance ‘disappears’ again.
If malonic acid is injected as a sample or is used as the terminating ion, while no
chromate zone is created before the malonate, a similar behaviour to that found with the
chromate is found, as shown in Fig.6.42 (4 and 5). If both chromate and malonate are
present, and the chromate zone may be very small, the typical behaviour of the electrodes
can be recognized only during the passage of the chromate zone, as shown in Fig.6.42(6).
If we look more closely at the isotachopherograms in Fig.6.42, in which chromate and
malonate are used as terminating ions, a typical shape can be seen in both traces. Even
sulphate, when used as a terminating ion, shows this behaviour if the recording of the
sulphate step is scaled up. The shape has three different and clearly distinguishable parts:
an overshoot, a slow decrease and an increase towards a constant value.
The following explanation can be put forward. The anionic constituent present in a
zone, at the concentration and pH determined by the operating conditions chosen, may be
the cause of a change in behaviour from a polarized micro-sensing electrode partially to
a charge-transfer electrode, (during the passivation of the electrode by the chromate ion).
This always causes an overshoot, because the concentration must decrease if a current is
applied to give an electrode reaction in addition to the electrophoretic transport, in order
to fulfil the isotachophoretic condition and the mass balance of the buffer. Especially if
oxygen is generated, for instance as a result of the electrode reaction (which means
passivation of the electrode), the gas diffuses into the metallic structure. We found that
passivated electrodes record the conductivity of a particular electrolyte with an apparently

ADDITIVES TO THE ELECTROLYTES
1 2 3 4 5 L
6
Fig.6.42. The a.c. recording of isotachophoretic zones of chromate and malonate. In spite of the
addition of Mowiol (O.l%), electrode reactions of these types could not be prevented. In later experi-
ments, in which the thickness of the micro-sensing electrodes was reduced, and in which Pt-Ir has been
used, these electrode reactions disappeared. In traces 1-3, the electrode reaction caused by the chromate
ion is shown. The final step height of the glutamate (terminator) is not constant, but depends on the
amount of chromate injected into the system. In trace 3, chromate itself was taken as the terminating
ion. Similar behaviour was found with malonate (4 and 5). When both chromate and malonate were
present (6), the typical effect was only found during the passage of the chromate ion. R = Increasing
electric resistance; t = time.
lower impedance than non-passivated or even activated electrodes. The full explanation
of the trace can be given as follows. Owing to the change in behaviour of the electrode, the
isotachophoretic zone is recorded with an overshoot; owing to passivation, the conduc-
tivity of the zone is recorded with an apparently higher value (simultaneously the
electrode reaction stops, which means that the real conductivity of the zone between the
micro-sensing electrodes increases); the oxygen is adsorbed more strongly to the electrode
surface and a new equilibrium is established, which is why the trace shows a different
inclination. This inclination is not observed if hydrogen can be produced as a result of
an electrode reaction (activation of the measuring electrodes).
During an isotachophoretic separation and recording by means of the a.c. method,
these effects are often difficult to observe, because the zone length is often too small. In
187
R

188 DETECTION SYSTEMS
order to prove that electrode reactions of all types influence the recording of the
conductivity, a leak current (lo4 A) was created artificially. The leak current is small
compared with the direct driving current (lo4 A). The result is shown in Fig.6.43. Again
the test mixture of anions (Fig.6.15) was separated in the operational system at pH 6
(Table 12. I).
This isotachopherogram shows the separation as recorded if a leak current towards
earth is permitted. The isotachopherogram was obtained with the linearized a.c. conductim-
eter as described in this chapter. The W detector was mounted after the conductimeter
in this instance in order to check if the concentrations of the zones had really changed or
not.
Fig.6.43 shows that good construction and insulation of the conductimeter probe are
necessary. That the material of which the equipment is made plays an important role in
the resolution of the detector proves the following. When experiments were carried out in
Fig.6.43. Isotachopherograms of the test mixture of anions (Fig.6.15) in the operational system at pH
6 (Table 12.1). This figure shows the disturbance of conductimetric detection (a.c. method) if leak
currents towards earth are not prevented. The UV trace is given for comparison of the resolution. In the
experiment shown, a leak current towards earth of 10- A was created artificially. The isotacho-
pherogram is difficult to interpret, although from later experiments we know that, owing to the leak
current towards earth, a coating is deposited on the micro-sensing electrodes (the electrodes are
sensitive to the presence of doubly charged ions). R = Increasing electric resistance; A = increasing
UV absorbance; t = time.
I"

ADDITIVES TO THE ELECTROLYTES 189
narrow-bore tubes made of PTFE in combination with a conductivity detector made of
Perspex, sharp isotachopherograms were obtained only when a surfactant was added
(Fig.6.44). When the UV detector was used it was also found that an additive is necessary,
as can be seen in Fig.6.44.
When the conductivity detector was made of TPX, the additives showed much less
influence on the detection with the conductivity detector. Now, the wetting capacity of
Fig.6.44. Resolution in the absence (below) and presence (above) of surfactants. When no surfactants
were added to the electrolytes, the conductimetric detection had poor resolution. This fgure also
shows that additives need to be added when a UV detector is used. This proves that the electroendosmotic
profite is reduced by the addition of a surfactant, which increases the viscosity in the vicinity of the
wall. Sample: test mixture (Fig.6.15).

190
DETECTION SYSTEMS
TPX was found to be very poor, even if surfactants in high concentrations were applied.
Experiments in which the TPX was 'coated' with a small layer of silicone oil showed
improved resolution, which means that the TPX itself makes a large contribution to the
electroendosmotic flow, which is difficult to suppress.
TPX also show a poorer performance in methanol compared with Perspex, although
TPX must be used because Perspex is affected in a long run. No additives have so far been
found that can be added to methanolic electrolytes to give improved resolution.
The inhibition by the surfactants of electrode reactions, if the thickness of the
electrodes is too great or the insulation towards earth is not sufficiently suppressed, is
poor. Therefore, additives still need to be added for this purpose, or the electrode
reactions must be prevented in another way: low current densities and thin electrodes of
hard material (Pt-Ir). A disadvantage of most of the additives that are suitable for this
purpose is that most of them show UV absorption, which can be neglected if they need
to be added only in trace amounts, but they cannot be used as counter ions. No observable
difference in the current-voltage curve could be found if trace amounts of surfactants
were applied (even high concentrations gave a smaller effect than expected, as shown in
Fig.6.45. This aspect, however, will be discussed in more detail in section 6.7.
Fig.6.45. Current-voltage curve measured with the equipment shown in Fig.6.40. (a) Bright platinum
electrodes with the addition of 2% of Mowiol (polyvinyl alcohol). (b) Bright platinum electrodes
with no addition of surfactant. The curves were measured in a 0.1 Mpotassium chloride solution.
They show that the surfactant must reduce the electroendosmotic profile (compare the results shown
in Fig.6.44); the effect on the electrode reactions is small.

COATING OF THE MICRO-SENSING ELECTRODES
6.7. COATING OF THE MICRO-SENSING ELECTRODES
6.7.1. Introduction
The second means of preventing electrode processes is to apply a polymer coating to
the micro-sensing electrodes. The main problem is to find a method of coating that gives
a uniform layer. Two different methods were tested: (1) the electrophoretic coating
process and (2) the electrolytic coating process. In the electrophoretic process for
preparing coatings of polystyrene, acrylic and epoxy resins, both water and methanol
were used as solvents. The platinum metal is probably not suitable for these coatings, and
it is known that the metal plays a very important role in the coating process. In the
electrolytic process, the electrode metal is less important, and therefore only electrolytic
coatings are considered below.
6.7.2. Experimental
The anodic polymerization of aromatic amines was particularly successful. The more
aromatic rings present in the compound, provided that a sufficient amount could be
dissolved, the more stable the coatings were found to be. 1-Aminonaphthalene dissolved
in ethanol (saturated solution at room temperature) was employed in several experiments.
A few drops of this solution were added to 10 ml of 1 M potassium chloride solution and
water was then added to give a total volume of 100 ml. The solution was filtered in order
to remove undissolved 1-aminonaphthalene. The filtrate was approximately 0.01 Min
the aromatic amine. During anodic oxidation at 700 mV, a violet-coloured layer was
formed. The electric current was maintained at 0.1 mA for 5 min and an increase in the
electrode potential up to 2000 mV was obtained; the cell constant increased from 0.68
to 2.5 cm-' . The layer formed was cathodically very stable; even after drying and heating
(lOO°C), the quality of the electrode improved. While the results with the coating of
1-aminonaphthalene were satisfactory, the results with 1-aminoanthracene were even
better. The colour of the coating layer was yellow, and the cell constant increased from
0.68 to 3.5 cm-'.
For these electrodes, current-voltage curves were obtained in order to characterize
this quality. However, as it is difficult to estimate the thickness of the layers, experiments
in the isotachophoretic equipment were still carried out. A conductivity measuring probe
was used in which the micro-sensing electrodes were mounted axially as shown in Fig.6.12.
In order to discriminate between electrodes, only a selection of thin and thick coatings
was examined, depending mainly on the electric current applied during the coating
procedure. The isotachopherograms obtained with the a.c. method of conductivity deter-
mination were unusual for the separation of both anions and cations. In order to
demonstrate the difference between the a.c. and d.c. methods, the influence of different
coatings and the influence of changing the frequency of the measuring current and the
191

192 DETECTION SYSTEMS
Fig.6.46. Effect of a coating deposited on the micro-sensing electrodes on the final recording of the
isotachophoretic separation of the test mixture of anions (Fig.6.15) in the operational system at pH 6
(Table 12.1). 1 = A.c. method (4 kHz) with a phenol coating; 2 = a.c. method (4 kHz) with a coating
formed by ‘Kolbe electrolysis’ 3 = a.c. method (4 kHz) with a thin coating of 1-aminoanthracene;
4 = a.c. method (1 kHz) with a thick coating of 1-aminoanthracene; 5 = a.c. method (4 kHz) with a
thick coating of 1-aminoanthracene. In the analysis shown in 5, the same coating as in 4 was used
but the frequency of the measuring current was altered. Traces 6 and 7 are simultaneously recorded
isotachopherograms obtained by the d.c. method, corresponding to traces 5 and 1, respectively. No
surfactants were added to the electrolytes.

DETECTION LIMITS 193
electric driving current, a series of experiments was performed with the test mixture of
anions as described in Fig.6.15.
Also in this series of experiments, a non-linear a.c. conductimeter was used. The
operational system at pH 6 (Table 12.1) was chosen and the direct driving current was
stabilized at 80 PA, unless mentioned otherwise in the figure captions.
In Fig.6.46, a series of isotachopherograms are shown, which indicate that the ax.
method gives unusual results for the test mixture of anions before the coating. The
sensitivity (selectivity) of the combination of the a.c. method with coated electrodes
for the doubly charged sulphate ion was such that the following zone of chlorate was
measured with a negative step, which suggests that this zone has a higher conductivity
than the preceding zone. This is in contradiction to the isotachophoretic principle, if
these ions are involved. When the thickness of the coating layer was increased, this effect
also increased, as shown in Fig.6.46 (3 and 4). An increase in frequency of the measuring
current also demonstrates this effect. Even the acetate-adipate transition shown in
Fig.6.46 (5) is recorded with a negative step. In Fig.6.46 (3 and 4), the acetate-adipate
transition was recorded with a smaller difference than under normal conditions (without
a coating).
The difference between the simultaneous detection by the a.c. and d.c. methods of
determination of the conductivity, as already found in some instances when passivated
gold electrodes were applied, must be ascribed to the change in capacity of the conductivity
cell. In all experiments, the simultaneously performed d.c. method of conductivity
detection showed a normal isotachophoretic pattern, as does UV detection. Similar
behaviour was found if cations were separated, as can be seen in Fig.6.47. Hence the
coating layer is only selective for the difference between singly and doubly charged ions.
If, during an isotachophoretic run, a coating is deposited on the micro-sensing electrodes
by an electrode reaction due to a leak current or a change in the nature of the sensing
electrode from polarized to charge transfer due to the driving potential, similar effects
can be expected. The effect occurs especially when this coating layer is formed very slowly
after a series of experiments, even if the most stringent precautions are taken. Cleaning
must therefore be carried out from time to time.
That a coating is formed more quickly if a high current density of the driving current is
applied is shown in Fig.6.48.
Owing to the higher potential gradient, the electrode reactions typical of the chromate
zone are a function of the driving current. Also, the ratio of step heights is changed more
quickly than under normal conditions, for which a change in the ratio of step heights
could not be observed.
6.8. DETECTION LIMITS
6.8.1. Introduction
Although it is somewhat premature at the present stage of isotachophoretic develop-
ment, brief information will be given on detection limits in isotachophoretic experiments.
More research aimed at optimizing detectors, equipment and Operational systems will

194 DETECTION SYSTEMS
Fig.6.47. Influence of a coating deposited on the micro-sensing electrodes on the final recording of
the isotachophoretic separation of the cations. Ba2+, Ca'+, Na', Cd'+ and (C, H, l4 N+ in the operational
system at pH 5.39. K+ (0.01 N) was used as the leading ion, acetate was the counter ion and Tris'
was the terminating ion. The electric current was stabilized at 80 PA. 1 = No coating; 2 = with a thin
coating of 1-arninoanthracene (4 kHz); 3 = with a thick coating of 1-aminoanthracene (4 kHz).
certainly improve the detection limits in the future. Particularly when micro-scale
preparative equipment becomes available it will be possible to combine various specific
detection techniques with isotachophoretic equipment. Because isotachophoresis is still in
the development phase, it is impossible to determine the ultimate detection limit of this
technique.

DETECTION LIMITS 195
Fig.6.48. Influence of the current density of the driving current on the final recording of the test
mixture of anions (Fig.6.15) isotachophoretically separated in the operational system at pH 6
(Table 12.1). An addition of 0.05% of Nonic 218 was made to the leading electrolyte. The recording
was made by the a.c. method (4 kHz): 1 = 40 PA; 2 = 80 PA; 3 = 150 MA.
The following advances will give improvements in detection limits: if it will be possible
to reduce the diameter of the narrow-bore tube and the electroendosmotic flow can be
suppressed efficiently, the transitions of the zones will become smaller, which will improve
the sensitivity of the method (see Appendix B); and if the sensitivity (or selectivity) of
the detectors can be increased, smaller zones of ionic material at lower concentrations can
be recorded. Of course, some of these aspects overlap.

196 DETECTION SYSTEMS
Apart from the above areas of development of the method, we should consider the
following. (1) Are the chemicals available pure enough? (2) Is the operational system
well chosen, Le., such that the proportion of the electric current carried by the buffering
counter ion is small and the buffering capacity large enough, and is the solvent well
chosen? (3) Is the detector sensitive enough? An example of ths is the isotachopherogram
in Fig.6.32, where an enrichment of salicylic acid is shown by the W detector, whereas
the conductivity detector gives an ‘ideal’ mixed zone. (4) Is the time of analysis well
chosen? If, for instance, the difference in the effective mobilities of a given pair of
ions is small, a longer narrow-bore tube must be applied. If the difference in effective
mobility is critical, the counter flow of electrolyte will not give relief (this aspect is
discussed in Chapter 7). (5) Has the equipment for a special application been constructed
well?
Convincing evidence that demonstrates the variations in detection limits that can be
obtained is provided by the difference in resolution attained by thermometric, conduc-
tivity and W detectors. A low-resolution detector gives no information about the real
detection limits of the isotachophoretic separation process. The use of electrolytes at low
concentration limits the choice of pH and hence the operational systems to be used,
because of the increasing influence of OH and H ions on the electrophoretic separation
procedure. An increasing eluting effect will be the result and zone electrophoretic
effects can be expected. This means also that theoretically isotachophoresis is impossible.
However, other solvents may be explored, which alter these limits.
In the following discussion, some information is given on detection limits in isotacho-
phoretic separations carried out on the equipment developed in our laboratory with
commercially available chemicals, although purification was necessary, especially in
analyses at low concentrations. This aspect is dealt with in more detail in Chapter 10.
The detection limits in thermometric detection are discussed in section 6.2; they
provide no information on the detection limits of the isotachophoretic process. Special
attention is paid to UV and conductivity detectors with the electrodes mounted equi-
planar (0.01 mm, Pt-Ir) in direct contact with the electrolyte inside the narrow-bore
tube in combination with the linearized electronic measuring circuit as considered in this
chapter. For the W detector, a round slit of 0.3 mm diameter was used.
6.8.2. Experimental
In order to find the lower detection limit, a series of experiments was carried out with
ADP, because its ion can be detected by both W and conductivity detectors. Moreover,
it was found to be very pure and dissolved easily compared with other strongly W-
absorbing compounds available in our laboratory. The effect of the direct driving current,
temperature and the concentration of the leading electrolyte were studied. The experi-
ments were carried out at pH 4 (see the operational system at pH 4.5, Table 12.5), and
some results are given in Table 6.6. All of the data given are average values from three
separate experiments. The analyses were carried out for each concentration range of two
batches of electrolytes.
The minimal amount needed for detection by the W detector was found to be about
the same as that in the as. method of conductivity determination, although if two

DETECTION LIMITS 197
TABLE 6.6
SURVEY OF THE MINIMAL AMOUNTS THAT CAN BE DETECTED BY HIGH-RESOLUTION
UV AND CONDUCTIVITY DETECTORS
CLE = Concentration of the leading anion (M); Quv = minimal amount of ADP that can be detected
by the UV detector (pmoles); Qa.,. = minimal amount of ADP that can be detected by the a.c.
detector (pmoles); QKv = minimal amount of ADP necessary for qualitative and quantitative
detection by a UV detector (pmoles); and Qf,. = minimal amount of ADP necessary for qualitative
and quantitative detection by an a.c. detector (pmoles). The injected volume was 1 pl. By using the
dilution method (Fig.6.33), the UV detection limit can be further decreased.
CLE
0.01 25 25 150 150
0.005 20 20 130 130
0.001 15 15 100 120
0.0005 5 10 so 100
W-absorbing species were present the resolution was lower (Fig.6.49). This effect may
be due to the fact that a longer zone boundary is detected due to the parabolic
profile of the zones and the fact that the fan-shaped field lines of the a.c. detector are less
than 0.3 mm (the diameter of the slit). Table 6.6 shows that the minimal amount that can be
detected by diluting the concentration of the leading electrolyte is far less than expected.
Dilution by a factor of 10 decreases the limit by a factor of only about 2. The reason must
be ascribed to the poor development of the profile in the low concentration of electrolytes,
because the electroendosmotic flow cannot be suppressed sufficiently, and to the increase
in the electrophoretic transport by the more mobile ions (impurities, H?, OH).
Changes in the temperature of the thermostated narrow-bore tube have only a slight
effect, although a drastic change (from 20 to 4°C) occasionally decreases the separating
capacity by about 50%. This is to be expected because ionic mobilities increase by 2-3%
per 1°C change in temperature. Ths means that the differences between two ions that are
difficult to separate change in a similar way, although the effective separation length
increases.
The separating capacity was measured by comparing the times of analysis of the
standard mixture of anions (Fig.6.15), as follows. At 25"C, an amount of the standard
mixture was injected just such that no mixed zones were obtained. The mixed zones are
found especially in the beginning of the isotachopherogram, for zones at the rear always
have a longer time of separation compared with the zones at the front because the
detector is mounted at a fixed position. If the temperature is decreased, mixed zones soon
appear. The extra time required for complete separation, if a counter flow of electrolyte
is used, is taken as a measure of the separating capacity. Of course, one has to take account
of the fact that the counter flow of electrolyte always disturbs the profiles and that the
difference in effective mobility between the various ions is affected in a negative way by
the counter flow. This was checked by observing the profiles of dyes moving in the
narrow-bore tube with and without a counter flow of electrolyte. The influence of
temperature on the pK values of the counter ion and the sample ions was not taken into
account. The influence of the diffusion constant is negligible because the diffusion constant
is directly proportional to the absolute temperature.

198
t
I t.
DETECTION SYSTEMS
Fig.6.49. Isotachopherogram in the operational system at pH 4, with HCI (0.01 N) and e-aminocaproic
acid as the counter ion. The terminating ion was glutamate. While the UV detector (below) indicates
only one of the two UV-absorbing components injected, the conductimeter (above) shows the separa-
tion of the two components. In the trace derived from the conductimeter, a further component is
determined, which is an impurity from the electrolytic system. The current was stabilized at 80 PA.
R = Increasing electric resistance;A = increasing UV absorption.
The influence of variations in the direct driving current were small in the range studied.
In order to prevent the already discussed electrode reactions, this driving current has to
be limited to 150 PA. However, experiments at 300 pA showed that the standard mixture
of anions was separated in a few minutes, although the resolution decreased, The main
reason for this effect must be that high current densities increase the radialnon-uniformity
of the temperature profile inside the narrow-bore tube. This causes an increased parabolic
profile, especially for the zones that are situated towards the rear side. Also, the electro-
endosmotic profile increases.
The use of the dilution method of concentration determination improves the resolu-
tion of the UV detector (Fig.6.33) about 50-fold. This factor depends on the molar
extinction coefficients of the ionic species involved. The disadvantage of this method of
detection, as can be seen in the determination of salicylic acid in Fig.6.32, is that a small
change in the pH of the leading electrolyte may change the effective mobilities between
the two ions forming the mixed zone in such a way that an enrichment of one of the
components is soon detectable in the UV trace*. This sometimes makes an accurate
determination of the step height in the UV trace (quantitative determination) less
accurate. If a W-absorbing ion can be sandwiched between two non-W-absorbing ions,
minimal amounts of the UV-absorbing ion can be measured quantitatively because one
can make use of the parabolic profile of the consecutive zones. The W-absorbing ion can
be determined, in spite of its small zone length (e.g., less than 0.01 mm), because the
*A similar effect can be expected if the pK, values of the components involved differ much.

CONCLUSION 199
average length of a parabolic profile is commonly about 0.4 mm. This is shown later in
Fig. 17.3, in which the profiles are visible because coloured ions are used. Special calibra-
tion graphs must be prepared for each ionic species.
When using equipment in which high-resolution detectors are mounted, for compounds
present in the range from micromoles to nanomoles (average molecular weight loo), full
qualitative and quantitative results can be obtained; at the picomole level or even lower, in
special cases quantitative results can be obtained, as is discussed above.
6.9. CONCLUSION
The choice of the method of detection, especially in analytical isotachophoresis,
must be carefully considered. All of the types of detectors discussed in this chapter are
not always needed, and for many purposes only one type of detector (specific or
universal, with low or high resolution) is sufficient. Especially if one is interested only in
the amount and/or quality of a single component, a detector of very simple performance
can be chosen*. The choice of the method(s) of detection determines to a great extent the
final construction of the equipment, but also makes demands on the purity of the
chemicals used in the various operational systems.
To compare the various method of detection, Figs.6.50 and 6.51 can be considered.
In Fig.6.50, three isotachopherograms of the test mixture of anions (Fig.6.15), in the
-
a b ' c
Fig.6.50. Isotachophoretic separation of the test mixture of anions (Fig.6.15) in the operational system
histidine/histidine hydrochloride at pH 6 (Table 12.1). Detector used: (a) d.c.; (b) non-linear a.c.;
(c) thermometric. Speed of the recorder chart paper: (a) and (b) 2 cmlmin; (c) 5 mm/min. Average
time of analysis: (a) and (b) 15 min; (c) 45 min. The linear traces should be noted. R = Increasing
electric reisstance; T= increasing temperature; f = time.
*Moreover, the use of thin-wall narrow-bore tubes with a small I.D. (e.g., 0.2 mm) improves the
resolution (Appendix B).
T R

200 DETECTION SYSTEMS
f.
11 L 1’
1’
Fig.6.51. Isotachophoretic separation of the test mixture of anions (Fig.6.15) in the operational
system histidinelhistidine hydrochloride at pH 6 (Table 12.1). Conductimetric detection was carried
out with a linear conductimeter. The UV trace was derived from a UV absorption detector (not
chopped) at 256 nm. The speed of the recorder chart paper was 6 cm/min and the time of analysis
was 12 min. The electric current was stabilized at 70 PA. R = increasing electric resistance; A =
increasing UV absorption; t = time. 1 = Chloride; 2 = sulphate; 3 = chlorate 4 = chromate; 5 =
malonate; 6 = pyrazole-3,5-dicarboxylate; 7 = adipate; 8 = acetate; 9: p-chloropropionate; 10 =
glutamate; X = impurity, possibly propionate (a degradation product of p-chloropropionic acid).
operational system at pH 6 (Table 12.1), are shown. The traces of the linear signals
have been equalized photographically in order that a valid comparison of the results can
be made. The recording of the final sharpness of the zone boundaries and the difference
in the time of analysis with a high-resolution and a low-resolution detector can clearly be

REFERENCES 201
seen. Again it is clear that in order to observe a sample zone with a thermometric detector
the zone length must be greater (Table 6.2) than if a high-resolution detector is applied
(UV, conductimeter). Hence more sample has been introduced into the system and
consequently a longer time of analysis is needed.
In Fig.6.51, an analysis under conditions identical with those in Fig.6.50 is shown,
recording being effected with a linearized conductimeter (Fig.6.18) and a W-absorption
detector (256 nm) (Fig.6.26).
It should be pointed out that the various isotachopherograms shown in this chapter
are given mainly for pattern recognition, in order to show different effects such as
sharpness of the profiles, electrode reactions and impurities. It is difficult to compare
one isotachopherogram with another, although a single test mixture of anions was used.
In preparing the manuscript, the various isotachopherograms were treated photographically
in order to make comparisons simpler. In the Section Applications (Chapters 8-17)
the time axis is also given on the relevant isotachopherograms, so that the qualitative and
quantitative information can be deduced more easily.
Although electrode reactions may sometimes still occur during detection with a
conductimeter with the electrodes in direct contact with the electrolyte, its resolution is
lugh compared with that of other types of universal detectors. Moreover, possible coating
of the electrode can easily be observed by using a combination of an a.c. and d.c. detector
(see also Fig.8.1.). We recommend that the entire system should be cleaned from time to
time with a non-ionic surfactant, which can be purified by running it through a mixed-
bed ion exchanger, because all types of material may be adsorbed on walls made of
Perspex, TPX, Pt or even PTFE. When adsorbed, these impurities change the {-potential
and hence the electroendosmosis, and thus the resolution of both conductivity and UV
absorption detectors is decreased. For effective rinsing of the electrophoretic equipment,
we recommend the surfactant Extran (Merck, Darmstadt, G.F.R).
REFERENCES
1 F.M. Everaerts, Thesis, University of Technology, Eindhoven, 1968.
2 F.M. Everaerts and Th.P.E.M. Verheggen,J. Chromatogr., 53 (1970) 315.
3 F.M. Everaerts and Th.P.E.M. Verheggen, in P.G. Righetti (Editor), Progress in Isoelectric Focusing
and Zsotachophoresis, North-Holland, Amsterdam, and Elsevier, New York, 1975, p. 309.
4 F.M. Everaerts, J.L. Beckers and Th.P.E.M. Verheggen, Ann. NY. Acad. Sci., 209 (1973) 419.
5 F.M. Everaerts, Graduation Rep., University of Technology, Eindhoven, 1964.
6 F.M. Everaerts and Th.P.E.M. Verheggen, J. Chromatog., 91 (1974) 837.
7 F.M. Everaerts and P.J. Rommers,J. Chromatogr., 91 (1974) 809.
8 F.M. Everaerts and Th.P.E.M. Verheggen,J. Chromatogr., 73 (1972) 193.
9 F.M. Everaerts, internal report, University of Technology, Eindhoven, 972.
10 A. Vestermark and B. Sjodin, J. Chromatogr., 71 (1972) 588.
11 L. Arlinger and H. Lundin, Protides Biol. Fluids, Proc. Colloq., 21 (1973) 667.
12 A.J.P. Martin and F.M. Everaerts, Anal. Chim. Ac~Q, 38 (1967) 233.
13 A.J.P. Martin and F.M. Everaerts, Proc. Roy. SOC., Ser. A, 316 (1970) 493.
14 B.P. Konstantinov and O.V. Oshurkova, Sov. Phys.-Tech. Phys., 37 (1967) 1745.
15 I. Vacik, J. Zuska, F.M. Everaerts and Th.P.E.M. Verheggen, Chem. Listy, 66 (1972) 545.
16 F.M. Everaerts, J. Vacik, Th.P.E.M. Verheggen and J. Zuska, J. Chromatogr., 49 (1970) 262.
17 M. Coxon and M.J. Binder,J. Chromatogr., 101 (1974) 1.

202 DETECTION SYSTEMS
18 Publication No. 556 f424, AGA, Infrared Instruments Department, Lidingo, Sweden, 1974.
19 J.L. Fergason, Sci. Amer., 231 (1974) 76.
20 J.L. Beckers, Thesis, University of Technology, Eindhoven, 1973.
21 M. Demjanenko, J. Vacik and .I. Zuska, Chem Listy, in press.
22 Th.P.E.M. Verheggen, E.C. van Ballegooijen, C.H. Massen and F.M. Everaerts, J. Chromatogr.,
64 (1972) 185.
23 D.I. Shernoff, Rev. Sci. Instrum, 40 (1969) 1418.

Chapter 7
Instrumentation
SUMMARY
This chapter is devoted to the electrophoretic equipment developed for isotachophoretic
analyses. Many classifications can be considered but, of the series of equipment
resulting from the development of the instrumentation, only three clearly distinguishable
types have been selected. Because the various instruments generally are combinations of
injection systems, counter electrode compartments and detectors, these components
are considered insofar as they were not discussed in Chapter 6.
Special attention is paid to the counter flow of electrolyte during isotachophoretic
analyses. In particular, the optimal regulation of this counter flow of electrolyte and
simple and accurate means by which it can be achieved are considered.
7.1. INTRODUCTION
The design of isotachophoretic instruments, for analytical purposes is determined
mainly by the detection systems used. The stabilizing effect of narrow-bore tubes makes
the use of stabilizing agents, e.g., polyacrylamide, agar agar, arrowroot and dextran,
unnecessary. The shape of the narrow-bore tube, cylindrical or flat, has some influence
(Appendix B). So far, only narrow-bore tubes made of Pyrex glass, quartz glass, Perspex
(acrylic) and PTFE have been tested.
In this chapter, most attention is paid to the injection system, the counter flow com-
partment next to the compartment with the semi-permeable membrane, and the means by
which a counter flow of electrolyte can be achieved. Some instruments as constructed by
Verheggen and Everaerts and used in our laboratory are discussed.
7.2. INJECTION SYSTEMS
7.2.1. Introduction
The method of introducing a sample into equipment for isotachophoretic analyses
may have a great influence on the time of analysis and even the separation of the ionic
species involved.
The sample can be introduced sandwiched between the leading and terminating
electrolytes with aid of a sample tap, in which case the ionic species are separated from
the mobile leading ion and the less mobile terminating ion. The influence of both the
leading and terminating ions is minimal and also the pH of both electrolytes has virtually
no influence in the initial phase. The amount of sample, however, can be changed only by
203

204 INSTRUMENTATION
varying the concentration of the sample or by inserting a small piece of insulating material
in the bore of the tap. The latter procedure is very complicated.
Introduction of the sample with aid of a syringe seems to be the most commonly used
technique, because the sample size can be vaned quickly and usually a smaller amount
of sample is required. However, if the sample is introduced in the leading electrolyte,
mixed zones can be expected between the leading ion and the fastest moving ion of the
sample. If the sample is injected in the terminating electrolyte, ionic species with a low
pK value (cationic separation) or a high pK value (anionic separation) can be retarded so
much that considerable amounts of these ionic species can be missed or even lost.
Reproducible quantitative results can hardly be expected. Particularly if experiments are
carried out at low concentrations (0.001 N), special care must be taken in selecting the
concentration and the pH of the terminating electrolyte. If the sample is mixed with a
terminating electrolyte that has too high a concentration or an incorrect pH, both the
qualitative and quantitative results will be poor. In addition, the influence of impurities
in the electrolyte may play an important role, but this is not influenced by the method
of sample introduction. More attention is devoted to this aspect in the Section
Applications.
Of course, if a syringe is used for sample introduction, some of the sample will always
be mixed with the leading and terminating electrolytes if the sample is introduced at
the boundary between these electrolytes, as this boundary is never well defined.
7.2.2. Four-way tap
The principle of the four-way tap is shown in Fig.7.1. The mechanism is shown in
four alternative positions. In position 1 the narrowbore tube is rinsed and can be filed
with the leading electrolyte, in position 2 the terminating electrolyte can be introduced
into the reservoir for the terminating electrolyte, in position 3 the sample tap can be
rinsed and filled with the sample and in position 4 the sample is sandwiched between the
leading electrolyte and the terminating electrolyte. The analysis can be performed with
the tap in position 4, in which case the connections must fit exactly, because no dead
volumes can be allowed (gas bubbles may stick to these connections and if the dead
volume is located between the narrow bore and the sample tap the time of analysis is
adversely influenced). The other connections are not important in this respect, because they
are used only for rinsing and filling the various compartments of the electrophoretic
equipment .
The tap initially applied by us was made of Pyrex glass, although any other insulating
material can be used. A combination of Kel-F and Arnite can be particularly recom-
mended. The average volume of the tap applied by us was 20-100 pl. The volume of the
tap was sometimes changed by inserting a piece of insulating material, but this procedure
proved to be very complicated if good qualitative and quantitative results were to be
obtained.

INJECTION SYSTEMS
I!
I
n
Fig.7.1. Principle of the four-way tap for rinsing and re-filling the electrophoretic equipment and for
sample introduction. Position 1: the narrow-bore tube can be rinsed and re-fiied. Position 2: the
reservoir with terminating electrolyte can be rinsed and re-fiied. Position 3: the sample can be
introduced. Position 4: the analysis can be performed.
7.2.3. Six-way valve
Fig.7.2 shows the way a six-way valve is used, while Fig.7.3 shows an exploded view
of this valve in order to demonstrate its construction. The conical plunger is made of
Amite, while the plunger housing is made of Kel-F. This plunger housing is surrounded by
a brass hexagon for mechanical stability. Moreover, this hexagon prevents any shift of
the plunger and the plunger housing due to the weakness of the Kel-F and the forces
on the plunger so that a liquid-tight connection is obtained. Holes are drilled in the brass
hexagon for connection of the various components via the holes drilled in the plunger
housing and the plunger. In each of these holes in the hexagon, a threaded base is soldered,
so that with screw-caps and collars liquid-tight connections can be made, as shown in
Fig.7.4. It does not need further explanation that the liquid inside the various bores may
not have any electrical contact with the brass hexagon. By means of the special construc-
tion shown in Fig.7.3 (parts 5 and 6), the six-way valve can be turned only through 60°,
so that the three canals inside the plunger are always connected with the bores inside the
plunger housing. The connections with the narrow-bore tube and the piece of insulating
material that provides the connection with the injection block or directly with the
reservoir filled with the terminating electrolyte must fit exactly, otherwise a dead volume
will occur that will decrease the effective length for separation enormously.
205

206 INSTRUMENTATION
4
A 6
Fig.7.2. Principle of the six-way valve used for rinsing and re-filling the isotachophoretic equipment
and sample introduction (J. Vacik, Prague, private communication). In position A, the narrow-bore
tube is rinsed via an open Hamilton valve (1MM1) at the side of the counter electrode compartment;
(3) is the connection towards drain. The sample is introduced via the syringe (S), while (2) again is
connected with the drain. The reservoir of the terminating electrolyte can be rinsed via (6); (1) is
the reservoir for the terminating electrolyte. In position B, the valve is shown in the ‘running’ position.
By varying the central bore inside the plunger, the volume to be injected can be varied.
The tap constructed in our laboratory had a volume of 5 pl.
The narrow-bore tube is connected with the six-way valve without the use of any
adhesive, using a piece of insulating material that has an outside diameter such that it fits
exactly in a chamber made for it in the plunger housing. The length of tlus piece of
insulating material is about 2 cm. In order to make a liquid-tight connection with the
piece of insulating material, it must be smooth and flat on top. Moreover, an extra small
O-ring made of rubber is mounted on top of this cylinder of insulating material. In the
cylinder, a hole is drilled with a diameter equal to the outside diameter of the narrow-
bore tube in which the analyses are performed. The narrow-bore tube that is to be
mounted is first stretched over a length of about 4 cm to enable it to penetrate the
cylinder of insulating material via the central bore. The narrowbore tube is then pulled
through this central bore until it fits exactly. After allowing for shrinking (this piece of
insulating material with the narrow-bore tube is inserted in hot water), the narrow-bore
tube is cut with a lancet. The cylinder of insulating material with the narrow-bore tube
can be connected to the plunger housing by fitting a screw-cap over the threaded base.
A water pressure of at least 7 atm can be applied without any visible leakage at this
clamping piece. However, a pressure no higher than 6 atm could be applied, because at
this pressure the narrow-bore tube shows its porosity and droplets appear all over it.
The pressures applied for rinsing and re-filling are, of course, much lower. The
connection of the six-way valve with the injection block (or directly with the reservoir
filled with terminating electrolyte) is achieved with a cylinder of insulating material with

INJECTION SYSTEMS
Fig.7.3. Exploded view of the six-way valve. 1 = Brass hexagon with screw-caps for connection of the
various parts liquid-tight to the plunger housing (2), made of Kel-F; 3 = pins for locking the plunger
housing in the brass hexagon (1); 4 = Arnite plunger with three pardel canals; 5 = stainless-steel
screw-cap provided with a ridge for the exact determination of the position of the plunger in the
plunger housing, in combination with component (6); the plunger can be switched through 60" ;
7 = handle; 8 = narrow-bore tube. The clamping device of the nanowbore tube, provided with a small
O-ring (see section 7.2.3.), should be noted.
a bore of 1 mm. This cylinder of insulating material has two collars, and on both ends
it is flat and provided with two small rubber O-rings. Again, liquid-tight connections can
be made with screw-caps.
In practice, the tap is very reproducible in sampling, especially when various people
use the instrument. For the use of syringes, more ability is needed.
It need not be explained that a shorter length of narrow-bore tube is needed for
separation, because the sample is not mixed with the leading and terminating electrolytes.
This six-way valve was very useful particularly when experiments were carried out in
which the position of the sample is important, e.g., zone electrophoresis in narrow-bore
207

208 INSTRUMENTATION
\
6
A
Fig.7.4. Cross-section of the six-way valve in the ‘running’ position. 1 = Connection towards the
reservoir of the terminating electrolyte; 2 = connection towards drain; 3 = connection towards drain;
4 = narrow-bore tube in which the separation is performed; 5 = position where the syringe filed with
sample can be mounted; 6 = position where the syringe fiied with terminating electrolyte can be
mounted. Materials: a = brass; b = Amite; c = Kel-F; d = Kel-F.
tubes or movingboundary experiments. It can also be recommended for automation
purposes.
7.2.4. Injection block
A method for sample introduction with a micro-syringe is demonstrated in Fig.7.5, and
a photograph of the injection block is shown in Fig.7.6. .
The leading electrolyte can be introduced via an open tap at the side of the counterelectrode
compartment, not shown in the figure (see Fig.7.9). The tap between the
injection block and the connection towards drain (4) is opened during this procedure,
while tap (2) is closed. The tap at the side of the counter flow compartment is then
closed and tap (2) is opened. The terminating electrolyte can now flow towards drain. In
general, no suction need be applied. Next, tap (4) is closed and a ‘well’ defined boundary
is obtained between the leading and terminating electrolytes. A sample can now be
introduced via the septum (3) with a normal micro-syringe. The sample introduction can
be effected in the leading electrolyte, in the terminating electrolyte or at the boundary of
the two electrolytes, as desired.
/
2
\

INJECTION SYSTEMS
Fig.7.5. Injection block suitable for isotachophoretic analysis. 1 = Reservoir for the terminating
electrolyte; 2 = PTFE-lined Hamilton valve (1MM1); 3 = silicone rubber septum; 4 = tap provided
with a conical tip, which gives the connection towards drain; 5 = narrow-bore tube, provided with
a Perspex clamping piece (see section 7.2.3). This clamping piece is provided with a small O-ring for
a liquid-tight connection with the injection block.
Of course, as sharp a plug profile can never be obtained as when the sampling is performed
with a sample tap.
The connection with the narrow bore tube is made by the construction discussed in
209

210
INSTRUMENTATION
Fig.7.6. Photograph showing the injection block in Fig.7.5 and the six-way valve in Figs.7.2-7.4.

COUNTER ELECTRODE COMPARTMENTS 21 1
section 7.2.3. The injection block was made of Perspex and PTX. The conical tip of
tap (4) was made of silicone rubber (shape 90"). This tap is not available commercially
as a single piece. The other taps used were commercially available PTFE-lined Hamilton
(1MM1) taps (Hamilton, Bonaduz, Switzerland).
7.2.5. Simplified injection block
Because the construction of the injection block described in section 7.2.4 is rather
complicated, an injection block of much simpler construction (T-way) is shown in
Fig.7.7. Such injection blocks have been made of Perspex, TPX, Kel-F, PTFE and
polypropylene. The connection with the narrow-bore tube is similar to that described in
section 7.2.3.
A Hamilton (1MM 1) PTFE-lined valve is mounted between the injection block and the
reservoir containing the terminating electrolyte. Another tap, also a PTFE-lined Hamilton
(IMM1) valve, is mounted between the injection block and the drain. The equipment
can be rinsed and re-filled with leading electrolyte via a tap at the side of the counter
electrode compartment (not shown in Fig.7.7, but shown in Fig.7.16). Tap B is opened
during this procedure, while tap A is closed. The tap at the side of the counter electrode
compartment is then closed and tap A is opened, while tap B remains open. The termi-
nating electrolyte now flows towards drain. No suction or pump need be applied. After this
procedure, tap B is closed and the sample can be introduced via the septum with a standard
micro-syringe. In this method of sample introduction, the injection can be made only in
the leading electrolyte. A large amount of mobile ions of the leading electrolyte are
behind the sample introduced and these ions must overtake the sample ions during the
isotachophoretic separation procedure. This may be a complication, especially if the
concentration of the sample ions is hgh. Because some of the leading electrolyte is mixed
with the terminating electrolyte before the analysis, as a result of the introduction of the
needle of the micro-syringe, then after the injection of the sample has been made, tap B
must be opened in order to remove the leading electrolyte that is mixed with the
terminating electrolyte in the horizontal canal.
Because of its simple construction, this type of injection block can be recommended
in many instances, especially when some precautions can easily be taken with respect to
the leading and terminating electrolytes. The concentration of all of the sample ions must
not be too high.
7.3. COUNTER ELECTRODE COMPARTMENTS
7.3.1. Introduction
Because in narrow-bore tubing a stabilizing effect is obtained, in most experiments no
stabilizing agents (e.g., agar agar, polyacrylamide, arrowroot, dextran, agarose) are added
to the electrolytes. The counter flow compartment must therefore consist of a semi-
permeable membrane in order to prevent any hydrodynamic flow of electrolyte between
the two electrode compartments owing to the difference in levels in these compartments.

212
INSTRUMENTATION
Fig.7.7. Simpler injection block for electrophoretic equipment suitable for isotachophoretic analyses.
Two Hamilton (1MM1) PTFE-lined valves are applied: between the reservoir of the terminating
electrolyte and the I-mm narrow-bore tube (A), and between the 1-mm narrow-bore tube and the
drain (B). The sample can be introduced into the narrow-bore tube filled with leading electrolyte,
so that it is always mixed with the leading electrolyte. A considerable amount of the leading ion must
be behind the sample if no intern standard is applied.

COUNTER ELECTRODE COMPARTMENTS
Even if the narrow-bore tube is arranged in a horizontal position, this membrane is
needed. Moreover, gas will generally be produced at the electrodes as a result of the
electric current necessary for electrophoretic separations. These gas bubbles may also
introduce a hydrodynamic flow of electrolyte if the electrodes are not separated by a
semipermeable membrane from the narrow-bore tube in which the separation is performed.
It is of minor importance that the electroendosmotic profile is somewhat suppressed
by the semipermeable membrane, as discussed in Chapter 6. Moreover, mainly those
conditions such that electroendosmotic flow can be prevented must be sought. Although
semipermeable membranes need to be applied, one must bear in mind that their use
always causes a shift in pH on both sides of the membrane, due to the potential gradient
across the membrane and the difference in the ionic mobilities of the various ions through
the membrane. This shift in pH may disturb or minimally influence the analysis in a long
run, especially if a counter flow of electrolyte is applied.
7.3.2. Cylindrical counter electrode compartment
Fig.7.8 shows schematically a cylindrical counter electrode compartment. The main
parts of this electrode compartment should be made of material resistant to various
solvents; so far, Perspex, Kel-F, Arnite and Pyrex glass have been used. The membrane,
made of cellulose polyacetate, fits around the two cylinders that are provided with a
central bore. The membrane is fixed with Araldite, which in fact does not really stick
the membrane to the two central cylinders, but still prohibits any leakage from the
side on which the electrode is mounted towards the narrow-bore tube, or vice versa. The
cylindrical membrane is made by wrapping a sheet of cellulose polyacetate (0.1 mm)
around a rod with an external diameter equal to the external diameter of the cylinders
on which the membrane will finally be mounted. During the wrapping of the cellulose
polyacetate, acetone, in which the membrane is soluble, is applied. In order to remove
this acetone, the rod, with the wrapped sheet on it, is immersed in a stream of water. A
white, small-pore cylindrical membrane is the result, which is easy to remove from the rod
with aid of a sheet of abrasive paper. The mechanical stability of the membrane is very
high, the thickness being approximately 0.3 mm. The main advantage of a cylindrical
membrane is that during rinsing and re-filing of the instrument with leading electrolyte,
possible gas bubbles can easily be removed and do not stick to the wall. Also, the washing
of the entire system is very easily effected. If experiments with a counter flow of
electrolyte are performed, this procedure is normally carried out via the tap, a common
PTFE-lined Hamilton (1MM1) valve. The disadvantage is that the fresh electrolyte has to
pass the membrane, by which the existing pH jump is transported by the counter flow
quickly into the narrow-bore tube. Of course, the separation is influenced by this effect.
This has been partially overcome by the construction of a special connection for the
counter flow of electrolyte between the counter flow compartment and the narrow-bore
tube in which the separation is carried out. (In section 7.3.3, another counter flow
compartment is described that is much more suitable for experiments with a counter flow
of electrolyte.) The connection with the narrow-bore tube in which the separation is
carried out is similar to that described in section 7.2.3.
At the side on which the electrode is mounted, the counter electrode compartment is
213

214 INSTRUMENTATION
Fig.7.8. Cylindrical counter electrode compartment, provided with a semipermeable membrane.
1 = Piece of Perspex on one side of which the semipermeable membrane is mounted, provided with
an Wing; 2 = brass screw to clamp component (1) liquid-tight to the electrophoretic equipment;
3 = brass support for component (1); 4 = cap for the electrode compartment, provided with an O-ring;
5 = electrode (Pt); 6 = cylindrical semipermeable membrane made of cellulose polyacetate; 7 = wall
of the electrode compartment; 8 = bottom of the electrode compartment with a PTFE-lined
Hamilton (1MM1) valve, a connection for the currentstabilized power supply.

COUNTER ELECTRODE COMPARTMENTS 21 5
generally filled with double-distilled water in order to decrease any interference from
impurities formed by electrode reactions. Moreover, a rapid change from one operational
system to another is possible because the membrane is not ‘soaked’ with different types
of electrolytes. If another operational system is chosen, less attention needs to be paid
to the membrane compartment, as explained briefly below.
Suppose one is interested in anion separations and for a complete separation three
different operational systems are needed. In general, in all systems chloride will be chosen
as the most mobile ion, because it is pure, stable and cheap. Even if another anion is
chosen as the leading ion, this will not affect the analysis because it migrates through the
membrane in the direction of the anode. Of course, the buffering counter ions move in
the opposite direction and in a different operational system another counter ion must
be taken. If double-distilled water is placed in the reservoir surrounding the anode, no
buffering counter ion coming from this reservoir will be present. Therefore, the membrane
is not saturated with the buffering counter ions. In most instances, simple rinsing of the
system is sufficient for cleaning the membrane. The disadvantage when double-distilled
water is used in the electrode compartment with the semipermeable membrane is that a
large potential drop is caused. Especially if a conductivity detector is applied, this
potential drop may cause an electric leak towards earth because the detector electrodes
may finally reach too high a voltage for which the insulation is not adequate.
The disadvantage of the cylindrical construction of the counter electrode compartment
is that sometimes small leakages may arise because the Araldite employed to fix the
membrane does not really fix it. Also, if experiments with methanol are performed, the
Araldite becomes brittle and electrolyte may flow from the reservoir of the terminating
electrolyte towards the counter electrode compartment. If these leakages are small, they
are hardly noticeable, but ultimately there may be a decrease in resolution. Because a
decrease in resolution may have many origins [e.g., electroendosmosis by adsorbed
material on the wall and electrode reactions (if the a.c. method is used for conductivity
determinations), due both to the driving current (polarization) and to electric leakages
to earth], the hydrodynamic flow cannot be directly localized in the initial phase often.
7.3.3. Counter electrode compartment with flat membrane
A more advanced counter electrode compartment is shown schematically in Fig.7.9 and
a photograph is shown in Fig.7.10. The electrode vessel is separated from the narrow-bore
tube in which the analysis is performed by a flat membrane made, for instance, of
cellulose polyacetate (0.2 mm thickness). This membrane is clamped by two screws and
an O-ring. The tap used is a common FTFElined Hamilton (IMMI) valve that provides
the connection with the reservoir of the leading electrolyte. This reservoir is generally an
ordinary polypropylene syringe with a volume of 20 ml. If the entire bystem has to be
rinsed or re-filled with fresh leading electrolyte, the liquid applied flows as well along the
membrane as along the septum constructed for the experiments with a counter flow of
electrolyte. Because the bore is relatively large compared with the inside diameter of the
narrow-bore tube (2 mm), the potential drop in the canals is small. Therefore, a normal
metal syringe can be inserted for the experiments with a counter flow of electrolyte
without the risk that gas will be produced owing to polarization (Fig.6.36). In addition to

216 INSTRUMENTATION
8
1
u l7
2
8.
L
12

EQUIPMENT
the large bore, a more direct connection between the narrow-bore tube and the
electrode compartments exist, so that the potential gradient in the canal where the
syringe will be inserted is negligibly small. Of course if any gas were produced, it would
destroy the analysis. So far, no other electrode reactions have been observed. The
connection with the narrow-bore tube is as described in section 7.2.3,
The great advantage of this counter electrode compartment is that a counter flow of
electrolyte is permitted that does not pass the membrane. A considerable time is needed
for the pH jump at the membrane to enter the narrow-bore tube, where the analysis is
carried out, by the direct electric current, because again the bore, which forms a direct
connection between the PTFE narrow-bore tube and the membrane, is relatively large,
so that a small potential gradient exists in this bore. In addition, the surface area and
thickness of the membrane are small tie., the disturbance is relatively small) and,
moreover, the buffer capacity of the electrolyte present in the 2-mm bore is high, so
that any disturbance can be counterbalanced easily. A further advantage, of course, is
that no adhesive is used with the membrane, so that a membrane can be changed and
experiments in, e.g., methanol can be carried out more easily.
7.4. ECUIPMENT
7.4.1. Introduction
With the injection systems and the counter electrode compartments briefly discussed
in this chapter, and the detectors discussed separately in Chapter 6, many types of
instruments can be constructed. Moreover, the different components are connected in
such a way that no adhesive need be applied and therefore the different parts are
interchangeable. The means of thermostating can also be taken into consideration, e.g.,
the narrow-bore tube may be free-hanging in air that is thermostated with circulating
water, a thermostated aluminium block can be applied with the narrow-bore tube
mounted on it in a helix, or the narrow-bore tube may be thermostated directly with, e.g.,
circulating kerosene.
The development and combination of electrode compartments, injection systems and
auxiliary equipment has, of course, resulted in a continuous gradation of types of
instruments and modifications, and it is sometimes difficult to distinguish one type
from another. Therefore, in this section only three types of equipment will be discussed,
Fig.7.9. Counter electrode compartment with a flat semipermeable membrane and a septum for
experiments with a counter flow of electrolyte. 1, Perspex connection between the central bore of
the counter electrode compartment and the narrow-bore tube of the electrophoretic equipment,
provided with an O-ring; 2 = brass screw for clamping component (1); 3 = brass support for component
(1); 4, 14 = Perspex units for mounting the counter electrode compartment on a rail (see Fig.7.16) and
for clamping 8 and 11; 5, 15 = brass pen with screw-thread; 6, 16 = bolts; 7 = cap of the electrode
compartment, provided with a hole; 8 = electrode compartment; 9 = flat cellulose polyacetate
membrane; 10 = rubber O-ring; 11 = central housing with canals of 2 mm diameter that pass along the
flat membrane and the septum; 12 = septum; 13 = screw-head for clamping the septum in the central
housing; 17 = PTFE-lined Hamilton (1MMl) valve.
217

218
Fig.7.10. Counter electrode compartment with flat membrane.
INSTRUMENTATION

EQUIPMENT 219
belonging to three clearly distinguishable types in the development of the instrument&
tion of analytical isotachophoretic equipment with a narrow-bore tube.
7.4.2. Narrow-bore tube surrounded with a water-jacket
Fig.7.11 shows a photograph of the isotachophoretic equipment with which
experiments were carried out in the early days of isotachophoresis (1964). Instead of
the PTFE narrow-bore tube, as in Fig.7.11, a narrow-bore tube made of Pyrex glass was
used.
The equipment consists of a narrow-bore tube, which is fixed with adhesive (shellac
if a Pyrex or Araldite if a PTFE narrow-bore tube is used) in a type of Liebig condenser.
On the left-hand side a four-way tap is mounted, as discussed in section 7.2.2. The
electrode compartment, which contains the semipermeable membrane as discussed in
section 7.3.2, is not mounted as a counter electrode compartment as is usually done, but
is mounted behind the sample tap and contains the terminating electrolyte. The reason
for this is the fact that we still use this equipment for measurements of mobilities by
the moving-boundary method and it is easier to rinse if the membrane compartment is
mounted at the position shown in Fig.7.11.
By means of the cylindrical membrane electrode compartment, hydrodynamic flow
of electrolyte is prevented. The connection between the reservoir containing the
terminating electrolyte and the cylindrical membrane electrode compartment is made
with a PTFE tube and a PTFE-lined Hamilton (lMF1) valve in which the syringe fits.
In the Liebig-type condenser, there are three holes into which the thermocouples
(linear and differential) may be mounted. Because these thermocouples are so thin,
the narrow-bore tube is fured by a small spring, which fits the tube and which is fixed to
the wall of the Liebieg-type condenser with hot shellac. The thermocouples are finally
soldered on copper wires, also fixed to the wall of the condenser with hot shellac. In
Fig.7.11, three thermocouples, all of the linear type, are mounted.
In order to reduce the influence of temperature differences in the laboratory (due to
movement, draughts etc.), the holes in the wall of the condenser are sealed with adhesive
foil that is covered with aluminium foil. For optimal results, the whole equipment is
covered with a blanket of cotton-wool. Thermostated water is circulated through the outer
space of the condenser to give a constant temperature inside. The reference junction of
the thermocouple is therefore mounted with some adhesive on the inside of the condenser
wall, protected with a heat-sink compound. For thermostating in our work, a Hacke
thermostat with an accuracy of +O.l"C is used. However, if the variations in the
temperature of the laboratory are not too large, no thermostat is needed and the outer
space of the condenser can simply be filled with water. The capacity of the outer space
and hence the volume of water are large enough to keep the temperature constant in the
space where the thermocouples are mounted during the isotachophoretic run. The
procedure for filing and cleaning this equipment was described in section 7.2.2.
The total length of the narrow-bore tube of the equipment shown in Fig.7.11 is
approximately 1 m, while the thermocbuples are mounted at distances of 25, 50 and
75 cm. In our work, the signals derived from the thermocouples are amplified with a
hick amplifier (type A) and recorded with a potentiometric recorder. If only the linear

Fig.7.11. Electrophoretic equipment suitable for isotachophoretic analyses, constructed in 1964 by Everaerts. Detection is effected with thermocouples
(copper-constantan), wound aound the narrow-bore tube at three different positions and fixed with adhesive. I?le narrow-bore tube is mounted in a
type of Liebig condenser, filled with thermostated water. The injection can be made via a four-way tap (Fig.7.1.). On the left-hand side is mounted
the cylindrical electrode compartment with the cellulose polyacetate semipermeable membrane (Fig.7.8.), which was added at a later stage to the
equipment.

EQUIPMENT 221
signal of the thermocouple is needed, no amplification is necessary if a 100-mV poten-
tiometric recorder is available.
7.4.3. Narrow-bore tube thermostated with an aluminium block
Fig.7.12 shows the main difference between this equipment and that described in
section 7.4.2.
A PTFE narrow-bore tube (O.D. 0.75 mm, I.D. 0.45 rnm) is embedded in a groove in
an aluminium block, and is wound around the aluminium block in the form of a helix.
Fig.7.12. Exploded view of the basic components of an electrophoretic apparatus suitable for
isotachophoretic analyses in which the narrow-bore tube is mounted on a thermostated aluminium
block. 1 = Narrow-bore tube; 2,3 = thermocouples (copper-constantan); 4 = aluminium block;
5 = cap for the aluminium block; 6 = Pt resistor; 7 = load. Tap water can flow through the block
through four canals.

Fig.7.13. Electronic circuit for thermostating the aluminium block shown in Fig.7.12.
d
222
INSTRUMENTATION

EQUIF'MENT 223
No effect on the resolution of the fact that the narrow-bore tube is no longer straight
could be observed on the thermometric detector. Later experiments with high-resolution
detectors showed that the narrow-bore tube must be mounted as straight as possible,
especially the last 2 cm before the detector. If even a small kink was present just before
the detector, the resolution was decreased.
Gaps between the narrow-bore tube and the aluminium block are carefully filled
with a heat-sink compound (zinc oxide powder in silicone oil). Because the heat produced
in the narrow-bore tube is transferred so quickly to the aluminium block, a special
compartment (see Fig.7.12) is created where the thermocouples are mounted in order to
ensure that there will still be a signal to detect. If this compartment is too small, a noisy
baseline results because a very high amplification has to be used. Of course, a com-
promise must be sought, because if this compartment is too big a situation similar to that
described in section 7.4.2 results, and the narrow-bore tube is cooled only by thermo-
stated air surrounding it. The temperature of the reference junctions of the thermocouples
is always the same as that of the aluminium block, because a certain amount of heat-
sink compound is smeared on the junction that is insulated with a PTFE spray, which is
glued to the aluminium block in order to guarantee good thermal contact with the
aluminium block. The narrow-bore tube and the heat-sink compound are fixed by a thin
layer of shellac spray (Krylon). For thermostating the aluminium block, thermostated
water (accurate to +O.0loC) is used. A temperature sensor (100-L2 Pt resistor) is mounted
in the neighbourhood of the detector compartment. Also here gaps are filled with the
heat-sink compound. In the centre of the aluminium block a load of 60 W is mounted. The
Pt resistor and the load are both connected to the temperature control unit, as shown in
Fig.7.13. Rubber O-rings are employed to prevent contact of water, circulating inside
the aluminium block, with the electrical circuits.
The narrow-bore tube protruding from the thermostat is connected at one side with
a type of injection block as shown in Fig.7.7. The narrow-bore tube is connected to the
injection block, without adhesive, by means of the clamping device discussed in section
7.2.3. As the counter electrode compartment, the cylindrical construction shown in
Fig.7.8 was used.
The proportional temperature controller used in the thermostat is based on the
relatively high temperature coefficient of a Pt resistor, a Pt resistor of 100 52 at 0°C being
used. This Pt resistor forms an a.c. bridge (R5) together with the resistors R1, R2, R3 and
%. The temperature coefficients of all resistors in the a.c. bridge, apart from the resistor
R5, must be chosen to be as small as possible. The smaller these values are, the more
accurate will be the thermostatic control. Of course, one of the resistors of the a.c. bridge
is variable so as to permit the a.c. bridge to be balanced. If the bridge is unbalanced, a
signal, the result of the unbalanced position, will be fed to a pre-amplifier, the phase of
this signal being dependent on the polarity of the imbalance of the bridge. The pre-
amplifier generates a sinusoidal voltage and this will be transformed by a voltage limiter
into a symmetrical square wave. By means of the very high amplification of the pre-
amplifier and the voltage limiter, the amplitude of the bridge voltage is transformed into
a phase-shifted square wave. The leading edge of this square wave is amplified and triggers
two antiparallel-connected thyristors that control the amount of heat dissipated in the
load, which is mounted in the direct neighbourhood of the Pt resistor (Fig.7.12).

224 INSTRUMENTATION
If the bridge approaches its balanced condition, the output voltage of the bridge
decreases and the phase shift of the thyristor trigger pulse will thus be reduced. The
thryistor will trigger later and the heat produced in the load will also decrease, and a
steady state will result.
The temperature (T, "C) can be selected by the variable resistor & of the a.c.
bridge, according to the equation
T= 2.59 (R4 - 0.5835) (7.1)
(temperature coefficient Rs = 0.003916). Some possibilities are shown in the Table 7.1.
An RC filter in front of the input of the pre-amplifier corrects the phase of the trigger
pulse, and the proportional band (system gain) can be changed by varying the a.c. voltage
over the bridge. Incorrect temperature regulation may result if the temperature coef-
ficients of the other resistors of the a.c. bridge are poor, leading to instabilities if the
thermal resistance between the load and the Pt resistor is large or if the heat capacity of
the object to be thermostated is too high.
A photograph of the equipment with indirect thermostating via the aluminium block
is shown in Fig.7.14, which also shows the power supply. A pressure system has been
developed for rinsing and re-filling the different compartments of the equipment with the
chosen electrolytes. In order to prevent the dissolution of air in these electrolytes, the
surfaces of the electrolytes were covered with a layer of kerosene. An additional
advantage is that negligible amounts of carbon dioxide dissolve in the electrolytes if a
'high' pH is chosen in performing the analysis (even pH 7). If experiments are to be
carried out in parallel-mounted narrow-bore tubes, this method of mounting the narrow-
bore tubes on a thermostated aluminium block is recommended.
7.4.4. Equipment with high-resolution detectors
Fig.7.15 shows a schematic diagram of isotachophoretic equipment for use with a highresolution
W absorption detector and a conductimeter, and a photograph is shown in
Fig.7.16.
The equipment consists of a PTFE narrow-bore tube in which the analysis is performed,
the length being about 25 cm, although a longer tube can easily be mounted. It is
produced by Habia (Breda, The Netherlands), with O.D. c.a 0.7-0.75 mm and I.D. ca
TABLE 7.1
SOME VALUES FOR THE RESISTANCES (a) TO BE MOUNTED IN THE BRIDGE (FIG.7.13) OF
THE TEMPERATURE-REGULATING UNIT TO SELECT A TEMPERATURE RANGE
Resistors R, , R, and R, : 1%, 50 ppm/"C.
Resistance Temperature range ('0
0-50 0-125 0-300
R, =R, 120 150 180
R3 100 100 100
R, 50 50 100

EQUIPMENT 225
Fig.7.14. Electrophoretic equipment suitable for isotachophoretic analyses, with an injection system
comparable with the injection block described in section 7.2.5. A counter electrode compartment of the
cylindrical type (Fig.7.8) is used. The narrow-bore tube is wound around a thermostated aluminium
block (Fig.7.12.). Detection is performed with thermocouples (copper-constantan). A pressure system
is applied for rinsing and re-filling the various compartments. This equipment was constructed in 1968
by Verheggen and Everaerts.
0.4-0.45 mm. The variations in diameter per unit length are negligibly small. This narrow-
bore tube is clamped by a special clamping device, as discussed in section 7.2.3, in the
injection block, the counter electrode compartment and on both sides of the conductivity
probe. The narrow-bore tube is uninterrupted in the W detector.

226 INSTRUMENTATION
/
I
/,
\
/
I
1
d
/
Fig.7.15. Exploded view of more advanced electrophoretic equipment suitable for isotachophoretic
analyses in which the injection block shown in Fig.7.5 and the cylindrical counter electrode compart-
ment shown in Fig.7.8 are used. For the detection of the various zones, a UV absorption meter, a
potential gradient detector (d.c. method) and a conductivity detector (a.c. method) are applied (see
Chapter 6).

EQUIPMENT
Fig.7.16. Photograph of the more advanced electrophoretic equipment based on that shown in
Fig.7.15. This photograph shows the flexibility of the construction. Various injection systems,
detectors and counter electrode compartments can easily be combined, In the equipment shown,
the injection block shown in Fig.7.5 and the counter electrode compartment with a flat membrane
shown in Fig.7.9 are used. The six-way valve shown in Figs.7.2-7.4 is fitted in order to make a
simple introduction via either a micro-syringe or a tap possible. For the detection of the various
zones, a UV absorption meter, a potential gradient detector (d.c. method) and a conductivity meter
(a.c. method) are applied (see Chapter 6). The equipment was constructed by Verheggen and Everaerts.
227

228 INSTRUMENTATION
The W light is generated by a microwave-powered low-pressure mercury lamp. This
W light is transported by an optical quartz rod and fed into a cylindrical slit of width
0.05-0.3 mm. As already mentioned, the PTFE narrow-bore tube is not interrupted and is
clamped by the slit, which is made of brass, and the W light passes through the narrow-
bore tube and is again transported via an optical quartz rod to a set of filters (an
interference filter in combination with an end filter). The UV quanta then illuminate a
UV light-sensitive photodiode (S330; Hamamatsu, Hamamatsu City, Japan). The quality
of the PTFE narrow-bore tube is not sufficiently constant for the UV detector, because
the PTFE material itself has a high W absorption. On mounting a particular narrow-bore
tube, the amount of light that passes through it and finally reaches the detector may
vary by a factor of up to three compared with a previously used narrow-bore tube owing
to the difference in the thickness of the two tubes, if they are filled with a non-UV-
absorbing liquid. On one hand the high absorptivity of the PTFE material in the UV
range is a disadvantage, while on the other hand the dark current, i.e., the current that is
transported by the PTFE wall and reaches the detector without passing through the
narrow-bore tube, is reduced to a minimum.
The signals are handled electronically and result in a trace on a potentiometric recorder.
The trace does not have a continuous stepwise character if the isotachophoretic zones pass
the detector.
At the position where the conductimeter is mounted, the narrow-bore tube is inter-
rupted by a piece of insulating material (Perspex or TPX) in which the micro-sensing
electrodes are mounted (Chapter 6). As a result, there is always a slight difference in cell
volume between the conductimeter and the UV detector. The sequence of mounting
these two detectors was tested and it proved to be of no importance; experiments were
carried out to prove this only with components that were stable in the UV region, and
possible deleterious effects due to W light were not studied.
The conductivity detector can be applied for measurements of the conductivity
(a.c. method) or for measurements of the potential gradient (d.c. method) via two micro-
sensing electrodes (10-pm Pt-Ir foil) mounted axially and in direct contact with the
electrolytes inside the narrow-bore tube. In Fig. 7.16 can be seen the position where
the coil is mounted in order to give good galvanic separation of the high potential on
the micro-sensing electrodes from the circuit for measuring the conductivity (potential
gradient) at low potential. As discussed in Chapter 6, a leak current towards earth (even
lo-” A) must be prevented. For this reason the conductivity probe is surrounded by
PTFE insulation. Even the wires connecting the micro-sensing electrodes with the
electronic measuring circuit are provided with extra insulation by means of a PTFE
narrow-bore tube.
The signals derived from the conductivity probe are fed to a field effect transistor
(potential gradient measurement) or directly to a well insulated transformer for good
galvanic insulation. If the measuring electrodes are mounted equiplanar, only the
conductivity can be determined of course.
For the measurement of the conductivity (a.c. method) with axially mounted elec-
trodes, these electrodes must be separated from each other via a capacitor, otherwise an
electric current will flow, due to the potential gradient, and electrode reactions will
result, e.g. , coating or gas production. Even when the micro-sensing electrodes were

EQUIPMENT 229
mounted equiplanar, we found it advantageous to have this capacitor between the
measuring electrodes; possibly an exact equiplanar construction is not always possible.
Owing to the high potentials applied, some leak current will decrease the resolution
after a small series of experiments. The signals derived from the transformer are handled
electronically and result in a trace on the potentiometric recorder. This trace has a
continuous stepwise character if the isotachophoretic zones pass the detector. If the
trace does not have a continuous stepwise character, e.g, if a drift is obtained or dips
and/or overshoots are recorded, something is wrong. A drift of the base line is obtained
if, for instance, impurities are present that are more mobile than the leading ion, the
buffer capacity of the counter ion is not sufficient or some electrode reaction occurs at
the micro-sensing electrodes. Dips (or negative steps) can be expected if the buffer
capacity of the counter ion is not sufficient, if an enforced isotachophoretic system is
obtained or if the electrodes are coated with a polymer as a result of an electrode
reaction or the physical adsorption of any material. Overshoots can be expected if the
buffer capacity of the counter ion is not sufficient, if the temperatures of two adjacent
zones are too different or if an electrode reaction occurs.
A modified Brandenburg (Thornton Heath, Great Britain) power supply of the
alpha-series is used. It is modified in such a way that it not only can be applied as a
constant-voltage source (+30 kV), but for the isotachophoretic experiments can also be
applied as a current-stabilized power supply (lt30 kV). Various current-stabilized power
supplies, however, are commercially available nowadays, even up to 60 kV.
Between the injection block, shown in detail in Figs.7.5 and 7.6, a six-way tap
(Figs.7.2-7.4), whch can easily be removed without changing the narrow-bore tube if
necessary, is mounted. In the equipment shown, an injection can also be made with a
normal commercially available micro-syringe, as the sample can be introduced via the
tap. Thus this instrument combines all the advantages of taps and syringes. Also, instead
of the cylindrical counter electrode compartment (Figs.7.8 and 7.1 5), a counter electrode
compartment with a flat membrane (Figs.7.9 and 7.10) can be used. These electrode
compartments can easily be changed if necessary without any problems or the need to
fit a new narrow-bore tube.
All components of the isotachophoretic equipment shown in Fig.7.15 are replaceable
because no adhesive is applied, rubber O-rings and screw-threads being used for clamping.
We found that it is sometimes necessary to replace the narrow-bore tube as their life was
found to vary from several years to only a few months. A narrow-bore tube needs to be
changed if a decreased resolution cannot be improved.
Owing to differences in the behaviour of the various operational systems, or compounds
of the sample, even the ‘inert’ PTFE can become coated with material that is not easy
to remove, and the resolution may decrease. Impurities, possibly building up over a long
period, that are adsorbed on the walls of the injection system or the counter electrode
compartment have less influence on the detection or the separation itself, because the
bores in these parts of the instruments are considerably greater. The conductivity probe,
if washing with a non-ionic detergent gives no improvement, can easily be cleaned with
some metal polish and a cotton thread.

230
7.5. COUNTER FLOW OF ELECTROLYTE
7.5.1. Introduction
INSTRUMENTATION
Many of the papers describing electrophoretic techniques have dealt with the elec-
trolytic counter flow of electrolyte, and a large number of other papers could be cited,
especially relating to equipment filled with various stabilizing media. In ths field,
experiments are carried out to increase the length of the separation, especially for
improving the separation of isotopes, and it has been found that mainly enrichment
could be obtained. The electrophoretic techniques applied usually involved a moving-
boundary system, in whch a complete separation cannot be expected. However, if an
isotachophoretic system is chosen for the separation of isotopes, the separation procedure
is also a moving-boundary system that may result in a complete separation, ie., the
steady state.
In this section, some possible methods for regulated and non-regulated counter flows
are given, and a newly developed pumping system is described in which the gas produc-
tion is used for pumping hydrodynamically the liquid needed for the counterflow of
electrolyte. The driving current can be regulated by signals from the isotachophoretic
equipment, by means of which the zones can be stopped in the separation chamber (the
narrow-bore tube) if counter flow is applied. Of course, it is beyond the scope of this book
to discuss all possible systems for electrolytic counter flows.
We can consider a regulated counter flow in terms of the main basic principles, as
follows. (a) The electric current is constant during the analysis, and the hydrodynamic
counter flow of electrolyte is regulated and controlled by signals derived from the electro-
phoretic apparatus. (b) The hydrodynamic counter flow of electrolyte is constant during
the time the counter flow of electrolyte is required, and the electric current is adjusted
to this counter flow by means of signals derived from the electrophoretic equipment.
During the detection, the electric current is stabilized again. (c) The electric current is
constant in the initial phase and the hydrodynamic counter flow of electrolyte is started
as soon as a pre-set value of the voltage of the current-stabilized power supply has been
reached. The counter flow of electrolyte is then adjusted until no further increase in
voltage is obtained. If for any reason a lower pre-set value is reached, the counter flow of
electrolyte is stopped.
It should be pointed out that although the length of separation is generally increased,
the counter flow of electrolyte disturb the electrophoretic separation (Chapter 17).
The method of producing the counter flow can vary widely, and syringe pumps,
peristaltic pumps, level differences or ‘gas pumps’ can be applied.
Two main reasons can be given for wanting a counter flow of electrolyte, both
originating from the fact that the narrow-bore tube is not long enough for a particular
separation: (1) the concentration differences between the ions to be separated are too
laIge; and (2) the differences in (effective) mobility between the ions of interest are small.
Of course, these two factors may be combined in a specific instance.
In those instances when the difference in (effective) mobility is minimal, the use of a
counter flow of electrolyte will generally fail. More research needs to be carried out in
order to determine the effect of the ‘disturbance factor’. It is not unlikely that in specific

COUNTER FLOW OF ELECTROLYTE 231
instances this factor may be zero or even that the separation may be positively influenced.
Also, when the difference in effective mobility is minimal, one cannot expect always a
complete separation because in some cases the ions have a mutual adverse influence on
the pH of the mixed zone and give a poorer separation. Once the ions are separated they
will form discrete zones owing to the difference in the pH values in the two zones.
We found the counter flow of electrolyte to be successful especially when samples need
to be separated with large concentration differences between the various zones. The
use of a counter flow of electrolyte has also proved of value in elucidating whether a
separation is completed or not (i.e., mixed zones are present or not).
Although the use of a counter flow of electrolyte in isotachophoretic experiments
can be seen to be a valuable tool, it also has disadvantages. If a counter flow of electrolyte
is to be considered, the chemicals must be of the highest purity available, and even then
they often are not pure enough. The impurities may sometimes be collected betwetn the
leading and terminating zones and influence the analysis. Sometimes the zone still
undergoes a small migration and cannot be stopped owing to impurities present. The
impurities in the leading electrolyte and/or in the terminating electrolyte must be
removed by recrystallization, zone refining or electrophoretic procedures, etc., if
Eqn. 7.2 relates to the leading electrolyte and eqn. 7.3 to the terminating electrolyte,
where meff,T, meff.,I and are the effective mobilities of the terminating ion,
impurity and leading ion, respectively.
7.5.2. Counter flow with level regulation
Fig.7.17 shows schematically the equipment with which a counter flow of electrolyte
can be applied, and the circuit for the regulation of the counter flow of electrolyte is
shown in Fig.7.18.
The moment at which the counter flow is to be started can be selected with the
lO-kf2 potentiometer. The switch A is provided in order to have the possibility of
selecting a high potential on the side of the injection block of chosen polarity. It has to
be borne in mind that for optimal functioning of the conductimeter, the probe must be
at a ‘low’ potential, Le., less than 10 kV. As soon as the voltage selected by the lO-kS2
potentiometer has been reached, the level is controlled by the plunger (Fig.7.17) by means
of a coil. Before the experiment, this level is adjusted approximately to the level in the
compartment of the terminating electrolyte, such that the sample zones still migrate in the
appropriate direction by means of the electric field strength (possibly a small flow in the
direction of the movement of the zones is permitted). Owing to the construction of the
counter electrode compartment, the pH jump across the membrane is of minor importance.
Experiments with a counter flow of electrolyte showed that it is important that the
compartment in which the driving electrode is mounted should contain electrolyte also.
The electrolyte, containing buffer ions, decreases the potential at the measuring electrodes
of the conductivity probe and diminishes the pH jump across the membrane.

232
INSTRUMENTATION
Fig.7.17. Equipment for producing a counter flow of electrolyte via level regulation. 1 = Electronic
regulation circuit (shown in Fig.7.18); 2 = coil with which a movement of the plunger can be
effected; 3 = set of detectors.
The counter flow is stopped by switching the PTFE-lined Hamilton valve, mounted in
the plunger reservoir, to the closed position, and the device for regulating the counter
flow of electrolyte can then be switched off. It should be noted that the coil is fed by
a rectified electric current that is not smoothed by a capacitor. It has been found
experimentally that the vibration of the plunger by the unsmoothed current eliminates
the mechanical friction that could disturb the analysis at the moment the regulation is
started by an abrupt lowering of the plunger.

COUNTER FLOW OF ELECTROLYTE
p High V
lOOMR
50
Fig.7.18. Electronic circuit for regulation of the counter flow of electrolyte via level regulation in
isotachophoretic analyses. The resistances are given in kn unless stated otherwise.
This method of regulation can also be applied if a counter flow of electrolyte is
required that is regulated by signals derived from the detectors mounted directly on the
narrow-bore tube. In this instance the zones are stopped at the regulating detector,
which usually gives a better result.
7.5.3. Counter flow with light-dependent resistor regulation
This method of producing a regulated counter flow of electrolyte is shown schematically
in Fig.7.19 and the circuit for regulation is given in Fig.7.20.
A light-dependent resistor (LDR) is attached in series with the narrow-bore tube and
a constant potential gradient is applied over the narrow-bore tube and the LDR. Because
in isotachophoretic experiments the total potential gradient over the equipment is higher
than 300 V, which is the LDR limit, a series of LDRs is used so that one is able to work
at 10 kV. The series of LDRs can be considered as a voltage source. The amount of light
given by a lamp (see Fig.7.20) is regulated by a thermocouple (copper-constantan)
mounted around the narrow-bore tube.
A change in the temperature of the narrow-bore tube will automatically involve a
change in the electric current through it. As is normal in isotachophoretic analyses, the
2
233

234 INSTRUMENTATION
Fig.7.19. Equipment for producing a counter flow of electrolyte with regulation via a light-dependent
resistor. 1 = electronic circuit shown in Fig.7.20; 2 = light-dependent resistor; 3 = set of detectors.
total resistance of the electrolytes inside the narrowbore tube will increase in time owing
to the progress of less conductive zones. The increase in the resistance of the narrow-bore
tube during the analysis will produce a decrease in the current if a constant voltage is
applied over it, and this decrease in current will cause a decrease in the temperature of
the narrow-bore tube (quadratic relationship.) This decrease in temperature is recorded by
the regulating thermocouple. Hence the total resistance of the LDR and indirectly the
voltage drop over the narrow-bore tube are controlled by this thermocouple and a
stabilized current will be the result.
A means of obtaining a more stable regulation of the current is to arrange a resistor in
series with the narrow-bore tube. The potential drop over this resistor can be used for
current stabilization, and slowly moving concentration fronts, such as pH disturbances,
will not influence the regulation of the current.

COUNTER FLOW OF ELECTROLYTE
Fig.7.20. ElecQonic circuit for stabilizing the electric current by signals derived from a thermocouple
mounted around the narrow-bore tube in which the electric current flows. This circuit can also be
applied for regulation of the various zones moving isotachophoretically in the narrow-bore tube in
experiments with a counter flow of electrolyte. 1,2 = connections for the thermocouple (copper-
constantan); 3 = + 15 V; 4 = common terminal; 5 = - 15 V,
Therefore, during the counter flow of electrolyte, a thermocouple mounted around the
narrow-bore tube is applied and during the detection of the zones the current is stabilized
by an extra resistor mounted in series with both the LDR and the narrow-bore tube.
The counter flow of electrolyte can be produced in various ways, although only the
syringe pump is shown in Fig.7.19. Particularly if the counter flow is produced by a
difference in levels, a complication can arise because the level is not controlled, and the
counter flow will thus change with time. As will be discussed later, there are two limits
for the counter flow and if at a certain moment the lower limit is exceeded the counter
flow of electrolyte is no longer able to stop the zones. For a counter flow over a long
period of time, the electrode compartment that contains the counter flow electrolyte
must be very large and it is preferable to use a pump, especially that discussed in
section 7.5.5.
It wdl be noticed immediately that the adjustment of the electric current as described
here will automatically result in an oscillation of the zones around the regulating thermo-
couple. For thermometric recording, the zone must have passed the thermometric
detector by about 1-2 cm for complete qualitative and quantitative determination, but
for the regulation a much lower signal is needed. We found that this method of regulation
gives a negligible oscillation; the experimental conditions were checked with coloured
ions for which the sharpness of the boundaries was studied.
Fig.7.21 shows two isotachopherograms for the separation of formate and acetate
with and without a counter flow of electrolyte in order to demonstrate LDR regulation
235

236 INSTRUMENTATION
-
Time
Chloride
min.
Fig.7.21. Isotachophoretic separation of formate and acetate without (a) and with (b) a counter flow
of electrolyte. The experiments were carried out in the operational system at pH 6 (Table 12.1) with
glutamic acid as terminating electrolyte. Detection was carried out with a thermocouple mounted at
a distance of about 50 cm from the injection point. The electric current was stabilized by the circuit
shown in Fig.7.20. The thermocouple for regulation was mounted at a distance of about 25 cm
from the injection point. The traces show that the electric current decreases (lower temperature) if
a hot zone passes the regulating thermocouple. As soon as the terminating ion is below the regulation
thermocouple, the electric current is stabilized at imin.. Trace (b) shows that equilibrium is achieved
between the counter flow of electrolyte and the electric current. Traces (a) and (b) show that the
isotachopherograms finally obtained are similar. These isotachopherograms are not given to show the
usefullness of a counter flow of electrolyte, but only to demonstrate current stabilization via LDR
and the possibility of using a counter flow of electrolyte (see Chapter 17).
and the isotachopherogram that can be expected. The recording is performed with a
thermometric detector (copper-constantan thermocouple). In this experiment, therefore,
two thermocouples were mounted around the narrow-bore tube, one at the beginning
of the narrow-bore tube, which was used for the LDR regulation during the counter flow
of electrolyte period, and the other at the end of the narrow-bore tube, which was used
for the detection. Because more advanced systems are considered later, further isotacho-
pherograms with this type of regulation will not be shown. The experimental conditions
for the isotachopherograms shown in Fig.7.21, however, will be given.
The analysis was performed in the operational system at pH 6 (Table 12.1) with
chloride as the leading ion and glutamate as the terminating ion. The aluminium block

COUNTER FLOW OF ELECTROLYTE 231
around which the narrow-bore tube was mounted (section 7.4.3) was thermostated at
18°C. A 2-pl injection was made, containing 0.02 mole of sodium acetate and 0.02 mole
of sodium formate. The current was 92 pA in the initial phase and the temperature of the
zone of the leading ion was used as reference for the current stabilization (about 25°C).
In the initial phase, the current is not yet stabilized, possibly owing to the movement of
ions ahead of the zones of formate and acetate, which increase the conductivity. Subse-
quently the real zones of formate and acetate reach the regulation thermocouple. Because
these zones are considerably hotter than the leading zone (see Fig.6.7), even if the
aluminium block is applied as a thermostat, the electrophoretic driving current will
decrease. Fig.7.21 shows clearly a drop in electric current from ima. = 92pA towards
imh. = 52 pA, because the temperature of the glutamate is used for stabilization of the
electric current. In Fig.7.21 b, from the initial phase a counter flow of electrolyte is
produced in such an amount that the zones still have a movement in the appropriate
direction. Fig.7.21 shows that the temperature of the formate zone does not give such a
low electric current that the zones are stopped by the counter flow chosen. Between the
formate and acetate zones, however, a temperature is attained such that the zones are
stopped. The counter flow produced was 200 pllh. After the counter flow of electrolyte
has stopped, the current decreases further to the in,h. value and the experiment is
completed, as shown in Fig.7.21a.
It need not be explained that the thermocouple used for the detection of the various
zones must be mounted as far as possible from the regulating thermocouple, otherwise
some material may pass the recording thermocouple too soon, especially if components
are present in the sample that normally have a temperature in the zone lower than that
at which equilibrium is obtained.
7.5.4. Counter flow with direct control on the pumping mechanism via the power supply
A counter flow of electrolyte can be obtained in this way if the initial and end voltage
over the narrow-bore tube are known. If both of these values are known, the position of
the zones as a function of the potential gradient and the approximate counter flow
required in order to stop the zones can be calculated (Fig.7.22). A circuit such as that
shown in Fig.7.23 can be applied, with which it is possible to select a voltage of the
current-stabilized power supply at which the pump is started. Because the counter flow
to be produced is calculated only roughly, a counter flow must be selected such that the
zones are stopped and slowly pushed back. If the counter flow is insufficient, the zones
are not stopped by the pump and finally reach the detector, while if the counter flow
matches the movement of the zones the pump will be in action continuously.
If the zones are pushed back, the voltage across the narrow-bore tube will decrease. As
soon as a chosen lower limit has been reached, the counter flow of'electrolyte is stopped
and the zones will again move in the required direction. It needs no further explanation
that the range of voltage in which the operation of the pump is planned must be very
small. Experiments with coloured ions showed that the zone boundaries are less sharp
during the period when they are being pushed back, but as soon as the pumping was
stopped sharp boundaries were recorded very rapidly.

238 INSTRUMENTATION
Fig.7.22. Equipment for producing a counter flow of electrolyte by means of on-off regulation of
the pumping mechanism by signals derived from the current-stabilized power supply. 1 = Electronic
circuit shown in Fig.7.23; 2 = set of detectors.
7.5.5. Counter flow with no regulation
This method of producing a counter flow is comparable with the method discussed
briefly in section 7.5.3. In this instance also the initial and end voltages must be known,
and the current-stabilized power supply must have a voltage limiter. The procedure is
demonstrated in Fig.7.24.
A represents an experiment with no counter flow of electrolyte. The electric current
is constant and the potential gradient increases continuously with time, because fewer
conductive zones move and occupy more of the narrow-bore tube. This potential
gradient does not, of course, increase regularly, because the bore of the injection block
does not have a diameter identical with the inside diameter of the narrow-bore tube and
the bore of the conductivity probe.

COUNTER FLOW OF ELECTROLYTE
p high V
i 1OOGR r-
Fig.7.23. Electronic circuit for the on-off regulation of the pumping mechanism in isotachophoretic
experiments with a counter flow of electrolyte. The common terminal should not be mounted such
that the stabilization of the current in the narrow-bore tube is influenced (see Fig.7.26).
In B an experiment with a counter flow of electrolyte is shown. The voltage of the
current-stabilized power supply is limited to V1, which is higher than the initial voltage
and much lower than the final voltage. As soon as V, has been reached (after a time tl),
the power supply is no longer able to keep the electric current stabilized and as a result
the current will decrease. Depending on the magnitude of the counter flow produced, an
equilibrium current (Ieq, 1) can be reached at which the zones are stopped after a time
(tl*). After the counter flow of electrolyte has stopped (after a time tl**), the voltage is no
longer limited and a stabilized current will be the result.
In C, a similar experiment is shown with a limited voltage V, and a greater counter
flow of electrolyte. If a well chosen counter flow of electrolyte is applied, i.e., a flow such
that the zones will move in the appropriate direction if the current I, has been chosen
and the zones can be stopped before the detector, no further regulation need be used.
Nevertheless, we found this method to be difficult to apply in practice, particularly
because the position at whch the zones are stopped is influenced by the size and compo
sition of the sample. If the sample consists of many ions with a high effective mobility,
the increment in voltage is not great initially. If the regulation is not performed at a ‘low’
voltage, t!ie zones may be stopped if they have already passed the detector.
239

240 INSTRUMENTATION
V t
“2
Fig.7.24. Isotachophoresis with a counter flow of electrolyte without direct regulation of the process.
A, Experiment without a counter flow of electrolyte. The voltage increases because less mobile ions
enter the narrow-bore tube. In practice, this increment is not as smooth as is shown here. The electric
current is kept constant during the experiment at I, B, Experiment in which a counter flow of
electrolyte is applied. The electric current is stabilized at I, up to V, (fJ, then the voltage is
stabilized (limiter). This results in a decrease in the electric current to ieq, (t;). At time ff*, the
counter flow of electrolyte is stopped, the current is stabilized at I, again and the voltage can
increase steadily. C, Experiment with a greater counter flow of electrolyte t bn in B. The electric
current is stabilized at I, up to V, (t2), then decreases to ieqz) (tz). The counter flow of electrolyte
is stopped at fz* and the electric current is stabilized again at I,. This figure does not relate to actual
experiments, all values being chosen arbitrarily. For further explanation, see text.

COUNTER FLOW OF ELECTROLYTE 24 1
7.5.6. Counter flow regulated by the current-stabilized power supply; the membrane
Pump
This method is the most accurate and simple, and is therefore discussed in more detail.
The principle is shown in Fig.7.25.
Fig.7.26 can be used to explain the principle of the method and also the principle by
which the electric current through the narrow-bore tube (Ic) is stabilized.
If through the narrow-bore tube, filed with a suitable electrolyte (leading electrolyte),
an electric current is stabilized at Ic, the total voltage needed (V,) is then increasing during
T I
1
Fig.7.25. Equipment for producing a counter flow of electrolyte regulated by the currentstabilized
power supply. This method of pumping and also the regulation were found to be optimal in
combination with the narrow-bore tube, in spite of the fact that the membrane pump does not have
linear characteristics. 1 = Electronic circuit shown in Fig.7.29; 2 = set of detectors. If the counter
flow of electrolyte is also to be applied for micro-preparative purposes, another means of pumping
can be sought (e.g., an electroendosmotic pump).

242 INSTRUMENTATION
Fig.7.26. Principle of regulation of a counter flow of electrolyte, via a membrane pump (as shown
in Fig.7.25). Attention should be paid to the common terminal and the earth, which prevent distur-
bances to the current stabilization (electrophoretic driving current) by the counter flow regulation.
This could destroy, or at least obscure, the final result.
the isotachophoretic run. If gas is now produced in the electrolysis cell of the membrane
pump, the volume of this electrolysis cell tends to expand. The volume can expand easily
because between the electrolysis cell and a cell filled with leading electrolyte, mounted.
next to it, a thin membrane (e.g., a rubber contraceptive) is mounted. This membrane
is mounted with pre-stressing.
In Fig.7.27, the construction of the membrane pump is shown in more detail, and a
photograph is shown in Fig.7.28.
The flow of liquid caused by the production of gas in the electrolysis cell of the
membrane pump counteracts the increment in V,. This gas is produced by an electric
current I,, in an electrolyte (e.g., 0.01 NKC1). Because V, is of the magnitude of kilovolts,
V, is reduced to a value B V, with the aid of two resistors of 100 Ma and 56 ka. An
electronic circuit (Fig.7.29) compares this value B V, with an adjustable V, If BlV,l<
V,, (V,> 0), then I, = 0. If BlV,l> V&., then I, # 0 and the increment in V, is counter-
acted. The regulation is such that I. will reach a value such that BIG1 becomes and
remains approximately equal to VEf..
Thus the relationship betweenI,,, BI V,l and V, is:
BI Vc I Q Vmf.
then I,, = 0, and
BI VCl > V,f.
then I, = A (BI V, I - VXt).
The optimal value for the amplification factor, dl,/dl V,l = BA, is dependent, among
other factors, on the electrolytic system chosen (operational system) and on the cross-
(7.4)
(7.5)

COUNTER FLOW OF ELECTROLYTE
I I
31 I
Fig.7.27. Detailed diagram of the membrane pump. The pump can be used in isotachophoretic
experiments with a counter flow of electrolyte. 1 = Cap for closing the electrolysis cell; 2 = electrolysis
cell filled with a suitable electrolyte, e.g., 0.01 M KCl; 3 = nuts; 4 = the gas-producing electrodes;
5 = rubber O-ring; 6 = cap for closing the electrolysis cell; 7 = PTFE-lined Hamilton (1MM1) valve;
8 = cap for closing the compartment filled with leading electrolyte; 9 = rubber O-ring; 10 = electrode
that can be used, if required, such that during the time the counter flow of electrolyte occurs, the
electrode that is connected with the current-stabilized power supply is not separated from the narrow-
bore tube in which the analyses are performed by a semipermeable membrane; 11 = central body of
the compartment filled with leading electrolyte; 12 = bolts for clamping components (11) and (2)
together (in total four bolts and nuts are applied); 13 = needle; 14 = rubber membrane; 15 = rubber
O-ring.
section of the narrow-bore tube, which may vary if a replacement narrow-bore tube is
used. If BA has a too high a value, then the regulation will be unstable, while if BA is too
small, the accuracy of the regulation will not be sufficient.
Because the current, Z,., through the resistors of 100 MR and 56 ki2 may not influence
the current through the narrow-bore tube (Ic), the resistors must be mounted as shown in
Fig.7.26. Of course, the input current, Zi, of the electronic regulating circuit must be
negligibly small compared with Z,. A galvanic separation of the electrodes of the electroly-
sis cell of the membrane pump with earth is arranged, because otherwise part of Z, would
flow through the thm membrane. The membrane was found to be permeable for small
243

244 INSTRUMENTATION
Fig.7.28. Photograph of the membrane pump illustrated in Fig.7.27.
ions in a long run. In addition to the leak of the electric current, ions from the electrolyte
of the electrolysis cell may also interfere if they can pass through the membrane due to
poor galvanic separation of the electrodes of the electrolysis cell towards earth. The opera-
tional amplifiers (see Fig.7.29) ICl0, ICll and IClz form a differential amplifier with a
high input impedance. The amplification factor of this differential amplifier is unity.
With aid of a switch ‘polarity’, the output signal of the differential amplifier is always
kept positive, depending on the polarity of V,. By means of a ten-turn potentiometer,
the reference voltage Vref, can be adjusted. The trim potentiometer of 10 kn must have a
value such that the output voltage of ICI3 is equal to zero if I V,I = 10 kV and V,, has its
maximal value. If the absolute value of V, is greater than the selected value of VEf., a
negative output voltage of IC13 is obtained. The amplification factor of ICI3 is constant
within 3 dB up to approximately 3 Hz. This frequency is sufficiently high to make stable
regulation possible. By the low-pass characteristic of the amplifier, the eventual dis-
turbance of the electric mains (50 Hz) is sufficiently suppressed.
The transformer T, forms an oscillator with the two npn transistors. If the input
voltage of ICI4 is negative, the sum of the average collector currents of both transistors
is proportional to this voltage. The average value of the rectified current through L3, the
electric current I, needed for the electrolysis cell of the membrane pump, is approximately
proportional to the input voltage of IC14. If this voltage is positive, I, is zero. By means
of a resistor of 4.7 kSl between the connection points 4 and 5 of IC14, the offset voltage
of ICI4 is changed in such a way that I, is certainly zero if the input voltage of IC14 is
zero, in the case of manual regulation.

COUNTER FLOW OF ELECTROLYTE
max. 15 kV
hishVl-+
*
- common
a: 2 N 4 124
Fig.7.29. Electronic circuit that can be used for the regulation of the electrolytic counter flow in
isotachophoretic experiments, with aid of the membrane pump shown in Figs.7.27 and 7.28.
Components IC,,, IC,, , IC,, , IC,, and IC,, are all of the type rA741. All diodes are 1N4148 or
1N914. The resistances are given in kR unless stated otherwise. The specifications for the transformer
are: L, = two times 10 turns; L, = two times 50 turns; L, = 65 turns. For the wires enamelled
copper Wire, diameter 0.4 mm, is used. The potcore is of the type P 36/22,3B7, ~e (permeability)
= 2030.
By means of a switch ‘auto-manual’, automatic or manual regulation of the membrane
pump can be selected. The maximal value of I, is approximately 2 mA, and the voltage
needed is low (+ 3 V).
The amplification factor BA of the circuit (Fig.7.29) for experiments in the operational
system at pH 6 (see Table 12.1) with the equipment as described in section 7.4.4. (a
PTFE narrow-bore tube of I.D. 0.4-0.45 mm and O.D. 0.7-0.8 mm and a total length
of approximately 30 cm) is approximately 0.1 5 mA/V. We can therefore calculate that
I Vcl changes by approximately 14 V if I, changes from 0 to 2 mA. Of course, other values
can easily be taken, although we found the above values to be optimal.
245

This Page Intentionally Left Blank

APPLICATIONS

This Page Intentionally Left Blank

Chapter 8
Introduction
SUMMARY
In ths chapter some practical information is given on the Section Applications, and a
scheme is given for ‘trouble-shooting’.
8. INTRODUCTION
The Section Applications contains almost all of the practical information about
isotachophoretic separations in narrow-bore tubes. In this section, applications and results
are given for separations classified according to chemical compounds that belong to clearly
distinguishable classes.
The separations were carried out in so-called operational systems in which the electro-
lytes were shown to give optimal results. The operational systems are listed in tables, in
order to make a comparison between them possible. The systems listed were chosen
somewhat arbitrarily; many more possibilities could be given. Also, the separations con-
sidered were mainly chosen arbitrarily: many real problems from industry or hospitals
proved to be much simpler. The separations are shown in order to indicate their possi-
bilities and to make patterns recognizable.
When a specific operational system is chosen, one always has to bear in mind that the
pH in anionic separations by isotachophoresis tends to increase, while in cationic separa-
tions it tends to decrease, going from the leading zone towards the terminating zone.
Therefore, the pH must always be chosen such that the optimal effect of the buffering
counter ion is used. In some instances a buffering counter ion is not necessary, while in
other instances two or more counter ions with overlapping buffer regions are needed.
A difference of 0.5 pH unit can give an operational system that has completely
different characteristics for a specific analytical problem, as shown in the following
example.
If a separation of anions is sought and the operational system at pH 6 (Table 12.1) is
found to be suitable, one can adjust the pH of the leading electrolyte from its initial
value of 6; the buffering capacity of histidine is sufficient until a pH of ca. 7. If, however,
an anion is present with a very low effective mobility, which needs a terminator with an
even lower effective mobility than the anion to be separated, it may be preferable to
adjust the pH of the leading electrolyte to 5.5. If the pH of the leading’electrolyte is
decreased too much, sometimes difficulties can arise because too few counter ions are
present and the buffering capacity may not be sufficient (see Fig.9.5). Experimentally, we
found it best to adjust the pH of the leading electrolyte, with the chosen counter ion, to
the selected value as accurately as possible and to check the pH of the solution again the
following day. In most instances the pH is shifted (kO.1-0.2 pH unit).
Step heights listed in the various tables are proportional to the effective mobilities of
249

The analysis can he
carried out
c
NO
The analysis of me zest mixture of
anions or cations shows that
the step heights are not constant
and/or the resolution is bad, and the base.
line has a drift.
The micro-sensing electrodes
are coated. Depolarize the
For the UV detector and the conduc-
I tivity detector (ax. method).
I
I NO NO
YES
electrodes in 0.1 N HNOl.
If this is not effective apply
aqua regra.
If this is not effective dismount
the probe and apply metal polish.
Wash the equipment after each
procedure with a non-ionic detergent
and thoroughly with douhledistilled
water.
NO
Perform an analysis
detector (a.c. method).
The narrow-bore tube is
For the conductivity
c
detector (n.c. method)
only i
- For the UV detector only
1
with a non-ionic detergent
and rinse the entire equipment
-
If after several washings the analysis stlll does not improve, the entire
equipment must be dismounted. Polish the various narrow bores with
a suitable polish and use another narrow-bore tube.
Before an analysis can he carried out, the entire equipment must be washed
with a non-ionic detergent and rinsed with double-distilled water.
This easily can he checked. If the time hetween the start of the analysis
and the appearance of the first sample zone varies (in our equipment
dewrihed in section 7.4.4 the time is shorter). it indicates that a leak
is present. Generally the time is constant within 10 sec in an average
time of analysis of 15 min.
1
4
1
If only the resolution is had:
(1) there IS not a liquid-tight connection
between the Hamilton (1MM1) valve and the
counter electrode compartment or between
the counter electrode compartment and the
narrow-bore tube;
(2) the plunger of the Hamiltori (1MM1) valve
is loose and leaks;
(3) the semi-permeable membrane has a leak.
4
2
YES
‘The instrument must be waslie; liiorougldy
with a non-ionic aurfartant and then
thoroughly rinsed with double.distilled water.
Fig.8.1. Flowsheet for ‘troubleshooting’. It is assumed that the electronics of the conductivity detector and UV absorption detector are perfect, and
the electrolytes of the operational systems me pure and stable. For ‘trouble-shooting’, the electronics of the conductivity detector, the UV absorption
detector and the UV source are as discussed in section 6.4.3 for the d.c. method, section 6.4.5 for the a.c. method, section 6.5.2 for the UV source and
section 6.5.3 for the UV absorption detector. Later research shows that the sensitivity of the conductivity detector is improved if the complete equip-
ment is rinsed with a solution of 5% silicon grease in pentadecane, followed by a normal washing procedure.

INTRODUCTION 25 1
the various ionic species in the operational systems and they indicate which ions can be
separated in a given length of narrow-bore tube, assuming that the concentration differences
are not too great. If the concentration differences are too great, a longer narrowbore
tube or a counter flow of electrolyte must be used (see Chapter 17). It should be
noted that no corrections for the temperatures of consecutive zones have been made to
the results presented from either the thermometric or conductivity detector.
If the influence of the counter ion chosen is great (high effective mobility in the opera-
tional system chosen), greater differences in effective mobility between the sample ions
are needed for a complete separation. If the pH of the consecutive zones increases
regularly (in anionic separations) or decreases regularly (in cationic separations), small
differences in effective mobility are often sufficient because once the ions diffuse into
the zone where the pH is higher (anionic separations) or lower (cationic separations), they
may attain a greater effective mobility owing to dissociation. More attention is paid to
this phenomenon in Chapter 9.
It is sometimes easier and quicker to apply two or more operational systems, as the
equipment can be rinsed in a few minutes and is then ready for another operational
system, than to try to carry out a complete separation in one operational system. More
attention is paid to this aspect in Chapter 11. Sometimes only the concentration of the
operational system needs to be changed. The sulphate ion, for instance, in the operational
system at pH 6 (Table 12.1) moves behind the chloride ion (concentration of the leading
ion, C1- = 0.01 N), while it has a greater mobility than the chloride ion if the concentra-
tion of the ‘leading’ chloride ion is changed to 0.001 N. Another example is given in
Fig.12.7. In principle, we do not recommend the use of too long a narrow-bore tube,
because the voltages needed are too high and electroendosmosis may dominate the
separation.
If two types of detectors are available, in many instances the analysis can be carried
out, in spite of stable mixed zones (see Fig.6.33), in one operational system. From time
to time the equipment used in isotachophoretic analyses must be washed well with a non-
ionic detergent followed by thorough rinsing with double-distilled water. Adsorption of
many types of compounds may influence the detection (see Chapter 6), because amongst
other effects the {-potential may be changed. Because the resolution of both the W
absorption detector and the conductivity detector (ax. method) decreases in such
instances, an electrode reaction only must be rejected. We prefer the a.c. method to the
d.c. method because the detector indicates more quickly if something is going wrong and
measures can be taken directly (see Chapter 6). We recommend that, before a series of
analyses, a test mixture of anions or cations should be examined, because it can be seen
very quickly if the resolution and reproducibility are adequate. If the test mixture indi-
cates that non-reproducible data can be expected, appropriate measures must be taken,
as shown in Fig.8.1.
In our analyses, we pay special attention to the pH and the concentration of the
terminating electrolyte, although from various papers one may obtain the impression that
this is unnecessary. If too high a concentration of the terminating electrolyte is chosen,
eluting effects due to the impurities can be expected, the sample zones may still migrate
if a 100% counter flow of electrolyte is present (while the regulation is made via the

252 INTRODUCTION
current stabilizing power supply*) and sample ions can be flushed in the terminating
electrolyte if the counter flow of electrolyte occurs too soon (Chapter 17). Moreover,
quantitative results are non-reproducible if the injection is made at the boundary of the
leading and terminating electrolytes because some of the sample is always mixed with the
terminating electrolyte. Particularly if experiments are carried out at low concentrations
(0.001 N C1-) problems can be expected (see Chapter 10).
A wrongly chosen pH of the terminating electrolyte can cause eluting effects by H’
and OH- ions. Non-reproducible results can also be expected, especially if weak acids or
weak bases are present in the sample and some of the sample is mixed with the terminating
electrolyte.
In the following chapters, much data are given on thermometric detectors, but also
data obtained with conductivity and UV detectors are given. Data obtained with the
thermometric detector can be applied directly, if a conductivity detector is available.
However, the opposite is not true in some instances, because the resolution of the conductivity
detector (and the W detector) is so much greater. So far, the information
obtained with UV absorption detectors does not have qualitative uses.
The electrolytes applied in the various compartments must not, of course, contain gas
bubbles. Especially when the leading electrolyte is prepared, a surfactant (0.05% of
Mowiol) is added, and as a result small gas bubbles can easily be formed. For de-gassing
the electrolytes, in our laboratory we use an ultrasonic bath and still have a ‘trip unit’ on
our current-stabilizing power supply, which cuts off the electric current immediately
(faster than 0.1 sec) if the voltage increases too quickly.
It was determined experimentally that the micro-syringes often need to be cleaned,
because many impurities found in the isotachopherograms originate from dirty syringes.
For washing the equipment and cleaning the syringes, we recommend the detergent
Extran (E. Merck, Darmstadt, G.F.R.), which we purify by running it over a mixed-bed
ion exchanger. The syringes are cleaned with this surfactant in an ultrasonic bath.
*For an extensive discussion, see the sections 7.5.5 and 17.1.

Chapter 9
Practical aspects
SUMMARY
In experimental work on isotachophoresis, unusual effects are sometimes obtained.
These effects can be caused when not all of the conditions that are required in order to
obtain an isotachophoretic system are fulfilled. In this chapter, some of these phenomena
are discussed and a method for comparing and converting results obtained with different
types of apparatus is described.
9.1. INTRODUCTION
In Chapter 8, some practical information was given concerning the use of the operational
systems and the data presented in the Section Applications. It is difficult to give a
complete survey of all phenomena that may obscure or disturb the analysis. It will be
clear that, especially if narrow-bore systems are chosen, gas bubbles may affect the
analysis if they occupy too much of the tube. Once present above a critical size, the
temperature will increase, the gas bubbles will expand, and so on. Also, if small gas bubbles
are present and if the narrow-bore tube is mounted horizontally instead of vertically,
these gas bubbles may migrate, especially at the boundaries of two adjacent zones with a
large temperature difference.
Many obscure results in both qualitative and quantitative determinations can be expect-
ed if the operational systems are used in the wrong way and for the wrong application,
e.g., if the buffer does not have a sufficient buffering capacity. Another possibility is that
a leading electrolyte may be chosen of which the composition is not constant with time,
e.g., owing to an increasing amount of carbonate in operational systems at high pH (if no
precautions are taken), or an increasing amount of formic, acetic or propionic acid if the
leading electrolyte consists of formaldehyde, acetealdehyde or propionaldehyde, respectively.
This chapter summarizes some important general disturbances that can be found in
almost all operational systems; specific disturbances are discussed in the chapters to
which they belong.
9.2. DISTURBANCES CAUSED BY HYDROGEN AND HYDROXYL IONS
9.2.1. Disturbances from the terminator zone in unbuffered systems
Sometimes disturbances can be caused by the presence of a large amount of H+ at low
pH, especially in unbuffered systems. An unbuffered system for the separation of cationic
species in isotachophoresis can consist of a strong acid as a leading electrolyte (e.g.,
hydrochloric acid) and a terminator such as Tris. After the introduction of a sample and
25 3

254 PRACTICAL ASPECTS
the separation of the sample ionic species, a series of zones is obtained containing one
ionic species of the sample.
Two kinds of separation boundaries can be distinguished, viz., a separation boundary
between the leading ions (H') and the zone with ions of the sample (Ml), with the highest
mobility (we shall call this boundary the 'HI-MI boundary'), and a separation boundary
between two zones of sample cations (the 'MI-M,, boundary'). These two types of
separation boundaries have different characteristics and are discussed below.
9.2.1.1. HI-MI boundary
The zone of the cations M; will always contain H' ions, so that it is essentially a mixed
zone of M; cations and H+ ions. The H+ ions are more mobile than the Mi ions and will
therefore pass the HI-MI boundary. (In a buffered system they will be removed by the
buffer, according to the equilibrium state.)
Those H' ions which pass this boundary migrate into the leading electrolyte (hydro-
chloric acid) zone and create an H+ zone between the leading electrolyte zone and the
first sample zone, Mi. Evidently the extra H+ zone has the same H' concentration as the
leading electrolyte zone. In fact, this is a moving-boundary procedure. For the Mf zone,
the isotachophoretic condition is no longer valid. The speed of this zone is lower than
that of the leading electrolyte zone and the step heights will be smaller owing to the
effect of the H+ ions. If the H' concentration in the M; zone is low, the effect mentioned
above is very small and almost no disturbances can be expected. If the pH is low in the
M; zone, the original H' zone is elongated and the result is longer detection times and
smaller step heights.
Figs.9.la-9. Id show electropherograms for the situation with A13' as terminator
after 0.01 N hydrochloric acid as the leading electrolyte in methanol, as obtained in
practice. Fig.9.la shows the original situation, viz., the original leading ion zone H'(1)
and the terminator solution A13+(3), which also contains H+.
In Figs.9.lb-9.1 d, an increasing amount of H+(2) between the original solution of
H'(1) and the mixed zone A13+-H+ is obtained after a longer time of analysis. The
original concentration boundary, which will also be present, is neglected.
9.2.1.2. M,-M,, boundary
Now two mixed zones are close together, both consisting of a cation of the sample and
H' ions. The H' ions of the M;, zone will pass the boundary and will migrate into the M;
zone. Calculation of the pH relationship for the two zones (for hypothetical values),
including the mass balances for the H+ and OH- ions and the dissociation constant of
water, gives imaginary data, assuming a stationary state. Hence no stationary state will
exist.
If the pH is about 7, the influence on a stationary situation wil be small and almost
no disturbances can be expected. If the concentrations of H' or OH- are high, elution
phenomena will be dominant. If the pH of the second cation zone is low, the H+ concen-
tration will pass the boundary and a mixed zone of Mf and the H+ coming from the Mi,
zone is created.

DISTURBANCES CAUSED BY H+ AND OH 255
rt
Fig.9.1. (a)-(d): Simplified electropherograms of the leading electrolyte (HCl) and the terminator
(A13') with methanol as solvent, obtained after different times. (e)-(h): Simplified isotachopherograms
of the leading electrolyte (HCl) and the terminator (A13+), when a sample of K+ is introduced. Again
the experiment is carried out in methanol and various phases are shown. (i)-(1): Simplified isotacho-
pherograms of the leading electrolyte (KC1) and the terminator (A13+), when a sample of Na' is intro-
duced. Again the experiment is carried out in methanol and various phases are shown. T = increasing
temperature; t = time.
The step height in the electropherogram will decrease, which results in two zones of
the cation M;, viz., the original M; zone and the mixed zone of H+ and M;. After some
time, the H+ coming from the Mi, zone covers the whole Mi zone.
A situation as described was obtained using a leading electrolyte of 0.01 N hydrochloric
acid in methanol and a terminator of A13+. The sample K+ was introduced. Fig.9.le shows
the original situation. The first zone is the leading zone consisting of H'( l), the second
the original K' zone (2) and the last zone contains A13' plus H' ions (3).
In Fig.9.lf the H+ ions have partially penetrated the K'(2a) zone, whereas in Fig.9.lg
the H+ ions have nearly reached the leading zone. In Fig.9.lh an enlarged leading zone
(la) can be seen. Zone 2 fits the isotachophoretic condition, while zone 2a does not.
In Figs.9.li-9.11 a similar procedure is shown for a leading electrolyte of potassium
chloride (l), a sample containing Na'(2) and a terminator of A13+ (and H') (3). The H+
ions coming from the A13+ zone enter the Na' zone (2a) and finally reach the K' zone (la).
In order to check the influence of a low pH in the terminator quantitatively, experi-
mental values are compared with theoretical values, as calculated with the model as
described in Appendix A. As a terminator, mixtures of hydrochloric acid and potassium
chloride at different pH values are used with a leading electrolyte of 0.01 N hydrochloric

256 PRACTICAL ASPECTS
acid. The current was 70 PA. The ratios fL/tCi are taken as a check*.
In Fig.9.2, the relationship between the pH of the terminator and the rL/tU ratio is
given for theoretical (solid line) and experimental (individual points) values. Good agreement
is obtained, showing that a moving-boundary model provides a better description
than isotachophoresis.
If the influence of background electrolytes such as H' is too great, elution phenomena
will appear after a certain time. The zone boundaries become less and less sharp and after
a long time they release each other. The elution effects are often caused by electrode
reactions when the electrode compartments are not renewed in time; using C1- as a
counter ion in methanol (95%, wlw), the following reactions can be expected:
2C1- * Clz + 2e
+HZO HOCl
HC1+
+CH30H CHjOCl
In experiments with an unbuffered system, the H+ ions produced disturb the analyses.
As an example, the separation of caesium, sodium and lithium with the leading electrolyte
0.01 N hydrochloric acid and terminator cadmium chloride is shown in Fig.9.3a.
In Fig.9.3b, the separation of the same mixture in the same system after the terminator
Fig.9.2. Theoretical (line) and experimental (points) relationship between the pH of the terminator
solution and the relative detection times for solutions of KCl applied as terminator, in a moving-
boundary system.
*t~' Time of appearance of a sample zone if no ionic species with the same charge as the species
studied passes the separation boundary; tU = time of appearance of a sample zone if an ionic species
with the same charge as the species studied passes the separation boundary. If tL/ru = 1, an isotacho-
phoretic zone is obtained; if tL/tu< 1, a moving-boundary zone is obtained.

DISTURBANCES CAUSED BY H+ AND OH 25 I
T
1
5 4 3 2 1 5 4 3 2 1
Fig.9.3. Separation of a mixture of cations in an unbuffered electrolyte system. (a) With fresh termi-
nating electrolyte; (b) with old solution the pH of which is changed by the electrode reaction. 1 = H';
2 = Cst; 3 = Na+; 4 = Li+; 5 = Cd". T = increasing temperature; r = time. A thermometric detector was
used.
electrolyte has not been renewed for some time is shown. The terminator solution became
increasingly acidic and a flow of rmigrates through all zones towards the cathode
compartment.
From the phenomena described above, it can be concluded that it is not advisable to
work with unbuffered electrolyte systems, where regular renewal of the electrode compartments
is necessary. The use of terminator solutions at low pH is undesirable in cationic
separations.
A similar disturbance can be expected in anionic separations in unbuffered systems as
a terminator of high pH is used. A flow of OH- from the terminator zone penetrates all
7nn~r rniirino rlirtiirhanrpq RP Clirriirwrl nhnvp
9.2.2. Disturbances from the leading zone in unbuffered systems
In the previous section, disturbances caused by the presence of H' and OH- from the
terminator zone and penetrating all preceding zones have been described. Sometimes
disturbances can also be caused by H' and OH- from the leading zone penetrating all
proceeding zones. Although it is sometimes difficult to recognize whether the disturbances
originate from the terminator or from the leading zone, these disturbances have different
characteristics. In order to recognize the difference, two detectors have to be used, and
from the two signals obtained it can be concluded from which side the disturbance is
coming.
In this section we discuss the disturbances from the leading zone. In an anionic separa-
tion, this disturbance is due to a flow of H', whereas in a cationic separation it is caused
by a flow of OH-. Some examples of the latter situation are considered below.
If cations are separated in an unbuffered system, a leading electrolyte of, e.g., hydro-
chloric acid could be used. In such a case, hydrogen will be evolved at the cathode. OH-,
possibly formed in the cathode compartment and migrating in the direction of the anode,
will meet the H' ions of the leading electrolyte and will be neutralized because the

25 8 PRACTICAL ASPECTS
concentration of H' in the leading zone (normally about 0.01 N hydrochloric acid) is
rather high. No disturbances can be expected. However, if a leading electrolyte that con-
sists of a metal chloride, e.g., potassium chloride, is used, hardly any H+ is present in the
cathode compartment and it can therefore be expected that OH- will be formed in the
cathode compartment according to the equation
2Hz0 + 2e + Hz + 2 OH-
The OH- ions formed will migrate in the direction of the anode but will not meet H+ for
neutralization to occur (the leading electrolyte is potassium chloride) and a flow of OH-
through the whole capillary tube, and all zones, will be the result. Of course, this distur-
bance will be visible if the concentration of the OH- formed is fairly high and if the time
of analysis is sufficient for OH- to reach the detector.
For a leading electrolyte of lithium chloride, an increase in pH from 6.7 to 10.7 in the
cathode compartment could be measured after some experiments. In such a case, distur-
bances can be expected and renewal of the cathode compartment is necessary. An example
of such a disturbance is shown in Fig.9.4.
The isotachopherogram shows the disturbances of a flow of OH- from the cathode
compartment, moving in an opposite direction to the migration of the sample zones. The
* rim.
Fig.9.4. Disturbance due to the presence of OH- formed in the cathode compartment in cationic
separations. The leading electrolyte is KCl and the terminator is LiCl (unbuffered system). Thermo-
couple (2) is mounted closer to the cathode compartment and therefore records the disturbance by
OH- first (marked with an arrow). The amplification of the signal of thermocouple (1) is twice that
of thermocouple (2). T = increasing temperature.

DISTURBANCES CAUSED BY H+ AND OH- 25 9
leading electrolyte was potassium chloride and the terminator was lithium chloride. The
first thermocouple (close to the anode compartment) first detects the step height of
lithium, but after some time this step height decreases because the flow of OH- has
reached this thermocouple. A second thermocouple (close to the cathode compartment)
first detects a decreasing step height of the leading electrolyte because OH- reaches this
thermocouple first and after some time the step height of lithium appears. It is clear that
the disturbance is coming from the cathode compartment, in contrast with the disturbances
described in section 9.2.1. A similar disturbance can be expected in the separation of
anionic species if H+ is formed in the anode compartment.
Fig. 9.5 shows an example in which H' moves in the opposite direction to the anionic
zones. Creatinine (pK = 4.88) was used as the 'buffering' counter ion. The leading electro-
lyte was 0.01 Nhydrochloric acid (pro analysi grade), adjusted to pH 4 by the addition of
creatinine. Glutamic acid was used as the terminator. No sample was introduced. A UV
absorption detector (256 nm) and a conductivity detector (a.c. method), both described
in Chapter 6, were applied. The UV absorption detector was mounted closer to the
reservoir of the terminating electrolyte, so that the isotachophoretic zones reached this
detector first.
As is well known, the W absorption of creatinine is influenced by the pH if it is
approximately at its pK value, which is the reason why a disturbance by H' can be made
visible. The length of the narrow-bore tube between the anode and cathode compartments
e-- pH

260 PRACTICAL ASPECTS
is important and the ratio ZJZC is well chosen in order to show the effect as illustrated in
Fig.9.5. Because the buffer capacity of the creatinine is not sufficient, a front of H+ moves
towards the cathode and hence reaches the conductivity detector first. It increases the
conductivity of the leading electrolyte and is therefore recorded as a dip. If the H+ ions
reach the UV absorption detector, it isindicated by a change in absorption of the creatinine.
Shortly after this moving pH front has passed the UV detector, the glutamate reaches the
detector, already adjusted to the ‘new’ leading electrolyte (mixed zone). For this, the pH
of the glutamate is less than 4, which is abnormal if the conditions are chosen well (see
Fig.S.9). It will be clear that the conductivity as measured under these circumstances may
differ from experiment to experiment, because the disturbance is not exactly reproducible.
By changing the length of narrowbore tube between the anode compartment and the set
of detectors, another electropherogram could easily be obtained in which the glutamate
has passed the UV detector before the H‘zone.
9.2.3. Disturbances due to the presence of hydrogen and hydroxyl ions in buffered systems
In sections 9.2.1 and 9.2.2, disturbances due to the presence of H’ and OH- at high
and low pH for anionic and cationic species in unbuffered systems have been described.
Disturbances can also be expected sometimes in buffered systems, especially at low pH
for cationic species and at high pH for anionic species. The disturbances arise because at
these low and high pH values the H+ and OH- ions, respectively, are present in such large
amounts that they can carry the electric current and hence low step heights are obtained
that are almost identical for all ionic species. Under such conditions, the isotachophoretic
condition is no longer valid, the zones can release and a type of zone electrophoresis is the
result. Some examples are given below for cationic species.
In section 4.3.3, we mentioned that sometimes no real values for pH, could be
obtained because the isotachophoretic conditions were lost at low pH in cationic and at
high pH in anionic separations. This can be caused because the pH increases in anionic
separations and decreases in cationic separations until values at which ‘water’ acts as a
background electrolyte. This phenomenon was observed when analyzing nucleic bases,
which have low mobilities and low pK values. The step heights of some substances have
been determined with a leading electrolyte consisting of a mixture of potassium acetate
and acetic acid at different pH values (Table 9.1).
In Table 9.1 it can be seen that at low pH of the leading electrolyte (in the sample
zones the pH is even lower) all substances have the same step heights; some substances
have double peaks. At higher pH, the substances have different step heights but the
differences are too small to separate all of them together. Moreover, the step heights are,
in fact, step heights of mixed zones of the substances obtained at a high concentration of
H+, as the pH in the sample zone can be decreased substantially. It can be concluded that
substances with low pK values and low mobilities cannot be separated at low pH.
Some experiments were also carried out with amino acids, and similar results have
*I,= Length of narrow-bore tube between the point of injection of the sample and the detector;
2, = length of narrow-bore tube between the semipermeable membrane in the counter electrode com-
partment and the detector.

DISTURBANCES CAUSED BY H+ AND OH 26 1
TABLE 9.1
STEP HEIGHTS (mm) OF SOME CATIONS WITH A LEADING ELECTROLYTE OF 0.01 N
POTASSIUM ACETATE AND ACETIC ACID AT DLFFERENT pH VALUES
Cations 4.0 4.3 4.5 4.91
Adenine
Adenosine
-
12
30
12+37
69 + 90
85 + 120
~
156
Guanine 11.5 13 85 -
Uridine 12 12 77 119
Cytidine 12 12 t 40 103 145
Guanosine 12 12 80 120
TABLE 9.2
CALCULATED pH VALUES OF THE SAMPLE ZONES FOR CATIONS WITH THE LEADING
ELECTROLYTE (A) POTASSIUM FORMATE (0.01 m-FORMIC ACID AND (B) SODIUM
FORMATE (0.01 N)-FORMIC ACID AT DIFFERENT pH VALUES
Leading Cations PHL
electrolyte Mobility 3.25 3.5 3.75 4.0 4.1 4.2 4.5 5.0
A 50
30
10
30
30
30
30
30
B 38.8
30
20
(10-5cm2/V - sec) pK
14
14
14
6
5
4
3
2
14
8
8
- 3.34 3.61 3.87 3.98 4.08 4.38 4.88
- - - 3.55 3.68 3.80 4.12 4.65
- - - 3.54 3.67 3.79 4.10 4.49
-
-
-
-
-
-
3.48
-
3.60
-
3.70
-
3.93
3.38
4.13
3.51
3.068 3.368 3.637 3.896
- - 3.458 3.749
been obtained. At low pH, the amino acids had the same step heights. Van Hout [ 11 also
found the same step heights for an electrolyte system at pH 5 (see also Chapter 13).
Calculated pH values in the zones for some cationic species are shown in Table 9.2; where
no pH is given, no real zero points were present.
From Table 9.2, it can be seen that monovalent substances with low pK values (2 or 3)
cannot be separated by isotachophoresis, whereas completely ionized cationic species can
be analyzed even at low pH. In order to see what happens when separating these cations,
experiments were carried out with Tris' as a terminator and Li' as a sample ion with
leading electrolytes consisting of 0.01 N potassium formate-formic acid and 0.01 N
sodium formate-formic acid at different pH values. In Table 9.2, Li' (mobility 38.8 - lo-'
cmZ/V - sec) always shows real zero points with the leading electrolyte sodium formate-
formic acid, whereas Tris' (mobility about 30 * lo-' cmZ/V * sec) does not show real zero
points at pH 3.25 and 3.5.

262 PRACTICAL ASPECTS
lim.
Fig.9.6. Electropherogram for Li' between Tris' (terminator) and Na'. The leading electrolyte was
prepared by adjusting NaOH (0.01 N ) to the pH values shown by addition of formic acid. The isotacho-
pherograms show what happens if the counter ion does not buffer sufficiently. T = Increasing tempera-
ture. A thermometric detector was used.

DISTURBANCES DUE TO CO, 263
Electropherograms are given for these systems in Fig.9.6. Li' at these pH values has
normal step heights,but Tris+ at pH 3.5 shows a retardation and at pH 3.25 a large and a
lower step height are present between the Li' and Tris' zones (zone electrophoresis).
Note that the traces at pH 4.25,4.0 and 3.75 are nearly identical, but at pH 3.5 and 3.25
the step heights of Li' are about the same whereas those of Tris' decrease owing to the
presence of H+. Tris'already shows no real zero points at pH 3.75 in the system potassium
formate-formic acid, and indeed at this pH the isotachopherogram (see Fig.9.7) shows
a large and a low step height between Li' and Tris'. Similar results can be obtained at
high pH in anionic separations.
9.3. DISTURBANCES DUE TO THE PRESENCE OF CARBON DIOXIDE*
In section 9.2, some disturbances due to the presence of H+ and OH-, migrating through
all zones according to the moving-boundary principle, have been described. A flow of
HCO; ions can also cause such a disturbance, because of the presence of carbon dioxide
in air and solvents (even if all carbon dioxide is removed from the solution, it can still
diffuse through the capillary walls into the solution). Carbon dioxide can react in an
aqueous solution as follows:
COZ + HzO =+ HzCO3
HzCO3 + H,O =+ H30+ + HCO;
K, = 2.6 - 10-~
Kz = 1.72 *
(9.1)
(9.2)
Fig.9.7. Electropherogram of Li' between Tris' (terminator) and K'. The leading electrolyte was
prepared by adjusting KOH (0.01 N) to pH 3.75 by addition of formic acid. T = Increasing temperature.
A thermometric detector was used.
*See also Chapter 13.

264 PRACTICAL ASPECTS
The overall K as generally used is given by
K,=KlK2=4.47- (9.3)
According to the last equilibrium constant, carbonic acid will be characteristic of weak
acids. It must be noted, however, that although the reaction between carbonic acid and
water is instantaneous, the reaction between carbon dioxide and water is slow, which
accounts for the well known ‘fading’ of the colour of phenolphthalein in the titration of
an aqueous carbon dioxide solution.
At higher pH, carbon dioxide can also react with OH- :
C02 + OH- + HCO;
This last reaction is faster than that with water. Because carbon dioxide is always present
in water, especially at high pH, disturbances can be expected owing to the presence of
HCO,. The ionic mobility of HCO; ions is about 44 - cm2/V - sec at 25°C. Especially
for the separation of amino acids and proteins, which are normally carried out at high pH,
disturbances due to this effect can be troublesome. At low pH, the effective mobility of
HCOJ ions is very low and their presence is not troublesome. The step heights for
carbonate zones generally have a typical form and are not as sharp as those of other
substances owing to the slow equilibrium adjustment of eqn.9.1. In fact, carbonic acid
does not migrate as a single homogeneous zone, but as a continuous series of zones of
slightly different compositions.
9.4. ENFORCED ISOTACHOPHORESIS
(9.4)
The pH of the zones depends strongly on the pK values of the buffer ions and the
sample ions. When separating strong acids the pH is nearly equal to the pH,, but for weak
acids large pH shifts can occur. Problems can be expected when the pH of the zones does
not increase regularly.
When the pH of a zone is lower than the pH of the preceding zone in anionic separa-
tions, the effective mobility of the ionic species of the preceding zone will be smaller in
that zone, ie., if some of the ions are left behind* they cannot reach their own zone and
the self-correction of the sharpness of the front is lost. In course of time the zone length
will decrease and mixed zones are the result.
An example of this phenomenon [2] is the HCO; zone before a zone of cacodylic
acid. The pH in the cacodylic acid zone is lower than the pH of the HCO; zone, and the
effective mobility of HCO; is higher in the pure HCOJ zone than in the cacodylic acid
zone. The HCO; zone vanishes.
Further, a pH can be chosen such that the ionic species in a particular zone has an
effective mobility higher than that of the leading ion, but a smaller effective mobility in
the leading electrolyte zone. The ionic species cannot pass the boundary with the leading
electrolyte (pH shift) but has a larger effective mobility and therefore a smaller step
height. Ths can be called an enforced isotachophoretic system, because the zones do not
occur in order of decreasing effective mobility.
*The pH is lower, so their extent of dissociation and hence their effective mobility decreases.

ENFORCED ISOTACHOPHORESIS 265
t T TI
- -
t f
a b
I
I
I
3 2 - 1
Fig.9.8. Electropherogram of a hydrogen carbonate zone between the leading electrolyte potassium
acetate (0.01 “-acetic acid (pH 4.75) and cacodylate. (a) Analysis by a second thermocouple about
15 min after (b). T= Increasing temperature; t = time. 1 = Acetate; 2 = hydrogen carbonate; 3 = caco-
dylate. The sample was introduced via a four-way sample tap (Fig.7.1).
An example of such a system is a leading electrolyte consisting of a mixture of 0.01 N
potassium acetate and acetic acid at pH 4.75 and a sample of a hydrogen carbonate. The
effective mobility of HCO; is higher than that of the acetic acid, but the pH of the leading
electrolyte is such that the HCO, cannot pass that boundary.
In Fig.9.8, electropherograms are given for a system showing both effects at two
different times of detection. It can be seen that the step heights of the HCO; zones are
smaller than the step heights of the leading zones, where its zone length decreases with
time, because of the lower pH of the cacodylate zone.
9.4.1. Disc electrophoresis
In disc electrophoresis [3], the first stage of the separation consists of an isotacho-
phoretic system whereby the sample introduced is concentrated in small zones. Generally
the leading electrolyte consists of an acid (e.g., acetic acid) that buffkrs at a low pH
(4.75) and a buffering counter ionic species (e.g., Tris) that does not act as a buffer in
the leading zone (pK = 8). The counter ions buffer only in the following zones and they
create high pH values where the proteins have a sufficient mobility. In the literature [4-71,
different treatments for the calculation of the pH in the proteins zones have been con-
sidered and from comparisons with weak acids, pH values of 8-9 are assumed in the zones.
We calculated for some weak acids (hypothetical pK values) the pH values in the sample
zones for a system as described above at a pH, of 4.75; the pH values in the zones as a

266
9
I I I 1
10 20 30 40
PRACTICAL ASPECTS
d
M59m.ff. *
Fig.9.9. Relationship between the pH in the sample zones and the effective mobilities of the sample
ionic species for different pK values of the ionic species. The leading electrolyte was 0.01 N potassium
acetate adjusted to pH 4.75 by addition of acetic acid. Tris (pK = 8) was used as the counter ion. pK
values of ionic species: a = 10; b = 9; c = 8; d = 7.

WATER AS TERMINATOR 267
function of the effective mobilities are shown in Fig.9.9 for several pK values of the ionic
species. It can be seen that the pHzone depends strongly on the pK values and on the
mobilities of the ionic species. Especially for mobile ionic species, large pH shifts can be
obtained, while for low mobilities the shift in pH is not as high as is assumed in the
literature. For some acids with charges of -10 to -100 and pK values of 7-8, pH values
were calculated to be 5.72-6.4. If it is permissible to use the model for the calculation of
the pH values, then the proteins do not move in an isotachophoretic way, but are in fact
pushed along by the terminator solutions, where a high pH is present. This can also be
called enforced isotachophoresis.
9.5. WATER AS TERMINATOR
As already described in section 9.2.3, disturbances can be caused by the presence of
large amounts of H+ and OH- at low and high pH, even in buffered systems. These ions
can carry the electric current, act as a background electrolyte, the isotachophoretic con-
dition is no longer valid and zone electrophotetic phenomena can be the result.
As H’ and OH- can carry the electric current and as they are present in all zones, the
question arises of whether it is possible to use the ‘water’ as a terminator solution, with-
out the presence of another substance. The advantage is clear. A suitable terminator is
sometimes difficult to find owing to the requirements of mobility and purity, but if
‘water’ could be used these problems would be solved. Of course, it only can be used
between certain pH values. If, for instance, with cationic species the pH is rather high,
then ‘water’ cannot be used as its ‘effective mobility’ would be too low. At too low a pH,
disturbances can be expected, as shown in section 9.2.3. In order to show the possibility
and to determine the pH values if ‘water’ were used as a terminator, some experiments
were carried out. In Table 9.3, step heights are given for the terminating zone ‘water’ on
top of those of the leading zone. The step heights of Li’ and/or Tris’ are also given for
comparison.
TABLE 9.3
SOME STEP HEIGHTS FOR ‘WATER’ AS A TERMINATOR
Leading electrolyte: (a) 0.01 N potassium formate-formic acid; (b) 0.01 N potassium acetate-acetic
acid.
Leading PH Step height (in: lo-’)
electrolyte
Water zone Lit zone Tris’ zone
a 4.1 16 20.5 35
4.2 16 21 35
4.25 20 ~
31
b 4.25 40 -
35
4.5
4.15
5 .o
64
I5
81
23
21
-
39
-
-

268 PRACTICAL ASPECTS
It can be seen from Table 9.3 that the step heights of the Li’ and Tris’ zones are
nearly constant (at this pH they are both strong ions), whereas the step heights of the
terminating zone ‘water’ increase considerably with increasing pH. At pH values below
about 4.25 (depending on the type of leading electrolyte used), ‘water’ cannot be used as
a terminator because disturbances will occur. At these pH values, the step height of the
pure ‘water’ zone is lower than that of, e.g., Tris’ and zone electrophoretic phenomena
can be expected. Also, above a pH of about 5-5.5, ‘water’ cannot be used as a terminator
because its effective mobility is too low. Therefore water can act as a terminator in the
approximate pH range 4-5.2. In a similar way, water can act as a terminator in the
approximate pH range 9-10 for anionic separations.
Van Hout [ 11 used water as a terminator* in the separation of, e.g., amino acids at pH
9.2. An example is shown in Fig.9.10 for the separation of glutamic acid, taunne, serine,
&cine, tryptophan and sarcosine. The terminator was water and the leading electrolyte
was 0.01 N hydrochloric acid-ethanolamine at pH 9.2. Barium hydroxide was added to
the terminator ‘water’ in order to suppress the amount of hydrogen carbonate present in
the solution and to raise the pH in the terminating reservoir. In Fig.9.10, however, a small
zone of hydrogen carbonate can be seen (see also Chapter 13).
9.6. PURIFICATION OF THE TERMINATOR
As already described in section 5.4, terminating electrolytes have to be very pure,
because if small amounts of impurities are present in the terminating zone (a large voltage
gradient), with higher mobilities than that of the terminating ions, they will be pushed
forwards through all preceding zones until they reach a zone boundary in accordance
with their effective mobilities. This procedure is a type of moving-boundary procedure
and can cause disturbances. In addition to chemical methods for purifying terminators,
such as recrystallization and distillation, an isotachophoretic method can also be used.
TABLE 9.4
SOME STEP HEIGHTS MEASURED WITH TWO DIFFERENT THERMOCOUPLES
The metals were measured in the operational system WKCAC: and the anionic species in the system
MTris/HCI*i(A). The step heights are given in millimetres, and relate to 0 PA.
Species
Formate
Acetate
Caprylate
Stearate
Cacodyla te
Salicylate
Butyrate
Palmitate
Thermocouple 1
162
186
223
26 0
298
172
202
253
Thermocouple 2
170
197
234
27 6
312
183
216
272
Species
+ Li
Tris‘
Ni*+
cu2+
Pb’+
BaZ*
Na’
(CJ N
Cd’+
*WKCAC is listed in Table 11.4 and MTris/HCI in Table 12.4.
Thermocouple 1
233
305
201.5
22 1
24 3
169
184
270
223
Thermocouple 2
*Later experiments show that often a terminating ion can better be added to the water (section 13.1.3).
24 I
321
214
24 1
262
180
195
285
236

PURIFICATION OF THE TERMINATOR
7
4
9 /
t
Fig.9.10. Isotachopherogram of the separation of a series of anions at high pH with ‘water’ as the
terminating electrolyte. 1 = Chloride; 2 = hydrogen carbonate; 3 = glutamate; 4 = taurine; 5 = serine;
6 = glycine; 7 = trypthophan; 8 = sarcosine; 9 = OH-. A thermometric detector was used. T = Increas-
ing temperature; r = time.
For a certain time, the terminator is allowed to migrate after the leading electrolyte, with
no sample ionic species present. During this period, the impurities will migrate forwards,
out of the terminator solution. The terminating electrolyte will remain pure in a series of
separations provided that it is not renewed.
269

210 PRACTICAL ASPECTS
9.7. CONVERSION OF DATA MEASURED WITH DIFFERENT DETECTORS
When different apparatus are used at different times, it is sometimes difficult to compare
the results. For thermocouples and conductivity detectors, however, a simple method can
be used to interconvert the results. We can utilize the situation that the signals obtained
from one detector, when represented graphically as a function of the signals of another,
give a nearly straight line. After constructing a calibration graph, all results can then be
converted by means of the conversion graph. This graph is independent of the different
operational systems as it simply represents a conversion of measured temperatures and
conductivities, and so it can be used for all systems.
-
D
Ir,
150 200 250 3&0
t.c.2
Fig.9.11. Conversion graph for two different thermocouples (t.c.1 and t.c.2). * = Anionic species
given in Table 9.4; O= cationic species given in Table 9.4.

REFERENCES 271
AS an example, the conversion graph for two different thermocouples is shown in
Fig.9.11. The line was constructed by measuring the step heights for several substances in
different operational systems with two thermocouples in two different pieces of apparatus.
It can be seen that a nearly straight line is obtained. All of the step heights used for con-
structing this conversion graph are listed in Table 9.4.
REFERENCES
1
2
3
4
5
6
7
P. van Hout, Graduation Rep., University of Technology, Eindhoven, 1972.
R. J. Routs, Thesis, University of Technology, Eindhoven, 1971.
L. Omstein, Ann. N. Y. Acud. Sci., 121 (1964) 321.
R. A. Alberty, J. Amer. Chem. SOC., 72 (1950) 2361.
E. B. Dismukes and R. A. Alberty, J. Amer. Chem. SOC., 76 (1954) 191.
J. C. Nicho1,J. Amer. Chem. Soc., 72 (1950) 2367.
J. C. Nichol, F. B. Dismukes and R. A. Alberty, J. Amer. Chem. SOC., 80 (1958) 2610.

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Chapter 10
Quantitative aspects
SUMMARY
A method is described for determining a calibration constant that simplifies quan-
titative determinations considerably, as a calibration graph for each ionic species need
not be constructed. The calibration constant is a constant for each operational system,
assuming that the electric current is precisely constant between the analyses considered.
The reproducibility of both the equipment with a thermometric detector (section
7.4.2) and the equipment with a high-resolution detector (section 7.4.4) has been
determined experimentally. The use of a calibration constant has been verified
experimentally.
10.1. INTRODUCTION
In Chapters 1-5, the theory of isotachophoresis was described and quantitative aspects
were considered. Experiments have been carried out to check both the accuracy and the
reproducibility of this analytical method, using both a thermometric and a conductivity
detector (a.c. method) as their resolutions are different [ 1, 21 . For the experiments in
which a thermometric detector was used, the equipment with the four-way tap
(section 7.4.2) and the equipment with the injection block (section 7.4.3) were applied
in order to compare the differences between the four-way tap and sampling via a micro-
syringe. For the experiments with the conductivity detector, the equipment described in
section 7.4.4 was applied. In the equipment, the six-way valve was also used as the
injection block, mounted both as a single unit and in combination. No differences were
found in the isotachopherograms if they were both mounted in the equipment so that
there is therefore no mutual effect.
In the thermometric experiments, the reproducibility was found to be better if the
sampling was performed via a micro-syringe instead of with a four-way tap. Most of the
differences found must be ascribed to the better construction of the equipment described
in section 7.4.3. Moreover, the use of an injection block facilitates work with different
concentrations. The six-way valve was found to be excellent, especially when different
workers inject an identical sample. The reproducibility was found to be better than 99.5%,
which means that the differences found were less than 0.5%. The six-way valve was found
to be better than sampling via a micro-syringe and the injection block (section 7.2.4).
It should be noted that the concentration of the terminating electrolyte must be prepared
more accurately if the injection is made via a micro-syringe. The order of arrangement of
the UV absorption detector and the conductivity probe did not influence the quantitative
results for the ions tested.
Because the thermometric detector needs a minimum zone length of approximately
100 sec for a full qualitative and quantitative analysis, relatively large samples must be
213

214 QUANTITATIVE ASPECTS
introduced compared with experiments in which a high-resolution detector is applied.
The greatest linearity and reproducibility were found, however, when small amounts of
sample were introduced in low concentrations, resulting in small zone lengths. For the
experiments in which small amounts of sample are introduced in the narrow-bore tube,
an instrument was applied for automatic recording of the zone lengths, which enabled
small time intervals to be used between successive peaks, as found in the differential
traces of the linear conductivity trace (0.3 sec). We did not follow the principles of
chromatography, where internal standards are sometimes applied, because the accuracy
was found to be high enough without such a procedure. Moreover, the variations in
sample size, if any, can be compensated for by using the sample taps.
It will be recalled that if a UV detector is available for ionic material that has W
absorption, the so-called mixed-zone method can be applied. The sample to be analyzed
is diluted with a non-W-absorbing ion with an effective mobility that is the same, in the
operational system chosen, as that of the W-absorbing ion of interest. The step height
in the trace of the W absorption detector will give the necessary quantitative informa-
tion, after calibration.
10.2. THEORETICAL
For the quantitative determination of ionic species by isotachophoresis, calibration
graphs can be obtained experimentally. Theoretically, there should be a linear relation-
shp between the length of the zone of a specific ionic species and the amount of the
ionic species introduced. Calibration graphs for all ionic species present in a sample that
are required to be separated must be measured, however.
The introduction of a calibration constant, characteristic for all ionic species in the
chosen system, simplifies the quantitative determinations considerably. The calibration
constant can be determined as follows. The amount of an ionic species introduced into the
apparatus is given by
Q= y.c (10.1)
where Q (mole) is the total amount of the ionic species, Vj (ml) is the volume of the
sample injected and c (mole/ml) is the concentration of a particular ionic species present
in the sample. The amount of a particular ionic species in the narrow-bore tube will
therefore be
Q= Ac*L (10.2)
where A (cm2) is the cross-sectional area of the narrow-bore tube, c* (mole/ml) is the
actual concentration of the ionic species in the zone that is adapted to the leading
electrolyte and L (cm) is the zone length of a particular ionic species.
Combining eqns. 10.1 and 10.2, we obtain
y. C
A =- (1 0.3)
c*L
or

THERMOMETRIC MEASUREMENTS 215
vj c
Kcl= c*L"
(10.4)
where Kcd is the calibration constant and L* (sec) is the zone length as detected between
two successive signals obtained from the equipment if a zone boundary passes the detector. By
means of a computer program (Chapter 4), the actual concentrations of the ionic species
in the zone can be calculated in a well defined operational system. This means that once
the calibration constant is known, the concentration of all ionic species in a sample can
be calculated from the zone length. Not all calibration graphs for each ionic species have
to be measured separately.
In order to check the reproducibility and to determine simultaneously the calibration
constant, K,,, quantitative experiments were carried out in different electrolyte systems
with water as the solvent. The calibration constant is not a constant for all systems. Some
factors, such as variations in the concentration of the leading electrolyte, temperature
and changes in the current density, result in different potential gradients and hence affect
the migration speed in the system. This effect produces different zone lengths for the
same amounts of ionic species in different systems.
10.3. THERMOMETRIC MEASUREMENTS
10.3.1. Reproducibility
In order to estimate the reproducibility, the zone length of formic acid (injected volume
3 p1 of a 0.05 Nsolution) was measured ten times in different experiments. The leading
electrolyte was 0.01 Nhistidine and 0.01 N histidine hydrochloride. The pH of the
solution was thus 6.02. The current was stabilized at 70 FA. The terminator was 0.005N
glutamic acid (adjusted to pH 6 by addition of Tris).
The average zone length found was I* = 31 1 sec from ten experiments and the
average deviation was 4 sec. Owing to the asymmetry of the step response, the zone length
depends on the terminator used. Some experiments were therefore carried out with
the same sample but with a different terminator (acetic acid). The average zone length
then found was i* = 307 sec from five experiments and the average deviation was
3 sec. No significant differences were found when the values found in the experiments
with glutamic acid and acetic acid were compared. Glutamic acid was therefore used
as the terminating ion in the other experiments.
Later experiments showed that one has to be careful if glutamic acid is used as a
terminating ion because it is a strong acid and hence mixed zones can easily be formed,
which cannot be separated further.
10.3.2. Calibration constant
The calibration constant was determined from experiments carried out with histidine
(0.01 N)/histidine hydrochloride (0.01 N) (pH 6.02) as the leading electrolyte. The
current was stabilized at 70pA. All zone lengths are given in Table 10.1. The third

276 QUANTITATIVE ASPECTS
TABLE 10.1
CALIBRATION CONSTANTS, K,,l,AND ZONE LENGTHS WITH HISTIDINE/HISTIDINE
HYDROCHLORIDE AS THE LEADING ELECTROLYTE
The experimental values were measured with a thermometric detector.
Ionic species Concen- Concen- Injected Detected Calibra- Deviation from
tration tration volume zone tion average KCa1
in the in the 011) length constant
sample zone (set) (~,,,.10~) AKC,1.1O6 %
(mole/l) (rnolell)
Succinic acid 0.01 0.0051 4 163 0.481 2 -1.73 -3.5
Acetic acid
Adipic acid
Formic acid
Iodic acid
Lactic acid
p-Chloro-
propionic acid
Succinic acid
Sulphamic acid
Tartaric acid
Acetic acid
Adipic acid
Iodic acid
Maleic acid
Tartaric acid
Acetic acid
Formic acid
0.05
0.025
0.05
0.05
0.031
0.05
0.01
0.05
0.025
0.05
0.025
0.05
0.05
0.025
0.05
0.05
0.0085
0.0046
0.0093
0.0085
0.0081
0.0081
0.0051
0.0090
0.0048
0.0085
0.0046
0.0085
0.0057
0.004 8
0.0085
0.0093
1
1
358.5
335
311
350
222
370
119
335
3 20
234
223
231
349
21 3
120
105
0.4923
0.4867
0.5186
0.5042
0.5172
0.5005
0.4943
0.4975
0.4883
0.5028
0.4874
0.5093
0.5027
0.4891
0.4902
0.5120
-0.62
-1.18
2.01
0.57
1.87
0.20
-0.42
-0.10
- 1.02
0.43
-1.11
1.08
0.42
-0.94
-0.83
1.35
-1.2
-2.4
4.0
1.1
3.7
0.4
-0.8
-0.2
-2.0
0.9
-2.2
2.2
0.8
-1.9
Average 0.4985 0.93 1.9
-1.7
2.7
column in Table 10.1 shows the actual concentrations of the ionic species, calculated
with the computer program given in Chapter 4. The last two columns show the deviations
from the average K values. Reasonable values were obtained, which can be improved
al
if more accurate vafues are available for the ionic mobilities.
A similar determination of the calibration constant was carried out with imidazole/
imidazole hydrochloride (0.01 N) at pH 7.05 as the leading electrolyte (with water as the
solvent). The current was stabilized at 70 HA. All zones measured are given in Table 10.2.
The last two columns show the deviations from the average K,, value. Reasonable
constancy of the calibration constant was obtained. It should be remembered that the
influence of the activity coefficients is neglected in the calculations of the actual concen-
trations of the ionic species.
From our experiments, we can state that a minimum detectable zone length in the
PTFE narrow-bore tube (I.D. 0.45 mm, O.D. 0.7 mm) is about 5 mm, using a thermo-

THERMOMETRIC MEASUREMENTS
TABLE 10.2
CALIBRATION CONSTANTS, Kcal, AND ZONE LENGTHS WITH IMIDAZOLE/IMIDAZOLE
HYDROCHLORIDE AS THE LEADING ELECTROLYTE
The experimental values were measured with a thermometric detector.
lonic species Concen- Concen- lnjected Detected Calibra- Deviation from
tration tration volume zone tion average Kca,
in the
sample
in the
zone
(bl) length
(set)
constant
w~~,. to4) A K ~ 106 ~ ~ % -
(mole/l) (mole/l)
Ace tic acid
Adipic acid
Formic acid
Hydrofluoric acid
Iodic acid
Lac tic acid
Maleic acid
Tartaric acid
Acetic acid
Formic acid
Maleic acid
Acetic acid
Formic acid
0.05
0.025
0.05
0.05
0.05
0.0343
0.05
0.025
0.05
0.05
0.05
0.05
0.05
0.0075
0.0042
0.0087
0.0087
0.0074
0.0069
0.0046
0.0042
0.0075
0.0087
0.0046
0.0075
0.0087
1
1
467
407
398
409
465
340
735
416
308
255
491
154
129
0.4283
0.4388
0.4327
0.4215
0.4364
0.4386
0.4437
0.4290
0.4329
0.4508
0.4428
0.4329
0.4455
-0.82
0.23
-0.38
-1.50
-0.01
0.21
0.72
-0.75
-0.36
1.43
0.63
-0.36
0.90
277
-1.9
0.5
-0.8
-3.4
0.0
0.5
1.6
-1.7
-0.8
3.3
1.4
Average 0.4365 0.64 1.5
metric detector'. This value can vary, depending on the heat production in the adjacent
zones, the electric current, the type of solvent used and the cross-section of the narrowbore
tube. The concentration of an anionic species in the narrow-bore tube is about
0.01 g-equiv./l under the conditions used and the cross-section of the narrow-bore tube
is about 1.6 - 1 0-3 cmz . This means the minimum amount of an ionic species that can be
detected is about 8 *
g-equiv. If the volume of the sample injected is 3 pl, the
minimum concentration in the sample that can be detected is about 2.7 * g-equiv./l.
In order to illustrate this, the separation of a mixture of 0.005Noxalate, 0.01 N
formate, 0.01 N acetate and 0.01 5NP-chloropropionate in the system described above
at pH 6.02 was carried out. The results are shown in Fig.lO.1. Traces (a), (b) and (c)
correspond to injected volumes of 1,2 and 31.11, respectively. The amounts detected were
5 * and 1.5 - lo-' g-equiv., respectively, for the different anions, when
1 1.11 was injected. It can be stated that a complete separation of the mixture is obtained,
both qualitatively and quantitatively, in traces (b) and (c). All quantitative information
can be deduced from trace (a), for it should be remembered that for quantitative analyses
the transition of zone boundaries is required, once the sequence is known. Trace (a) shows
-0.8
2.1
*This means that a difference of about 100 sec is needed between two successive peaks, the differential
trace of the linear temperature response.

II
I
7
1
7
L
h
I
c- time
QUANTITATIVE ASPECTS
Fig.lO.1. Isotachopherogram of the separation of some anions in the operational system at pH 6 (see
Table 12.1). Volumes injected: (a) 1, (b) 2 and (c) 3 p1 of a solution of 0.005N oxalate, 0.01 N
formate, 0.01 Nacetate and 0.015 NP-chloropropionate. Detection was carried out with a thermo-
metric detector.
a complete separation of a mixture of anions. It should be remembered that the smaller
the amount of ionic species introduced into the separation chamber, the shorter is the
time of analysis required for a complete separation. If the isotachopherogram in Fig. 10.1 a
should have been a chromatogram, it should have represented an incomplete separation.
Of course, normally an isotachopherogram as shown in Fig.lO.la would not have any value.
The detection limit can be decreased by using a leading electrolyte with a lower
concentration. If the concentration of the leading electrolyte is decreased to
the minimum detectable amount of an ionic species will theoretically decrease by a
N,

CONDUCTIMETRIC MEASUREMENTS
factor of 10. Other factors, e.g. , electroendosmosis and temperature profiles, place
limits on the dilution of the leading electrolyte. Moreover, the pH range in which the
analysis can be carried out will be considerably smaller if dilute solutions are applied.
Elution effects due to OH and H’ soon appear. Also impurities, especially those
present in the terminating electrolyte, may play a dominant role. For correct sampling,
especially with a micro-syringe, the concentration of the terminating electrolyte must
be adjusted to the low concentration of the leading electrolyte. More attention must
be paid to the pH*.
The detection limit can be decreased by injecting a larger sample, and a sample tap is
particularly suitable for ths. Of course the availability of a counter flow of electrolyte
can also decrease the detection limit.
The time required for the analyses depends on the length of the narrow-bore tube
needed for separation, the electric current used, the type of leading and counter io;r;s
present, the pH, the differences in effective mobility of the most difficult pair of ions
that need to be separated, the volume injected, the concentrations of the various sample
ions, etc. The time required for the analyses discussed above was cu. 45-60 min.
10.4. CONDUCTIMETRIC MEASUREMENTS
10.4.1. Reproducibility
Experiments are often carried out at low concentration regions in which it is impossible
to use a thermometric detector. Experiments with thermometric detectors have shown,
however, that the greatest reproducibility and linearity are obtained if low concentra-
tions and small amounts of sample are used.
For the experiments with the conductivity detector, the improved injection block
(section 7.2.4) can be used and the injection of the sample made in the leading electrolyte,
the terminating electrolyte or at the boundary between them. The effects of the
terminators applied can be studied more precisely. The experiments showed that the
best terminator is a component with a suitable pK value and that its concentration must
be made as similar as possible to the adjusted concentration inside the narrow-bore tube.
Also, the pH must be adjusted to an appropriate value. However, all of these precautions
with respect to the terminating electrolyte need not be taken in all experiments.
Not only the injection block, but also the use of a high-resolution detector improves
the reproducibility. The experiments here were also carried out in the operational system
at pH 6 (Table 12.1.).
Again formic acid was injected ten times with glutamic acid and acetic acid as the
terminators. The terminator solution was carefully prepared, the pH being adjusted to
that of the leading electrolyte by addition of recrystallized Tris. The average zone length
was found to be L* = 65 sec for both series of experiments, with an average deviation of
0.4 sec.
*An isotachopherogram of a separation at a low concentration of the leading electrolyte is shown in
Chapter 6, where the thermometric detector is discussed (section 6.2.4, Fig.6.6).
219

280 QUANTITATIVE ASPECTS
10.4.2. Calibration constant
In Tables 10.3-10.5, the results are shown of analyses with nitrate, chlorate and
acetate. In addition to the pure components, mixtures of them were also injected. The
concentration was chosen such that a 0.5-pl volume could be injected each time. The
current was stabilized at 70 MA. Glutamic acid was applied as the terminator, at a
concentration of 0.01 N. The average results of two experiments are given.
The actual concentrations of the ionic species in the zones, in the steady state,
moving behind the leading electrolyte were again calculated with the computer program
given in Chapter 4. As expected, a linear relationship between zone length and amount of
component injected was found at low concentrations.
For the calculation of the calibration constant, an average of the values listed in
TABLE 10.3
ZONE LENGTHS FOUND EXPERIMENTALLY AT A CONCENTRATION OF 0.0125NIN THE
SYSTEM HISTIDINE/HISTIDINE HYDROCHLORIDE WITH A CONDUCTIVITY DETECTOR
Ion I. * K,,, .1 o4
NO,- 13.5 - - 14.1 - 13.8 14.1 0.448
Cl0,- - 13.5 - 13.8 14.1 - 13.8 0.461
CH, COO- - - 15.6 - 15.9 16.2 16.2 0.462
TABLE 10.4
ZONE LENGTHS FOUND EXPERIMENTALLY AT A CONCENTRATION OF 0.025NIN THE
SYSTEM HISTIDINE/HISTIDINE HYDROCHLORIDE WITH A CONDUCTIVITY DETECTOR
Ion L* K,d’ lo4
NO,- 21.9 - - 28.2 - 21.9 28.2 0.448
(30,- - 28.5 - 28.2 21.9 - 28.2 0.451
CH, COO- - - 32.1 - 31.8 31.8 31.8 0.459
TABLE 10.5
ZONE LENGTHS FOUND EXPERIMENTALLY AT A CONCENTRATION OF 0.05 N IN THE
SYSTEM HISTIDINE/HISTIDINE HYDROCHLORIDE WITH A CONDUCTIVITY DETECTOR
Ion L* K,~, 104
NO,- 51.3 - - 56.1 - 56.1 51.3 0.443
Cl0; - 56.1 - 51.3 51.6 - 51.9 0.452
CH,COO- - - 63.6 - 62.1 62.1 64.8 0.460

CONCLUSION 28 1
Tables 10.3-10.5 was taken. The deviations* from the average calibration constant
must be ascribed to the use of the micro-syringe for sample introduction. These values
are listed in Table 10.6.
The tables show that the zones do not have a mutual influence on each other, which
means that the buffer capacity of the counter ion chosen is sufficient.
TABLE 10.6
CALIBRATION CONSTANTS, Kca,, DETERMINED FROM THE VARIOUS EXPERIMENTS
LISTED IN TABLES 10.3-10.5
Ion Concentration K , - ~ lo4 Deviation Deviation
(N)
in KcaI * lo7 (%I
NO,- 0.0125
(30;
CH, COO-
NO,- 0.025
c10;
CH,COO-
NO,- 0.05
(30;
CH,COO-
Average
10.5. CONCLUSION
0.448
0.467
0.462
0.448
0.457
0.459
0.443
0.452
0.461
0.455
-7
t12
+7
With an automatic device for recording zone lengths, a high-resolution detector and
injection via a micro-syringe (a sample valve, as described in section 7.2.3, was even
better), good linearity can be obtained between the amount injected and the distance in
the isotachopherogram between the differential traces of the linear signal of the
conductivity detector.
If the experiments are carried out with care and good equipment is available, no
internal standard need be applied. If the detection of even smaller zones than those in
the tables is required, the profiles as shown in Fig.17.2 must be taken into account.
Among other factors, impurities and different profiles for different zone boundaries may
obscure the quantitative results. The zone boundary of, for instance, acetate with
glutamate is different from that of acetate with morpholinoethanesulphonate, which has
a considerably smaller effective mobility in the operational system at pH 6 (Table 12.1)
than has glutamate.
If a specific detector is available, e.g., the W absorption detector, one can use the
*As already mentioned in section 10.3.2, more accurate data, used for the calculation of the actual
concentrations of the various ionic species via the computer program in Chapter 4, will also improve
the accuracyof Kcal.
-7
+2
+4
-12
-3
+6
1.4
2.6
1.3
1.4
0.4
0.9
2.7
0.7
1.3
1.43

282 QUANTITATIVE ASPECTS
curvature of the boundary profile for the determination of very small amounts of UV-
absorbing material [3]. The W-absorbing compound must be sandwiched between two
non-UV-absorbing ions, the UV-absorbing ion ‘coating’ the profile with a small layer.
Because the parabolic profile is about 0.1-0.4 mm, even a layer of 0.01 mm of W-
absorbing component can be detected.
Of course, a calibration graph must be constructed for each component and distur-
bances by electroendosmosis of unwanted hydrodynamic transport influence the quan-
titative results enormously. Another problem may occur if one is interested in the quan-
titative determination of the ion which is the most mobile in the system, i.e., the leading
ion.
Reproducible measurements can be carried out if a sample tap is available. Because
the time of appearance of the leading electrolyte-sample boundary is constant (if the
injection of the sample is reproducible), the retardation in the time of the appearance of
the first sample zone, with an effective mobility smaller than that of the leading ion,
can be used to obtain the quantitative information. A reproducibility of better than 98%
can easily be obtained.
REFERENCES
1 J.L. Beckers and F.M. Everaerts, J. Chromatogr., 71 (1972) 329.
2 F.M. Everaerts, J. Chromatogr., 91 (1974) 823.
3 M. Svoboda and J. Vaci’k, J. Chromatogr., 119 (1976) 539.

Chapter 11
Separation of cationic species in aqueous solutions
SUMMARY
As mentioned in earlier chapters, the effective mobilities of cations can easily be
influenced. This can be an advantage, especially if the cations to be separated have the
same or almost the same effective mobilities in an electrolyte system. By changing the
system, it may be possible to separate such ionic species. In this chapter, the qualitative
simultaneous separation of some cations using both water and deuterium oxide as a
solvent are described, using buffered and unbuffered systems. Results obtained with
thermometric, conductivity and UV detectors are given. For these experiments, the
equipments described in sections 7.4.2 and 7.4.4 were used. The time of analysis, from
the start of the experiment to the detection of the last zone, is about 3045 min with the
thermometric detector and about 15 min for the high-resolution detectors.
1 1.1. SEPARATION OF CATIONIC SPECIES IN AQUEOUS SOLUTIONS USING A
THERMOCOUPLE AS DETECTOR
The experiments were carried out with the equipment described in section 7.4.2. The
sample was introduced via a four-way tap, as described in section 7.2.2. A constant d.c.
power source with a maximum potential of 20 kV was used. We used a Micrograph BD 5
recorder (Kipp & Zonen, Delft, The Netherlands), which is especially useful because of
its automatic zero suppression module.
The effective mobilities of some cations were sometimes very low and the increment
in electrical resistance during the analyses, due to the movement of the zones with small
conductivities, required the use of higher potentials than those which were available;
in this section, the term ‘not sufficiently mobile’ is used for these cations. Sometimes the
length of the capillary tube was too short for complete separation of the cations. Also, the
zones, although separated, sometimes could not be detected because the resolving power
of the thermometric detector was too low to detect small lengths of zones or small differences
in temperature between two zones; in these instances, the term ‘not separated’ is used.
The volume of the sample tap (about 20 pl) is rather large and corresponds to the
contents of about a 14-cm length of the capillary tube. If the concentration of the sample
ionic species is chosen to be too high, complete separation according to the isotacho-
phoretic principle cannot be expected. The average time for all analyses was about 45 min.
In Tables 11.1-1 1.5, the conditions of the systems used are listed; these are the so-
called operational systems. The abbreviations given in the tables are used in the text of
ths chapter.
For some systems, a scheme can be given to show which series of cations can be
separated simultaneously. The interpretation is as follows. Ions placed in one circle and
ions placed in circles directly connected by lines cannot be separated simultaneously,
283

284 SEPARATION OF CATIONIC SPECIES IN AQUEOUS SOLUTIONS
TABLE 11.1
OPERATIONAL SYSTEM AT pH 2 SUITABLE FOR CATIONIC SEPARATIONS
Solvent: H, 0.
Electric curent &A): Ca. 50-100.
Electrolyte
Leading Terminating
Cation H+ Tns+
Concentration 0.01 N Ca. 0.01 N
Counter ion c1- c1-
PH 2 Ca. 6
Additive 0.05% Polyvinyl None
alcohol (Mowiol)*
*For experiments with a thermometric detector, this additive is not necessary.
TABLE 11.2
OPERATIONAL SYSTEM AT pH 1.9 SUITABLE FOR CATIONIC SEPARATIONS (WHIO,)
Solvent: H, 0.
Electric current &A): Ca. 50-100.
Elec tr ol y te
Leading Terminating
Cation H+ Tris+
Concentration Ca. 0.01N Ca. 0.01 N
Counter ion 10,- (0.01 N ) c1-
PH 1.5 Ca. 6
Additive 0.05% Polyvinyl None
alcohol (Mowiol)*
*For experiments with a thermometric detector, this additive is not necessary.
e.g., in Fig.ll.1 (the system WHCl) BaZ+ and F%’+ cannot be separated because they are
placed in the same circle, and CaZ+ and A13+ cannot be separated because they are
connected directly by a line, whereas Ba" and A13+ can be separated because they are
not directly connected by a line. If BaZ+, CaZ+ and A13+ are present, they form mixed
zones together. I.i+ and (C, H5)4 N’ can be separated because they are not connected
by a line. AU step heights* found in the isotachopherograrns of the experiments in
*The step height in an isotachopherogram is a qualitative measure 101 the ionic species, where the
distance between two successive peaks (the differential signal of the linear thermocouple signal)
gives all necessary quantitative information.

SEPARATION USING A THERMOCOUPLE AS DETECTOR 285
TABLE 11.3
OPERATIONAL SYSTEM AT pH 5.4 SUITABLE FOR CATIONIC SEPARATIONS (WKAC)
Solvent: H, 0.
Electric current hA): Ca. 50-100.
Electrolyte
Leading Terminating
Cation K+ Tris+
Concentration 0.01 N Ca. 0.01 N
Counter ion CH, COO- CH,COO-
PH 5.4 Ca. 5
Additive 0.05% Polyvinyl None
alcohol (Mowiol)*
*For experiments with a thermometric detector, this additive is not necessary.
TABLE 11.4
OPERATIONAL SYSTEM AT pH 6.4 SUITABLE FOR CATIONIC SEPARATIONS (WKCAC)
Solvent: H, 0.
Electric current hA): Cu. 50-100.
Electrolyte
Leading Terminating
Cation K+ Tns+
Concentration 0.01 N Ca. 0.01 N
Counter ion (CH,),AsOO- CH,COO-
PH 6.4 Ca. 6
Addit.ive 0.05% Polyvinyl None
alcohol (Mowiol)*
*For experiments with a thermometric detector, this additive is not necessary
water measured by means of a thermocouple are given in Table 11.6. All these step
heights refer to 0 PA.
11.1.1. The system WHCl
~~ ~
The leading electrolyte used is hydrochloric acid in water and Tris in water is used as
the terminator. Many mono-, di- and trivalent cations have about the same step heights.
Their effective mobilities are nearly equal so that separations are impossible with the
apparatus available. Ions can be separated if they differ in step height by about 20 mm in
this system (see Table 1 1.6).

286 SEPARATION OF CATIONIC SPECIES IN AQUEOUS SOLUTIONS
TABLE 11.5
OPERATIONAL SYSTEM AT pH 7.4 SUITABLE FOR CATIONIC SEPARATIONS (WKDF)
Solvent: H, 0.
Electric current &A): Ca. 50-100
Electrolyte
Leading Terminating
Cation K+ Tris'
Concentration 0.01 N Ca. 0.01N
Counter ion I,-L-Tyr- (diiodo-L- CH,COO-
tyrosine)
PH 7.4 Ca. 7
Additive 0.05% Polyvinyl None
alcohol (Mowiol)*
*For experiments with a thermometric detector, this additive is not necessary.
Fig.ll.1. Simultaneous separation of some cations in the system WHCI. Guan = Guanidine; Im =
imidazole; S.C. = succinyl choline; Tea = (C,H,), N; Tma = (CH,), N.
Separations of mixtures containing cationic species with low pK values are difficult,
as explained in Chapter 9. Figure 11.1 shows which cations can be separated simultaneously
in this system. Fig.ll.2 shows the isotachopherogram for the separation of a
mixture consisting of l’, La3+, Ca*+, Fez+ CdZ+ and Liz, with H+ as the leading ion and
Tris' as the terminating ion.
11.1.2. The system WHI03
In this system, the leading electrolyte is M03 in water and the buffering effect is
small. The sequence of the step heights of the cations is similar to that in the system
WHCI. The most important shifts in step heights are those of Cr3+, Ce3+ and La3+. Pbz+
does not migrate noticeably.

SEPARATION USING A THERMOCOUPLE AS DETECTOR
TABLE 11.6
QUALITATIVE INFORMATION ON SOME CATIONS OBTAINED IN THE OPERATIONAL
SYSTEMS LISTED IN TABLES 11.1-11.5
The values given are the step heights as found in the isotachopherogram in the Linear trace of the
thermocouple signal. The values are given in millimetres and refer to 0 /.LA. About 20 mm is sufficient
for a complete qualitative and quantitative separation by isotachophoresis. The current was stabilized
at 100 MA.
Cation WHCl WHIO, WKAC WKCAC WKDIT
H+ .
K+
Na+
Li+
NH:
Ag+
TI+
(CH, 14 N’
(C, H, N+
(C, H, I., N+
Tris+
Imidazole’
cs+
Rb+
Guanidine+
Succinyl choline’
CO’+
Ni +
Mg2+
cu I+
Ca”
MnZ+
Cd’+
Fez+
Sn’+
Pb2+
Baz+
2n2+
Fe 4t
LaN
Ce
Cr 3+
AIw
60
216
292
352
215
-
217
302
400
n.s.m.
4 34
306
208
21 3
285
3 24
290
291
294
290
266
285
318
294
248 -2 70*
250
255
294
n.s.m.
241
246
312*
272
72
290
400
492
292
-
295
437
560
n.s.m.
625
412
-
-
391
450
416
415
408
4 04
372
412
420
410
-
n.s.m.
352
404
n.s.m.
366
368
498
3 80
-
220
302
378
220
260
-
340
432
n.s.m.
490
*Double step.
**Estimated value from experiment at a lower current density.
*The step height for imidazole at pH 6.53 is 472 mm.
5n.s.m. = not sufficiently mobile.
310
-
-
294
343
318
318
314
387
284
320
341
312
1276**
371
264
3 20
1128**
322
325
390
360
-
280
3 84
476
282
338
-
430
540
808
610
432-
-
-
372
434
407
403
396
442
362
420
446
508
n.s.m.
486
338
415
n.s.m.
402
416
n.s.m.
n.s.m.
287
-
300
410
504
303 .
n.s.m.0
-
442
568
-
680
551
-
-
399
477
-
n.s.m.
430
n.s.m.
388
440
n.s.m.
n.s.m.
-
n.s.m.
368
n.s.m.
n.s.m.
634
834
n.s.m.
-

288 SEPARATION OF CATIONIC SPECIES IN AQUEOUS SOLUTIONS
Fig.ll.2. Isotachopherogram of the separation of some cations in the system WHCl, using a thermometric
detector: 1 = W; 2 = lI+; 3 = La"; 4 = Caz+; 5 = Fez+; 6 = CdZ+; 7 = Li+; 8 = Tris+. T= Increasing
temperature; t = time.
11.1.3. The system WKAC
The leading electrolyte is a solution of potassium acetate in water, adjusted to pH 5.39
by adding acetic acid. This pH is chosen because in the following zones the pH of the
zones decreases almost to the pK value of acetic acid, producing a maximum buffering
effect. The differences in step heights of the cations, for a complete separation, must be
20 mm in this system. Fig.11.3 shows which ions can be separated simultaneously.
Comparing the step heights in this system with those of the other systems, some shifts
in the step heights must be explained. The most important are those of F'b2+, Ce3+, La3+,
A13' and Cr3+, which are all polyvalent ions. The reason for the shifts can be found in the
higher pH of the system and stronger complex formation.
Figure 11.4 shows the isotachopherogram for the separation of a mixture of Ba2+,
Ca'+, Na', Ni2+, Cd2+, Pbz+ and (C2H,),N+. K+is the leading ion and Tris+ the terminating
ion.

SEPARATION USING A THERMOCOUPLE AS DETECTOR 289
Fig.ll.3. Simultaneous separation of some cations in the system WKAC. Abbreviations as in Fig.11.1.
9
-
f
i,
Fig.ll.4. Isotachopherogram of the separation of some cations in the system WKAC, using a thermo-
metric detector. 1 = Kt; 2 = Ba”; 3 = Ca? 4 = Na’; 5 = Ni”; 6 = Cd2+; 7 = Pb2+; 8 = (C,H,), W;
9 = Trist. T = Increasing temperature; t = time.
11.1.4. The system WKCAC
The leading electrolyte is potassium hydroxide in water, adjusted to pH 6.37 by
adding cacodylic acid Tris’ is used as the terminating ion. This higher pH was chosen so
as to investigate the effect of increased pH.
In the system WKCAC, all ions have lower effective mobilities owing to the higher pH
(some cationic species have pK values between 5 and 7) and to complex formation. The
effective mobilities of N3+, Cr3+ and Fe3+ are too low. Imidazole shows a typical shift in
step height. It has a pK value of 6.95 and at higher pH its effective mobility will decrease.

290 SEPARATION OF CATIONIC SPECIES IN AQUEOUS SOLUTIONS
In order to check this effect, some experiments were carried out with the same buffer at
pH 6.53. For some cations of strong electrolytes the step heights were nearly identical,
while the step height of imidazole increased (see Table 1 1.6).
Figure 1 1.5 shows which ions can be separated simultaneously. In Fig.ll.6, the
isotachopherogram is given for the separation of the cations Ba2+, Ca2+. N$. Ni2+, Mnz+,
Cu2+ and (C2H,),N+. The leading ion was K'and terminator was Tris'.
Ni
Fig.ll.5. Simultaneous separation of some cations in the system WKCAC. Abbreviations as in Fig.11 .l.
Fig.ll.6. Isotachopherogram of the separation of some cations in the system WKCAC, using a thermometric
detector. 1 = K+; 2 = Ba2+; 3 = Ca"; 4 = Na+; 5 = Ni2+; 6 = Mn2*; 7: Cu"; 8 = (C, H 5) 4 N+;
9 = Tris+. T = Increasing temperature; t = time.

I
SEPARATION USING A THERMOCOUPLE AS DETECTOR 29 1
72 __
-1
12
i- 1
h -1
Fig.ll.7. Isotachopherogram of a test mixture of cations carried out in the operational system at pH
5.4 with water and deuterium oxide as solvents (Table 11.3). A conductivity detector (a.c. method) and
a W adsorption detector (256 nm) were applied 1 = K+; 2 = BaZ+; 3 = Na+; 4 = (CH,), N+; 5 = Pb2+;
6 = C,H,N+-CH,-CO-NH-NH, ; 7 = Tris'; 8 = histidid; 9 = creatinine*; 10 = bemidine+; 11 =
e-aminocaproic acid+; 12 = raminobutyric acid+. The amplification of both the UV absorption
detector and the conductivity detector was not changed. A = Increasing UV absorption, R = increasing
resistance; t = time.
TABLE 11.7
DIFFERENCES IN THE EFFECTIVE MOBILITIES OF SOME CATIONS IN THE SOLVENTS WATER
AND DEUTERIUM OXIDE
The values (h) refer to the step heights (mm) as measured in the linear trace of the conductivity detector.
Ionic species hHZO hDzO Ionic species hH,O hD,O
f,
K+ 0 0 Tris' 51.0 54.8
Baa+ 8.8 9.1 Histidine' 58.5 65.6
Na + 14.2 16.9 Creatinine' 65.6 78.0
Tma' 20.8 23.8 Benzidine' 81.3 98.1
Pb2+ 25.4 34.8 e-Aminocaproic acid+ 109.2 130.6
C,H,N'-CH,-CO-NH-NH, 39.4 44.8 y-Aminobutyric acid+ 115.6 149.2

292 SEPARATION OF CATIONIC SPECIES IN AQUEOUS SOLUTIONS
TABLE 11.8
RELATlVE STEP HEIGHTS OF CATIONS IN THE OPERATIONAL SYSTEM LISTED IN TABLE
11.3 WITH WATER AND DEUTERIUM OXIDE AS THE SOLVENTS
For the experiments with deuterium oxide, the same amounts of electrolytes as in the experiments
with water were dissolved. The accuracy is better than 4%. The values given are to be used only for
the identification of cations in isotachophoretic analyses in the operational systems given. The
potassium ion (leading ion) has a relative step height of 0, while the sodium ion has a relative step
height of 100. The current was stabilized at 90 pA. - - = No UV absorbance; - = not measured.
Ionic species
H*O
lOOh - uv
absorbance
hNa
D,O
lOOh uv
- absorbance
Amine, butyl
Amine, diethanol
Amine, ethanol
Amine, Zethylaminoethyl
Amine, octyl
Amine, triethanol
Amine, triethyl
2-Amino-2-methyl- 1,3-propanediol
Amphetamine
Arginine
Ammonium, tetrabutyl
Ammonium, tetraethyl
Ammonium, tetramethyl
Barium(I1)
Benzidine
Benzidine, 3,3-methoxy
Butyric acid, 3-amino
Cadmium(I1)
Calcium(I1)
Caproic acid, 2-amino
Chromium(II1)
Cobalt(I1)
2,4,6-Collidine
Copper(1I)
Creatinine
Diamine, trimethylene
Diamine, o-phenylene
Girard reagent D
Girard reagent P
Girard reagent T
Guanidine
His tidine
Hydrazine
Hy droxylamine
Imidazole
Iron(I1)
Lanthanum(II1)
Lead(I1)
225
29 2
159
51
367
290
290
343
350
422
635
3 04
147
59
576
814
81 3
142
86
772
175
114
315
169
462
35
557
280
277
280
83
411
59
134
119
108
109
179
_
-
165
-
350
-
-
313
335
409
590
29 1
142
54
580
-
881
154
81
768
197
1 24
290
237
460
20
550
260
265
252
95
388
64
132
122
128
138
210

SEPARATION USING CONDUCTIVITY AND UV DETECTORS
TABLE 11.8 (continued)
Lithium(1)
Lysine
Magnesiurn(I1)
Manganese(I1)
Nickel(I1)
Phenazone, 4-amino
3-Picoline
Piperidine
Piperidine, 1-methyl
Purine, 6,8-dihydroxy
Pteridine,2,4-diamino-6,7-dimethyl
F'yridine
Sitver(1)
Sodium(1)
Strontium(I1)
Tetramine, hexamethylene
Tin(I1)
o-Tolidine
Tris (hydroxymethy1)aminomethane
Zinc(1I)
11.1.5. The system WKDIT
HZ0
100 h uv
- absorbance
hN, (%I
194
400
102
114
115
1014
217
214
252
1051
439
24 2
18
100
73
461
1083
816
358
130
196
395
110
120
130
__
221 88
21 5 __
231 -_
432 98
232 92
100
476
-_
__
__
__
- -
- -
- -
- -
__
-_
- -
807 96
333 _-
134 -_
The leading electrolyte is potassium hydroxide in water, adjusted to pH 7.39 by
adding diiodo-Ltyrosine, and the terminator is Tris. At this pH, many ions do not
migrate at all and sometimes precipates are formed. While all possible step heights were
measured, the above effects were such that no separations could be achieved in this
system. For some special purposes, however, this sytem can be useful, e.g., in combina-
tion with other systems.
1 1.2. SEPARATION OF CATIONIC SPECIES IN WATER AND DEUTERIUM OXIDE
USING A CONDUCTIVITY DETECTOR (a.c. METHOD) AND A UV ABSORPTION
DETECTOR (256 nm)
As already shown in section 11.1, cations can be separated in aqueous systems and
the mobilities can be influenced by changing the operating conditions. Also, with these
types of detectors all types of systems can be prepared and analyses can be performed.
For the reasons mentioned in Chapter 8, only the operational system listed in Table 11.3
293

294 SEPARATION OF CATIONIC SPECIES IN AQUEOUS SOLUTIONS
(potassium acetate-acetic acid at pH 5.4), will be discussed*. Both water and deuterium
oxide were used as solvents. During a long series of analyses, it was found that, in spite
of the fact that the electric leak to earth of the conductivity probe can be neglected
(Chapter 6), a layer is formed on the micro-sensing electrodes (Kolbe electrolysis).
Owing to this layer, irreproducible results can be expected if no precautions are taken.
Therefore, between analyses, the electrodes were depolarized (by connecting the anode
of a power supply with the micro-sensing electrodes) for approximately 5 sec with an
electric current of approximately 5 PA. For a reproducible analysis, the base line must
show no drift (less than 1%).
In Fig. 1 1.7, a separation of cations in water and deuterium oxide is shown. This
mixture can be used as a test mixture of cations, as discussed in the Chapter 8 (Fig.8.1).
The differences obtained in the systems with water and deuterium oxide are listed in
Table 11.7.
Because the operational conditions are comparable, except for the solvent, the shift
is due to two main factors: the solvation and the difference in the pH and pD scale. In
Table 11.8, some step heights are listed for some cations obtained in the operational
system in Table 11.3 with water and deuterium oxide as the solvents.
*Many data are given from thermometric registration (Table 11.6), which can be used. The values given
in Tables 11.6 and 11.8 can be compared.

Chapter I2
Separation of anionic species in aqueous solutions
SUMMARY
Separations of anionic species in water and deuterium oxide solutions are discussed.
In the first section, two operational systems using a thermometric detector with measurements
in aqueous solutions are considered. In the second section, the results obtained
using a conductimeter (a.c. method) and a UV absorption detector are given, with
experimental data for separations in deuterium oxide.
The time of analysis with the thermometric detector is approximately 30-45 min and
with the high-resolution detectors approximately 15 min from the start of the experiment
till the detection of the last zone.
12.1. SEPARATION OF ANIONIC SPECIES IN AQUEOUS SOLUTIONS USING A
THERMOMETRIC DETECTOR
The results given in this section were obtained in the equipment briefly discussed in
section 7.4.2. The systems were measured in the two operational systems listed in
Tables 12.1 and 12.2.
Most of the organic acids have pK values not higher than about 5.5 and will therefore
be almost completely ionized at the pH values chosen, i.e., pH 6 and 7. In fact, only
separations according to mobilities are carried out, not according to the pK values.
12.1.1. Operational system histidine/histidine hydrochloride (pH 6)
Step heights* measured in this system are listed in Table 12.3 and refer to 0 PA.
Many anions have the same or almost the same step heights, because their effective
mobilities are almost identical, le., they cannot be separated. In our experiments we
found that anions can be separated in these systems if their step heights differ by about
10% when using a thermometric detector (thermocouple). It will be shown in section 12.2
that commonly, if a separation according to mobilities fails, a separation according to pK
values will be successful. In Fig. 12.1, a separation is shown of a mixture of anions carried
out in this operational system using a thermocouple as detector. Fig.12.2 shows the
separation of another mixture of anions. From the combination of anions chosen, it
will be clear that, e.g. , nitrate and sulphate cannot be separated under these conditions,
le., with this operational system and detector.
*The step height in an isotachopherogram is a qualitative measure for the ionic species, where the
distance between two successive peaks (the differential signal of the linear thermocouple signal) gives
all necessary quantitative information.
295

296 SEPARATION OF ANIONIC SPECIES IN AQUEOUS SOLUTIONS
TABLE 12.1
OPERATIONAL SYSTEM AT pH 6 SUITABLE FOR ANIONIC SEPARATIONS
Solvent: H,O and D,O.
Electric current &A): Cu. 50-100.
Purification: Morpholinoethanesulphonic acid (MES) is recrystallized three times and the
crystals are washed with acetone.
Electrolyte
Leading Terminating
Anion cl- E.g., MES-
Concentration 0.01 N Cu. 0.01 N
Counter ion Histidine' Tris+
PH 6.02 Cu. 6
Additive 0.05% Polyvinyl None
alcohol (Mowiol)*
*For experiments with a thermometric detector, this additive is not necessary.
TABLE 12.2
OPERATIONAL SYSTEM AT pH 7 SUITABLE FOR ANIONIC SEPARATIONS
Solvent : H, 0.
Electric current hA): Cu. 50-100.
Purification: Morpholinoethanesulphonic acid (MES) is recrystallized three times and the
crystals are washed with acetone.
Electrolyte
Leading Terminating
Anion c1- E.g., MES-
Concentration 0.01 N Ca. 0.01 N
Counter ion Imidazole+ Tris'
PH 7.05 Ca. 6
Additive 0.05% Polyvinyl None
alcohol (Mowiol)*
*For experiments with a thermometric detector, this additive is not necessary.
12.1.2. Operational system imidazole/imidazole hydrochloride (pH 7)
In this operational system, the absolute mobility of the counter ion (imidazole) is
greater than in the operational system listed in Table 12.1 in which histidine is the counter
ion. This means that all zone resistances are lower and consequently all step heights are
smaller. The resolution will also be decreased by the more mobile counter ion. Thls was
found particularly in experiments with a conductivity detector.

SEPARATION USING A THERMOMETRIC DETECTOR 29 7
TABLE 12.3
STEP HEIGHTS FOR SOME ANIONS OBTAINED IN THE OPERATIONAL SYSTEMS LISTED IN
TABLES 12.1 and 12.2
The values are the step heights found in the isotachopherograms in the linear trace of the thermo-
couple signal. The values are given in millimetres and refer to 0 fiA. The experiments were carried
out with a current stabilized at 70 @A.
-
Ionic species System* Ionic species System*
Adipic acid
Acetic acid
Acetylsalicylic acid
Allomucic acid
Azelaic acid
Benzoic acid
Benzoic acid, rn-amino
Benzoic acid, o-amino
Benzoic acid, p-amino
Benzyl-dl-aspartic acid
Benzoic acid, 5-bromo-
2,3-dihydroxy
Butyric acid
Cacodylic acid
Caffeic acid
Capric acid
Caproic acid
Caprylic acid
Carbonic acid
Chloric acid
Chromic acid
Cinnamic acid
Citric acid
Crotonic acid
Dichromic acid
2,4-Dihydroxybenzoic acid
EDTA
Formic acid
D-Galacturonic acid
Glucuronic acid
Glutamic acid
Glycolic acid
Glyoxylic acid
Guanidoacetic acid
Hippuric acid
Hydrofluoric acid
a-Hydroxybutyric acid
4,5-Imidazoledicarboxylic
acid
Indolylacetic acid
Iodic acid
Isovaleric acid
A B
334 252
366 281
474 380
319 250
365 274
430 340
454 340
408 -
454 350
531 440
460 316
428 356
620 400
526 420
51 1 400
478 386
510 400
520 sl.* 320 sl.
24 3 190
259 173
500 368
292 200
416 3 26
249 174
459 354
3 34 285
276 216
n.s.m.- -
509 420
476 386
360 286
399 290
n.s.m. n.s.m.
490 391
271 218
470 375
326 240
n.s.m. n.s.m.
358 290
460 360
2-K&ogulonic acid
Kynurenic acid
Lactic acid
Laevulinic acid
Maleic acid
dl-Malic acid
Malonic acid
Mandelic acid
Methacrylic acid
Molybdic acid
Naphthalene-2sulphonic
acid
Nicotinic acid
Nitric acid
rn-Nitrobenzoic acid
p-Nitrobenzoic acid
Nitrous acid
Orotic acid
Orthophosphoric acid
Oxalic acid
Pelargonic acid
Periodic acid
Peroxodisulphuric acid
Phenidon
Phenylacetic acid
o-Phthalic acid
Picric acid
Pimelic acid
Propionic acid, p-chloro
Pycrolonic acid
Pyrazine-2,3-dicarboxylic
acid
Pyrazole-3,5-dicarboxylic
acid
F'yrophosphoric acid
Pyrosulphuric acid
Pyrosulphurous acid
Salicylic acid
Succinic acid
Sulphamic acid
Sulphanilic acid
Sulphosalicylic acid
A B
496
470
391
430
312
286
280
456
4 04
335
496
436
220
440
442
217
454
408
236
494
358
212
n.s.m.
44 8
328
446
345
399
n.s.m.
298
301
-
224
224
408
3 04
3 04
420
283
395
383
314
-
216
222
204
364
312
185
358
342
174
340
345
170
310
266
180
393
250
162
n.s.m.
366
246
350
264
316
-
-
235
204
-
172
323
224
244
344
228
(Continued on p.298)

298 SEPARATION OF ANIONIC SPECIES IN AQUEOUS SOLUTIONS
TABLE 12.3 (continued)
Ionic species sys tem* Ionic species
A B
System*
A B
Sulphuric acid 224 169 Uric acid 424 360
Sulphurous acid 286 170 7-Oxoiminovaleric acid 466 382
Tartaric acid 280 216 Vanadic acid
320sl. 184
Tartronic acid 256 195 Vitamin C 510 390
Tiglic acid 410 332 Xanthurenic acid 484 353
Trichloroacetic acid 399 316
Trimethylacetic acid 470 363
*Systems: A = histidine/histidine hydrochloride; B = imidazole/imidazole hydrochloride.
*Ir
sl. = An indefinite step height was obtained. The signal slopes slowly to an end-point.
*n.sm. = Not sufficiently mobile.
n
Fig.12.1. Isotachopherogam of the separation of some anions in the operational system listed in
Table 12.1 (pH 6). 1 = Chloride; 2 = nitrate; 3 = oxalate; 4 = tartronate; 5 = formate; 6 = citrate;
7 = maleate; 8 = adipate; 9 = iodate; 10 = trichloroacetate; 11 = mandelate; 12 = ascorbate. The
current was stabilized at 70 rk T= Increasing temperature; t = time.
t

SEPARATION USING A THERMOMETRIC DETECTOR 299
Fig.12.2. Isotachopherogram of the separation of some anions in the operational system listed in
Table 12.1 (pH 6). 1 = Chloride; 2 = sulphate; 3 = chlorate;4 = chromate; 5 = malonate; 6 = pyrazole-
3,5-dicarboxylate; 7 = adipate; 8 = acetate; 9 = p-chloropropionate; 10 = phenylacetate; 11 = ascorbate.
The current was stabilized at 70 PA. T= Increasing temperature; t = time. This isotachopherogram
is used in several places in this book for comparison of the various detectors and the various solvents.
The smaller steps in the system imidazole/imidazole hydrochloride correlate correctly
with the calculated zone resistances (see section 4.5). Some step heights are low, which
can be ascribed to a dissociation that is more complete in this system. Some compounds
that show these very large shifts are citric acid (pK3 = 6.4), orthophosphoric acid
(pK, = 7.21) and chromic acid (pKz = 6.49).
Fig.12.3 shows aa isotachopherogram of the separation of some anions carried out in
the operational system listed in Table 12.2. Although the resolution is lower than in the
operational system at pH 6, in general anions can be separated if the step heights given
in Table 12.3 differ by about 10%. The disadvantage of this operational system is the
relatively high pH, which soon gives a disturbance due to carbonate.

300
I1
rc--
t
\
t
SEPARATION OF ANIONIC SPECIES IN AQUEOUS SOLUTIONS
n
Fig.12.3. Isotachopherogram of the separation of some anions carried out in the operational system
listed in Table 12.2 @H 7). 1 = Chloride; 2 = sulphate; 3 = oxalate; 4 = chlorate; 5 = formate;
6 = pyrazole-3,5-dicarboxylate; 7 = adipate; 8 = iodate; 9 = P-chloropropionate; 10 =nicotinate;
11 = ascorbate. The current was stabilized at 70 PA. If this isotachopherogram is compared with those
in Figs.12.1 and 12.2, it can be seen that the resolution is smaller, although separations are still possible.
12.2. SEPARATION OF ANIONIC SPECIES IN AQUEOUS SOLUTIONS USING A
CONDUCTIVITY DETECTOR (a.c. METHOD) AND A UV ABSORPTION DETECTOR
(256 nm)
12.2.1. Introduction
In this section, we consider isotachophoretic separations in four operational systems
with water and deuterium oxide. The pH values were chosen arbitrarily as 7.5 (Table 12.4),
6 (Table 12.1), 4.5 (Table 12.5) and 3 (Table 12.6). The results obtained in these systems
are listed in Table 12.7 for water and Table 12.8 for deuterium oxide.
The analyses discussed in section 12.2.2 were carried out mainly in these operational
systems, although the pH, for optimal separation, may be chosen to be higher or lower
than those given in the tables.
The analyses were carried out with the equipment described in section 7.4.4.

SEPARATION USING CONDUCTIVITY AND W DETECTORS 30 1
TABLE 12.4
OPERATIONAL SYSTEM AT pH 7.5 SUITABLE FOR ANIONIC SEPARATIONS
Solvent: H,Oand D,O
Electric current &A): CQ. 50-100.
Purification: Morpholinoethanesulphonic acid is recrystallized three times and the crystals are
washed with acetone.
Electrolyte
Leading Terminating
Anion a- MES-
Concentration 0.01 N Ca. 0.01 N
Counter ion Tris+ Tris+
PH 7.5 Ca. 6
Additive 0.05% Polyvinyl None
alcohol (Mowiol)
TABLE 12.5
OPERATIONAL SYSTEM AT pH 4.5 SUITABLE FOR ANIONIC SEPARATIONS
Solvent:
H, 0 and D, 0.
Electric current (PA): Cu. 50-100.
Purification: Morpholinoethanesulphonic acid (MES) is recrystallized three times and the
crystals are washed with acetone. c-Aminocaproic acid is recrystallized.
Electrolyte
Leading Terminating
Anion c1- MES-
Concentration 0.01 N Ca. 0.01 N
Counter ion COOH-C,H,-CH,N+H Tris'
PH 4.5 CQ, 4
Additive 0.05% Polyvinyl
alcohol (Mowiol)
None
12.2.2. Applications
As described in Chapter 11, we can change from one operational system to another if
a complete separation between various ionic species is sought. It is almost always more
convenient to perform the analyses in two or more operational systems than to lengthen
the narrow-bore tube, which involves the use of higher potential gradients.
Less attention is paid here to the influence of the activity on the effective mobility
(the concentration of the leading electrolyte must be changed), because if the concentra-
tion is decreased*, higher potential gradients must be used, the effect of electro-
endosmosis is difficult to suppress and higher demands are placed on the purity
*If the concentration is increased, many substances are no longer soluble.

302 SEPARATION OF ANIONIC SPEClES IN AQUEOUS SOLUTIONS
TABLE 12.6
OPERATIONAL SYSTEM AT pH 3 SUITABLE FOR ANIOMC SEPARATIONS
Solvent
H, 0 and D, 0.
Electric current (PA): Ca. 50-100.
Purification : p-Alanine must be purified by recrystallization from a methanol-water mixture
and the crystals are washed with acetone.
Electrolyte
Leading Terminating
Anion c1- E.g., CH,COO-
Concentration 0.01 N Ca. 0.01 N
Counter ion COOH-CH, -CH, WH Tris+
PH 3 Ca. 3
Additive 0.05% Polyvinyl None
alcohol (Mowiol)
of the chemicals used in the operational systems. An example of the use of two operational
systems is given in Fig. 12.4, where the separation of the oxidation products of
acetylsalicylic acid is shown. The oxidation was performed by heating the compound in
solution to 90°C and blowing air through it.
The separations shown in Fig. 12.4a and b were carried out in the operational system
at pH 6 (Table 12.1), while the analysis in Fig.12.4~ was performed at pH 3.2 (Table 12.6).
It is clear that a complete separation can be achieved at low pH.
The isotachopherograms in Fig.12.5 were obtained in the operational system at pH 6
(Table 12.1), with water and deuterium oxide as the solvents. This mixture is nowadays
applied as the test mixture of anions suitable for ‘trouble-shooting7 (Fig.8.1). The
sequence of the compounds is given in Table 12.9, where the shifts obtained by the use
of water and deuterium oxide are listed. Again, these shifts are due mainly to the
differences in solvation and differences in the pH and pD scales.
Large shifts occur for the components for which the pH of the operational system
chosen has a significant effect on the dissociation (pK values - pHoperational system).
Fig. 12.6 shows that solvation really plays a role. In these isotachopherograms, the separation
of nitrate and sulphate is shown in the operational system at pH 6 (Table 12.1). Wtiilc
a separation could be achieved only with difficulty when water was used as the solvent
(here the concentration of the leading electrolyte plays an important role), the separation
in deuterium oxide posed no problems. One should note the linear trace of the UV
absorption detector, which shows that nitrate has a small W absorption, clearly visible
in the experiments with deuterium oxide, while this W absorption is spread over the
total mixed zone when water was used as the solvent. The isotachopherograms in
Fig.12.6b and c are given in order to show the reproducibility.
In Fig.12.7, two important phenomena are shown. A research group working mainly
on peptide synthesis, asked us for very reproducible quantitative information about the
amount of chloride in their samples. As discussed in the conclusion of Chapter 10, this
can be achieved by measuring the retardation of the appearance of the first sample zone

SEPARATION USING CONDUCTIVITY AND UV DETECTORS 303
Fig.12.4. Isotachopherograms of the separation of the oxidation products of acetylsalicylic acid in
two different operational systems. (a), Reaction mixture before oxidation, analysis carried out in
the operational system at pH 6 (Table 12.1); (b), reaction mixture after oxidation, analysis carried
out in the operational system at pH 6 (Table 12.1); (c), reaction mixture after oxidation, analysis
carried out in the operational system at pH 3.2 (Table 12.6). The current was stabilized at 80 PA.
1' = Chloride; 2' = acetate; 3' = salicylate and phosphate; 4' = acetylsalicylate; 5' = MES; 1 = chloride;
2 = phosphate; 3 = salicylate; 4 = acetylsalicylate; 5 = acetate; 6 = propionate. A = increasing UV
absorption; R = increasing resistance; t = time.
with a smaller effective mobility than that of the leading ion, in this particular
instance the chloride ion. The reproducibility of these analyses, performed with a
sample tap, proved to be better than 2%. Nevertheless, a more mobile ion than chloride
was sought. The literature indicates that the Fe(CN6)& ion is a fast moving ion at infinite
dilution and is much faster than the chloride ion. For this reason, three operational
systems we re prepared :
i
for Fig.12.7a: 0.01 N Fe(CN6)4- with 0.02 N histidine;
for Fig.12.7b: 0.005 N Fe(CN6)" with 0.01 N histidine;
for Fig.12.7~: 0.001 N Fe(CNJ4- with 0.002 N histidine.
-6
-5
-4
-3
-2
-1

304
-t
H O
2
\
SEPARATION OF ANIONIC SPECIES IN AQUEOUS SOLUTIONS
-
15
~
15
I
I
I ii
YY-
c_
20 sec
Fig.12.5. Separation of a test mixture of anions in the operational system at pH 6 (Table 12.1) with
water and deuterium oxide as solvents. The sequence of the anions is listed in Table 12.9, where the
differences in step height are compared. The current was stabilized at 70 PA. A = Increasing UV
absorption; R = increasing resistance; r = time.
1

SEPARATION USING CONDUCTIVITY AND UV DETECTORS 305
An injection was made of an industrial sample that contained the chloride ion and the
isotachopherograms shown in Fig.12.7 were obtained. No attention will be paid to the
mixture of anions (x).
In Fig.l2.7a, it is clear that the chloride ion is more mobile than the Fe(CN6)4- ion.
This can be seen in the UV trace and the linear trace of the conductivity detector. -. It
should be noted that the concentration of the original Fe(CN6)& thistidine is not changed
after the passage of the chloride ion, which can be seen in the linear trace of the conductivity
detector and the linear trace of the W absorption detector [Fe(CN,)4- has a W
absorption]. In Fig.12.7b, a mixed zone between Cl- and Fe(CN6>e can be seen. In
Fig. 12.7c, Fe(CN6)4- is moving in front of Cl-.
2+3+
I--
"f
-
-+t
c ! : I
t
t I- 1"
r I b C
Fig.12.6. Separation of nitrate andsulphate in the operationalsystemat pH 6 (Table 12.1) withwater
and deuterium oxide as the solvents. The difference in solvation of these two solvents is clearly visible.
1 = Chloride; 2 = nitrate; 3 = sulphate; 4 = acetate. The current was stabilized at 70 PA. A = Increasing
W absorption; R = increasing resistance; t = time.

306 SEPARATION OF ANIONIC SPECIES IN AQUEOUS SOLUTIONS
3 @ 2+i 1 3 0 2
Fig.12.7. Isotachopherograms with Fe(CN,)4- as the leading ion at concentrations of (a), 0.01 N;
(b), 0.005 N; (c), 0.001 N. Histidine was used as the buffering counter ion. (a) shows that C1- is more
mobile than Fe(CN,)4-, but that the original concentration is not changed if this mobile chloride
passes the first separation boundary; (b) shows that a mixed zone is obtained; (c) shows that Cl- is
less mobile than Fe(CN,)4-. The current was stabilized at 70 MA. A = Increasing W absorption;
R = increasing resistance; t = time. 1 = Fe(CN,)4-; 2 = C1-; 3 = MES-; 4 = mixture of anions.
Quantitative analyses could easily be performed, although the leading electrolyte must
be freshly prepared each time as it is unstable. It should be repeated that the concentration
change in the leading electrolyte of the operational system may give this system
completely different properties.
The concentration adjustment is not influenced if impurities with a higher mobility
than that of the leading ion pass the first separation boundary.
The test mixture of anions (Fig.12.5) was also analyzed in the mixture urea-water
(1: 1). Differences similar to those in the isotachopherograms shown in Chapter 16 were
obtained. Because of the high concentration of urea, these operational systems are
difficult to work with, for practical reasons as the eqhpment soon becomes covered
with urea. Nevertheless, this system can be used, because many organic substances are
more soluble in it and it is not aggressive.

SEPARATION USING CONDUCTIVITY AND UV DETECTORS 307
TABLE 12.7
RELATIVE STEP HEIGHTS OBTAINED IN THE OPERATIONAL SYSTEMS LISTED IN TABLES
12.1, 12.4,12.5 and 12.6 FOR VARIOUS ANIONIC SPECIES
The accuracy is better than 4%. The values given are to be used only for the identification of anionic
species in isotachophoretic analyses in the operational systems considered. The chloride ion (leading
ion) has a relative step height of 0, while the chlorate ion has relative step height of 100. At pH
3.0 the current was stabilized at 90 wA; at pH 4.5 the current was stabilized at 80 MA; at pH 6.0
the current was stabilized at 70 PA; at pH 7.5 the current was stabilized at 80 MA. h = step height.
-_- - No UV absorbance; - = not measured.
Ionic species pH = 3.0 pH = 4.5 pH = 6.0 pH = 7.5
Aspartic acid, dl.
benzyl
Acetic acid
Acetic acid,
monochloro
Acetic acid, dichloro
Acetic acid, trichloro
Benzoic acid
Benzoic acid,
p-amino
Benzoic acid,
2,4-dihydroxy
Benzoic acid, p-
nitro
Butyric acid
Cacodylic acid
Capric acid
Caproic acid
Caprylic acid
Chlorate
Chromic acid
Chromic
acid, hi
Citric acid
Enanthic acid
Formic acid
E'umaric acid
Glucaric acid
Glucuronic acid
Glu tamic acid
Glycerinic acid
Glycolic acid
Gluconic acid
lOOh UV lOOh UV lOOh UV 100h UV
absorp- 7 absorp- 7 absorp- - absorphchlorate
tion chlorate tion chlorate tion hchlorate tion
-
3880
745
578
656
2810
6290
1460
1600
5280
n.s.m.*
n.s.m.
6520
7280
100
184
210
1370
6750
1010
980
-
2200
3750
1670
1620
-
Hippuric acid 1370
-
1090
516
537
699
1200
1780
930
890
1240
6610
6260
1930
2320
100
173
168
51 9
2040
285
338
-
1310
1510
760
587
-
1180
1150
484
487
542
620
726
836
803
771
751
1509
1486
905
1062
100
134
132
259
988
208
217
355
1033
927
629
456
1023
962
1139
466
465
5 24
596
7 23
768
734
759
725
940
1410
859
101 1
100
33
35
178
94 8
209
230
_
1019
887
61 1
469
-
412

308 SEPARATION OF ANIONIC SPECIES IN AQUEOUS SOLUTIONS
TABLE 12.7 (continued)
Ionic species pH = 3.0 pH = 4.5 pH = 6.0 pH = 7.5
Iodic acid
orKetoglutaric
acid
Lactic acid
Laevulinic acid
Maleic acid
Malic acid
Malonic acid
Me thacrylic
acid
Naphthalene-2-
sulphonic acid
Nicotinic acid
Nitric acid
Nitrous acid
Orotic acid
Oxalic acid
Pelargonic acid
Perchloric acid
Phenylacetic
acid
Phosphoric acid
Phthalic acid
Picric acid
F’imelic acid
Pivalic acid
Propionic acid
Propionic acid,
p-chloro
Pyrazine, 2,3-
dicar box ylic
acid
Pyrazole, 33-
dicarbox ylic
acid
Salicylic acid
Succinic acid
Sulphamic acid
Sulphanilic acid
Sulphuric acid
Sulphurous acid
Tartaric acid
Tartronic acid
lOOh uv lOOh uv lOOh w 100h w
absorp 7 absorp- 7 absorp- - absorp-
‘chlorate tion chlorate tion chlorate tion %hlorate tion
498
915
1910
4220
508
1460
750
3360
830
5040
20
32
970
400
5970
69
3680
840
1280
820
4330
7090
4480
1820
616
876
1060
2720
308
1260
33
364
1000
696
17
__
_-
_-
50
__
__
8
89
100
__
__
90
5
__
__
12
__
72
100
-_
__
__
__
73
65
30
__
__
100
__
--
__
-_
*n.s.m. = not sufficiently mobile.
500
505
1060
1390
491
5 24
469
1100
910
1570
21
31
810
120
1880
63
1210
750
5 00
890
1270
1910
1460
820
368
303
686
84 1
311
7 87
49
332
321
274
464
259
609
744
305
259
209
619
798
731
35
25
738
93
1150
75
823
614
382
766
450
859
635
6 29
296
293
647
301
294
688
60
284
233
157
460
250
593
719
207
253
169
6 24
779
667
33
19
720
83
1023
72
814
326
346
753
412
821
604
565
290
288
667
279
303
719
48
172
226
142

SEPARATION USING CONDUCTIVITY AND UV DETECTORS 309
TABLE 12.8
RELATIVE STEP HEIGHTS OBTAINED IN THE OPERATIONAL SYSTEMS AT pH 6
(TABLE 12.1) FOR VARIOUS ANIONIC SPECIES WITH WATER AND DEUTERIUM-OXIDE
AS SOLVENTS
The accuracy is better than 4%. The values are given for comparison of the two solvents. The
experiments with water were carried out at 70 PA and those with deuterium oxide at 80 PA*. In
both solvents the chloride ion (leading ion) has a relative step height of 0, while the chlorate ion has
a relative step height of 100. h = step height. -- = No UV absorbance.
lonic species
Aspartic acid, dl-benzyl
Acetic acid
Acetic acid, monochloro
Acetic acid, dichloro
Acetic acid, trichloro
Benzoic acid
Benzoic acid, p-amino
Benzoic acid, 2,4-dihydroxy
Benzoic acid, p-nitro
Butyric acid
Cacodylic acid
Capric acid
Caproic acid
Caprylic acid
Chlorate
Chromic acid
Chromic acid, bi
Citric acid
Enanthic acid
Formic acid
Furnaric acid
Glucuronic acid
Glutamic acid
Glycolic acid
Hippuric acid
Iodic acid
a-Ketoglutaric acid
Lactic acid
Laewlinic acid
Maleic acid
Malic acid
Malonic acid
Methacrylic acid
Naphthalene-2-sulphonic acid
Nicotinic acid
Nitric acid
Nitrous acid
Orotic acid
100 h
hchlorate
1150
4 84
487
542
620
7 26
836
803
771
751
1509
1486
905
1062
100
134
132
259
988
208
217
1033
927
456
962
464
259
609
744
305
259
209
619
798
731
35
25
7 38
uv
absorption
(%)
40
100 h
hchlorate
1244
519
526
583
669
7 86
923
873
840
818
1787
1814
998
1170
100
146
145
277
1099
217
225
1143
1015
503
1060
486
268
643
801
323
27 2
224
680
858
817
28
17
808
uv
absorption
(%I
(Continued on p. 310)

310 SEPARATION OF ANIONIC SPECIES IN AQUEOUS SOLUTIONS
TABLE 12.8 (continued)
Ionic species
Oxalic acid
Pelargonic acid
Phenylacetic acid
Phosphoric acid
Phthalic acid
Picric acid
Pivalic acid
Propionic acid
Propionic acid, p-chloro
Pyrazine, 2,3-dicarboxylic acid
Pyrazole, 3,5-dicarboxylic acid
Salicylic acid
Succinic acid
Sulphamic acid
Sulphanilic acid
Sulphuric acid
Sulphurous acid
Tartronic acid
HZ0 D*O
- -
lOOh uv lOOh W
hcMorate
absorption
(%)
&hlorate
absorption
(%)
93
1150
823
614
382
766
859
635
6 29
296
293
647
301
294
688
60
284
157
88
1252
915
677
410
819
972
699
701
321
316
703
314
313
736
57
319
167
*The step height is not influenced by this difference in electric current, if conductivity detection is
applied (a.c. method).
TABLE 12.9
DIFFERENCES IN THE EFFECTIVE MOBILITIES OF SOME ANIONS IN THE OPERATIONAL
SYSTEM AT pH 6 (TABLE 12.1) WITH WATER AND DEUTERIUM OXIDE AS THE SOLVENTS
The values (h) are the step heights (mm) that can be measured in the linear trace of the conductivity
detector.
Ionic species 'H,O hD,O Ionic species hH,O hD,O
Sulphate 5.6 5.6 p-chloropropionate 58.4 69.5
Chlorate 9.3 9.8 Benzoate 67.5 76.6
Chromate 12.4 14.2 Naphthalene-2sulphonate 74.0 83.7
Malonate 18.8 21.8 Glutamate 86.1 99.0
Pyrazole-3,5-dicarboxylate 27.5 30.8 Enanthat e 92.2 102.2
Adipate 37.2 42.8 Benzyl-dl-aspartate 107.4 121.6
Acetate 44.9 51.4
~~

Chapter 13
Amino acids, peptides and proteins
SUMMARY
The separation of amino acids in aqueous solutions at low pH, at high pH and at
‘neutral’ pH when propionaldehyde is added to the electrolytes is discussed. Experimental
data for the amino acids in several operational systems are given. The separation of
proteins in an operational system at neutral pH is discussed. The addition of a mixture of
amphiprotic substances, by which the proteins are diluted in their zone, stabilizes
proteins of high moleculer weight, although this technique deviates from the originiil
principle of isotachophoresis as discussed in Chapter 4. For small peptides, the addition
of amphiprotic substances is not necessary.
The time of analysis is approximately 15 min from the start of the experiment to the
detection of the last zone.
13.1. AMINO ACIDS
13.1.1. Introduction
The analysis of amino acids is extremely important and nearly all separation techniques
have been applied to them. Good results have been obtained by various research workers
who analyzed these substances by liquid chromatography [ 1,2] , gas chromatography and
electrophoresis. Many references can easily be found and they are not cited here because
only an incomplete list could be given. So far, little attention has been paid to the
separation of amino acids by isotachophoresis [3-51.
In this section, we discuss various systems in which amino acids can be analyzed by
isotachophoresis. The application of ths technique to amino acids is particularly
interesting because in theory they can be separated both as cations and as anions. The
possibility of achieving a complete separation according to pK values (Chapter 5) is, of
course, considered first. It is clear that no systems at a neutral pH can be chosen,
because most amino acids have their pZ values at neutral pH and hence will have a
negligible migration in an electric field. It is also well known that amino acids form
stable complexes, e.g, with metals and aldehydes. If such a complex is formed, not only
the molecular size and solvation change, but also the pK values, and the effective mobility
therefore changes in an operational system chosen.
Several operational systems are considered below in order to show complex formation
and the variations in the effective mobilities. Much more research, however, needs to be
carried out. In particular, solvents or a combination of solvents in which the amino acids
are more soluble than they are in aqueous systems must be sought. Unusual combinations
of systems may be obtained e.g., a combination of urea and water to increase the
solubility of the amino acids, to which an aldehyde must be added to decrease the pl
31 1

312
AMINO ACIDS, PEPTIDES AND PROTEINS
values of the amino acids (Schiff base formation) and methanol to stabilize the aldehyde
(e.g , propanal). In this section, however, these solvent systems are not discussed as
this topic is beyond the scope of this book.
The analyses were carried out with the equipment described in Chapter 7, using the
modified injection block, the counter electrode compartment with a flat membrane and
hgh-resolution detectors. The conductivity detector (a.c. method) and the UV absorption
detector (256 nm) were combined.
The operational systems considered represent only a few of many possible combina-
tions, and were chosen arbitrarily, although optimal characteristics were sought. Because
the operational systems in the equipment can be changed quickly (it usually does not
take longer than the normal rinsing and re-filling procedure) and the time of analysis is
relatively short (10-1 5 min), sometimes a complete separation between the amino acids
in a given mixture can be better achieved by applying two or more systems rather than
by optimizing a single system. It will be recalled that a small difference in effective
mobility will increase drastically the time of analysis and longer narrow-bore tubes or
a counter flow of electrolyte must be applied. The last technique is effective only if the
difference in the effective mobilities of the various amino acids is still sufficiently large
(see Chapter 17).
13. I .2. Separation at low pH values in aqueous systems
At low pH, most amino acids will migrate as cations. However, most amino acids also
have a low effective mobility, so that the pH of the amino acid zone will be lower than
that of the leading electrolyte zone. Soon so many H ions are present that a significant
proportion of the electricity is carried by the protons. As a result, the amino acids show
only small differences in step height as measured in the linear trace of the conducti-
metric signal*. Only the amino acids L-lysine, L-arginine and L-histidine have a sufficiently
high mobility that they can be separated without a visible disturbance of the protons.
It does not need further explanation that the pH of the leading electrolyte cannot be
decreased too far, because soon zone electrophoretic phenomena occur, e.g., ‘elution’ by
the protons.
For basic amino acids, an operational system is given in Table 13.1.
13.1.3. Separation at high pH values in aqueous systems
If the pH of the leading electrolyte is above 8, most amino acids will have an effective
mobility suitable for a separation according to the isotachophoretic principle. A disad-
vantage is that at such pH values disturbances from carbon dioxide from the air can be
expected. More information on this aspect is given in Chapter 9. If suitable precautions
are not taken, the carbonate (hydrogen carbonate) may even obscure the analysis. For
optimal results, we found that the electrolytes of the operational systems must be
prepared under an atmosphere of nitrogen and stored in polyethylene bottles under
*The step height is a measure of the qualitative information in isotachophoretic measurements. It
indicates whether a complete separation can be expected, as it is a measure of the effective mobility.

AMINO ACIDS
TABLE 13.1
OPERATIONAL SYSTEM AT pH 5.4 SUITABLE FOR CATIONIC SEPARATIONS
Solvent: H, 0.
Electric current @A): Ca. 50-100.
Electrolyte
-~
Leading Terminating
Cation K+ DL-Ala’
Concentration 0.01 N Ca. 0.01 N
Counter ion
PH
CH, COO-
5.4
OH- [added as Ba(OH),]
>I
Additive 0.05% Polyvinyl
alcohol (Mowiol)
None
nitrogen. Moreover, barium hydroxide of pro analysi quality must be added to the
terminating electrolyte in order to prevent any carbonate (hydrogen carbonate) from
penetrating into the narrow-bore tube via the reservoir filed with the terminating
electrolyte. The addition of barium hydroxide to the leading electrolyte did not improve
the separation, however. Because of the high mobility of Ba2+, less sharp boundaries
can be expected.
If suitable precautions are taken, some carbonate (hydrogen carbonate) may still be
detected if an anion with a lugher effective mobility is used as the leading ion. This
carbonate (hydrogen carbonate), however, did not obscure both the qualitative and
quantitative results. Only a leading ion with an effective mobility higher than or equal to
that of the carbonate (hydrogen carbonate) ion can be used, otherwise the carbonate
may no longer be visible but may still disturb or at least obscure the analytical result, if
it is supported continuously. The resolution will decrease and zone electrophoretic
phenomena can soon be expected e.g., ‘elution’ by the carbonate (hydrogen carbonate)
ions.
Several suitable buffers are commercially available that enable one to work at high pH.
Some of them, including bis(3-aminopropyl)amine, trime thylenediamine, L-arginine,
1-methylpiperidine, octylamine, ethanolamine and L-lysine, were tested for purity and
effective mobility*. Some of the compounds were found to be very impure and even
could not be purified satisfactorily by the usual methods such as distillation, recrystallization
or ion exchange. Some of the counter ions show unexpected phenomena, e.g.,
enforced isotachophoretic systems or undesirable complex formation. Of the compounds
mentioned above, only 1 -methyl piperidine, L-arginine, L-lysine, octylamine and
ethanolamine gave good results.
In Tables 13.2 and 13.3, two operational systems are given in which analyses were
performed and for which more data will be given later.
*If a counter ion (buffer) is too mobile, a considerable proportion of the electricity is carried by this
counter ion, which results in less sharp zone boundaries and a decrease in resolution.
31 3

314
TABLE 13.2
AMINO ACIDS, PEFTIDES AND PROTEINS
OPERATIONAL SYSTEM AT pH ABOUT 9 SUITABLE FOR ANIONIC SEPARATIONS
Solvent: H, 0.
Electric current (/.LA): CQ. 50-100.
Electrolyte
Leading Terminating
__-
Anion 5-Br-2,4-diOH-C6 H, COO- 0-Ala- (recrys-
tallized from
waterethanol)
Concentration 0.004 M CQ. 0.01 M
Counter ion HOC, H, N'H Baa+ [added as Ba(OH),]
PH 9.0, 9.2, 9.4, 9.6 Ca. 10.5
Additive 0.05% Polyvinyl None
alcohol (Mowiol)
TABLE 13.3
OPERATIONAL SYSTEM AT pH ABOUT 9 SUITABLE FOR ANIONIC SEPARATIONS
Solvent: H, 0.
Electric current (fiA): Ca. 50-100.
Electrolyte
Leading Terminating
Anion 5-Br-2,4-diOH-C6 H, COO- P- Ala- (recrystallized
from wa tere thanol)
Concentration 0.004 M Ca. 0.01 M
Counter ion L-Lys+ Ba2+ [added as Ba(OH), J
PH 9.1, 9.2, 9.4 Ca. 10.5
Additive 0.05% Polyvinyl
alcohol (Mowiol)
None
The leading ion in these systems was chosen because its effective mobility is almost
identical with the effective mobility of the carbonate (hydrogen carbonate) ion. In the
linear trace of the conductivity detector, the carbonate (hydrogen carbonate) step is no
longer visible*. The leading ion, however, has a W absorption. If too much carbonate
(hydrogen carbonate) is present, it is present in the UV trace, which is used only to mark
the UV-absorbing zones and has so far not been applied for qualitative determinations.
The carbonate (hydrogen carbonate), if present, is enriched just before the zones of the
isotachophoretic 'train', as can be seen in Fig. 13.1.
*This simplifies the qualitative information considerably.

AMINO ACIDS
8-
?-
6-
5-
4-
3-
2-
f-
I
20 5.c
Fig.13.1. Isotachopherogram of the separation of a mixture of amino acids obtakled with the
operational system listed in Table 13.3. 1 = Chloride; 2 = Asp; 3 = Cys; 4 = I,-Tyr; 5 = Asn; 6 = Ser;
7 = Tyr; 8 = Gly; 9 = Trp; 10 = Ile; 11 = P-Ala. All are L-amino acids. R = Increasing resistance;
A = increasing UV absorption; t = time.
1"
315

316 AMINO ACIDS, PEPTIDES AND PROTEINS
TABLE 13.4
STEP HEIGHTS (mm) FOUND IN THE LINEAR TRACE OF THE CONDUCTIVITY DETECTOR
SIGNAL IN THE ISOTACHOPHEROGRAMS OBTAINED WITH THE OPERATIONAL SYSTEM
LISTED IN TABLE 13.2.
Amino PH
acid
L-Asp
L-cys
L-Glu
I, -L-Tyr
L-Ser
L-Thr
DL-Tyr
DL-Met
Gly
L-His
L-Phe
L-Ma
L-Val
L-Trp
3-L-Hyp
L-Ile
L-Leu
P-Ala
9.00 9.20 9.36 9.55
40.5
51.5
49.5
76
112.5
113
135
133
138.5
145
147.5
180
184
191
185
203.5
205
239
33
39.5
40
63
88
98.5
114.5
116
119
124
127.5
154
159
161.5
162
172.5
172.5
210
30.5
35.5
34
59
84
90
107
112.5
106
118
121
147
151
157
151
162.5
164.5
20 1
38.5
34.5
35.5
58
85
94.5
105
117.5
110.5
123
126.5
149
155
161
154
167
170
190
The results obtained when using the operational systems specified in Tables 13.2
and 13.3 are given in Tables 13.4 and 13.5.
Differences in step heights of about 15-20 mm are sufficient for a complete separation
of the various amino acids. The differences found in the two operational systems
considered must be ascribed mainly to the difference in effective mobility of the counter
ion used. While in the operational system specified in Table 13.2 about eight amino
acids can be separated in a single run, in that specified in Table 13.3 about ten amino
acids can be separated simultaneously. L-Lysine has a very small effective mobility at the
pH of the leading electrolyte chosen, while the effective mobility of ethanolamine is
considerably greater.
The idea that pure water, adjusted to a high pH by adding barium hydroxide, can be
used as an optimal terminating electrolyte in operational systems at high pH is nearly
always wrong. If double-distilled water adjusted to a high pH is applied as the terminating
electrolyte*, one can expect the buffer capacity of the counter ion to be insufficient. If,
instead, a suitable terminator, e.g., p-alanine, is added to the water, also adjusted to a high
pH, the pH of the zone of the terminating electrolyte does not need to be increased so
*OH- may carry the electricity because the pH in the zone is increased sufficiently as water is a weak
acid in this electrolytic system. This, in combination with the high absolute mobility, will give OH-
a sufficient high effective mobility.

AMINO ACIDS 317
TABLE 13.5
STEP HEIGHTS (mm) FOUND IN THE LINEAR TRACE OF THE CONDUCTIVITY DETECTOR
SIGNAL IN THE ISOTACHOPHEROGRAMS OBTAINED WITH THE OPERATIONAL SYSTEM
LISTED IN TABLE 13.3.
Amino PH
acid
L-Asp
L-cys
L-Glu
I, -L-Tyr
L-Ser
L-Thr
DL-Tyr
DL-Met
GlY
L-His
L-Phe
L-Ala
L-Val
L-Trp
3-L-Hyp
L-Ile
L-Leu
p-Ala
9.01 9.22 9.42
32
40.5
37.5
61
93
95.5
122
118.5
135
122
130
176
169
171
170
188
190
24 0
29
36
33.5
58
82
87.5
111.5
110.5
124.5
128.5
121
165
160
162
161.5
180
178
233
27.5
31
33.5
51
78
88
104
106
117
109
116
155
153
156
152.5
171
170
205
much because 0-alanine is a stronger acid than water under the conditions chosen. Conducti-
metric recordings of analyses in which double-distilled water, adjusted to a high pH, and
analyses in which water plus a suitable terminator are applied, showed that the zone
boundaries were less well defined and that the conductivity finally attained is smaller.
An isotachopherogram obtained in the operational system specified in Table 13.2 is
shown in Fig.13.2. Fig.13.1 and 13.2 also show the differences that can be found in
Tables 13.3 and 13.4.
Fig.13.3 illustrates the possible mixed zones that can be expected if a mixture of
amino acids is analyzed. Two amino acids with close effective mobilities were injected in
the system specified in Table 13.3, at a pH of the leading electrolyte of 9. A black square
indicates that a mixed zone can be expected in a time of analysis of about 12 min (70pA).
A decrease in the concentration of the leading electrolyte did not result in substantial
differences in the effective mobilities of the various amino acids. An increase must be
avoided because the amino acids are not sufficiently soluble. So far in our laboratory no
research has been carried out to find electrolyte systems in which the amino acids are
more soluble. Combinations of non-ionic substances with water seem to have good
prospects, as the amino acids are not sufficiently soluble in methanol.

318 AMINO ACIDS, PEPTIDES AND PROTEINS
9-
1
8- - 20 Sm2
7-
6-
5-
4-
3-
2-
Fig.13.2. Isotachopherogram of the separation of a mixture of amino acids obtained with the
operational system listed in Table 13.2. 1 = Chloride; 2 = Asp; 3 = I, -Tyr; 4 = Ser; 5 = Tyr; 6 = Phe;
7 = Ala; 8 = Leu; 9 = p-Ala. All are L-amino acids. A = Increasing UV absorption; R = increasing
resistance: t = time.
13.1.4. Separation by use of complex formation
It is well known that amino acids form complexes with metal ions, e.g., Cu*+.
In an
aqueous solution of copper(I1) sulphate, the addition of various amino acids cause a
colour change, which indicates that a complex is formed. Isotachophoretic experiments
have shown that only a few of these complexes are sufficiently stable to be detected as
real complexes. An isotachopherogram of a copper-histidine complex is shown in
Fig.13.4C. In the isotachopherogram in Fig.13.4A, 0.2 pl of 0.01 M L-histidine solution
was injected, in Fig.13.4B 0.2 pl of 0.005 M copper(l1) sulphate solution was injected and
in Fig.13.4C 0.4 pl of the solution obtained by mixing equal volumes of these two solu-
tions was injected, with the operational system specified in Table 13.1.
When other amino acids were examined, however, their complexes were found to be
too unstable. No further research was carried out on this aspect. It may be that the field
strength applied in isotachophoretic analyses is too great or the complexation constants

AMINO ACIDS
Fig.13.3. Schematic diagram illustrating that mixed zones are found in isotachophoretic analyses if
the differences in effective mobilities are too small. A black square indicates that a mixed zone is
obtained between that pair of amino acids if they are present in one sample. The experiments were
carried out in the operational system listed in Table 13.3.
are too small. If so, a method may be found for determining complexation constants in
isotachophoretic analyses by varying the field strength in various experiments.
13.1.5. Separation in aqueous propanal solutions
It is well known that amino acids easily form complexes (Schiff bases) with aldehydes.
If amino acids are dissolved in a solution that contains an aldehyde, differences in
mobility can be expected because the solvent property changes, the amino acid molecule
is larger after complex formation and its pZ value changes because the complex is formed
with the amino group(s). The first two effects result in a small difference and the last
effect in a large difference in mobility. The last effect occurs at very low concentrations
of the aldehyde, assuming that the aldehyde character is great enough.
Experiments with sugars did not show substantial differences in the effective mobilities
of the various amino acids, especially if they were added in relatively low concentrations.
Formaldehyde, which has a strong aldehyde character, and acetaldehyde were found
to be very unstable. Even during analysis, formic acid and acetic acid, respectively, are
formed. Research is continuing to find a suitable combination of a non-ionic stabilizer(s)
in order to work reproducibly with formaldehyde and acetaldehyde.
319

320
/
c
Cu-His complex
/
-
t
B
AMINO ACIDS, PEPTIDES AND PROTEINS
A
His +
-
t
Fig.13.4. Isotachopherogram obtained with the operational system listed in Table 13.1. Species
injected: A, histidine; B, CuSO,; C, a mixture of the two. R = Increasing resistance; f = time.
With propionaldehyde, experiments could be carried out satisfactorily, although it
must be distilled several times under nitrogen. Even after distillation, propionic acid is
present in small amounts, but it was found that the amount of propionic acid did not
increase during the analysis. A similar disturbance to that discussed briefly in section
13.1.3, due to carbonate (hydrogen carbonate), can be expected; this disturbance does
not obscure the analytical results either qualitatively or quantitatively.
For optimal information, before the analyses were carried out, the pK values of the
various amino acids were determined in aqueous propionaldehyde solutions of various
concentrations, and the results are given in Table 13.6. It can be seen that the pK values
of the acidic groups decrease, because the amino groups are blocked.
The analysis of some amino acids was carried out in a solution containing 3% of
propionaldehyde, with the operational system specified in Table 13.7. The leading
electrolyte was adjusted to pH 7.2 because it was found that the pK value of ethanolamine
was 7.2 in a 3% propionaldehyde solution. Only measurements at ‘neutral pH: are possible
with an aqueous solution containing 3% of propionaldehyde. At a pH of the leading
electrolyte, which contains propionaldehyde, of above 8, a white insoluble component is
formed after some time.
In Table 13.8, some step heights of amino acids obtained in the operational system

AMINO ACIDS
TABLE 13.6
DETERMINATION OF pK VALUES IN AQUEOUS PROPIONALDEHYDE SYSTEMS
Amino
acid
L-His
L-G~U*
L-AS~*
L-Ile
L-Leu
L-Val
L-Phe
DL-Ala
L-Met
L-Ser
L-Thr
3-L-Hyp
L-Trp
L-Tyr
GlY
L-Arg
L-Lys
*PK~ value.
~
Propionaldehyde concentration (mole%)
0.0 2.5 4.0 8.0
9.18
9.47
9.82
9.758
9.744
9.719
9.24
9.866
9.21
9.15
9.73
9.39
10.07
9.778
12.48
10.53
7.81
8.53
8.87
8.38
8.83
8.57
8.17
9.03
8.10
7.60
7.28
9.36
8.50
8.95
7.62
8.42
7.70
8.47
8.58
8.33
8.41
8.45
7.90
8.50
7.90
7.35
7.23
8.95
8.24
8.05
8.31
7.22
8.20
321
7.93
8.38
8.50
8.15
8.26
8.20
7.88
8.37
7.90
6.35
6.15
8.10
8.00
8.17
7.98
specified in Table 13.7 are given. Table 13.8 shows that the analysis can be performed at a
relatively low pH. By this means, the disturbance due to the carbonate (hydrogen
carbonate) is prevented, although in its place a disturbance due to propionic acid has to
be dealt with.
For the operational system specified in Table 13.7, the possibility of the formation of
TABLE 13.7
OPERATIONAL SYSTEM AT pH ABOUT 7.5 SUITABLE FOR ANIONIC SEPARATIONS IN
AQUEOUS PROPANAL' SOLUTIONS
Solvent:
H, 0 + 3% C, H, CHO.
Electric current kA): < 50.
Electrolyte
Leading Terminating
Anion c1- DL-Ma-
Concentration 0.01 N c4. 0.01M
Counter ion HOC, H, N+H Tris+
PH 7.2, 7.8 c4. 7 (

322 AMINO ACIDS, PEPTIDES AND PROTEINS
TABLE 13.8
STEP HEIGHTS (mm) FOUND IN THE LINEAR TRACE OF THE CONDUCTIVITY DETECTOR
SIGNAL IN THE ISOTACHOPHEROGRAMS OBTAINED WITH THE OPERATIONAL SYSTEM
LISTED IN TABLE 13.7
Amino PH
acid
L-cys
L-His
L-Ser
L-Thr
L-Glu
L-Asp
L-Asn
L-Met
L-Ile
Gly
L-Val
L-Trp
L-Tyr
I, -L-T~I
L-Phe
DL-Ala
7.30 7.80
55
67.5
46
43
29.5
24.5
43.5
115
175
170
168
147
135
51
128
190
29.0
60
41.5
39
13.5
13
30
84
128
108
32
103
175
mixed zones has been studied. Pairs of amino acids with comparable effective mobilities,
determined experimentally, were injected simultaneously into the system at pH 7.2. The
results are given in Fig.13.5. This figure, amongst others, shows that histidine can be
separated completely from the other amino acids, which was not possible in the aqueous
systems considered.
The disadvantages of using propionaldehyde are its instability, the extra work involved
in preparing the operational system and its relatively low boiling point (48 C). Because
of the last property, one can work only with relatively mobile components or at low
current densities.
In Fig.13.6, an isotachopherogram is shown of some amino acids obtained in the
operational system specified in Table 13.7. The UV trace is of lower quality than those
with comparable systems in aqueous solutions because propionaldehyde shows a
significant absorption at 256 nm.
13.2. SEPARATION OF PROTEINS IN AMPHOLYTE GRADIENTS
13.2.1. Introduction
While small peptides can usually easily be analyzed by isotachophoresis, the analysis
of proteins is often troublesome. If the proteins are not denatured, they are stacked in
small zones that have a high density, which is not ideal for isotachophoretic separations.

SEPARATION OF PROTEINS IN AMPHOLYTE GRADIENTS
Fig.13.5. Schematic diagram illustrating the formation of mixed zones in isotachophoretic analysis
if the differences in effective mobilities are too small. A black square indicates that a mixed zone is
obtained between that pair of amino acids if they are present in one sample. The experiments were
carried out in the operational system listed in Table 13.7.
The detection of these zones with a thermometric detector is difficult, because a zone
length of at least 5 mm in the narrow-bore tube is required for a complete qualitative
and quantitative determination. Even the resolution of a conductivity or UV detector
(Chapter 6) is often not sufficient.
Moreover, the proteins adhere to the wall of the electrophoretic equipment, even to
the ‘inert’ PTFE wall of the narrow-bore tube, so that the electroendosmosis changes
and hence the profile of the boundaries may also change. Owing to this effect, the
profile finally detected may vary from one analysis to another if no precautions are
taken (a rigorous washing procedure between analyses) or if one is not aware that these
changes are taking place. Another drawback, if proteins are being analyzed, is that the
micro-sensing electrodes of the conductivity probe can easily become coated with a
film of proteins that is not caused by an electric leak to earth (see Chapter 6). If a film
of protein adheres tathe wall of the micro-sensing electrodes the final signals from the
conductivity detector (a.c. method) may give non-reproducible results that are difficult
to interpret. It will be recalled that the potentiometric detector (d.c. method) is less
sensitive to these effects (Chapter 6).
Special components can be added to the sample of the proteins, which may both
dilute the protein zone (carrier function) and space the protein zones from each other. If
323

3 24
6- i
5-
4-
3-
2-
1-
\
t--
20 Sec
I 1.
AMINO ACIDS, PEPTIDES AND PROTEINS
Fig.13.6. Isotachopherogram of the separation of a mixture of amino acids obtained in the operational
system listed in Table 13.7. 1 = Chloride; 2 = propionate; 3 = Asp; 4 = I,-Tyr; 5 = His; 6 = Met;
7 = Tyr; 8 = Val; 9 = Ala. All are L-amino acids. A = Increasing UV absorption; R = increasing resistance;
t = time.
these samples are analyzed, one can say that they are being analyzed in isotachophoretic
operational systems, but owing to the presence of the additives isotachophoresis as
strictly defined (see Chapters 2 and 4) does not take place. The proteins are much more
stable if these additives are included, because not only are they present as a single substance
with the counter ion in a specific zone (as is common in isotachophoretic analyses),
but also the density is much lower. Hence the electric current can pass much more easily
and excessive temperatures do not occur. These two effects were verified experimentally.
Ampholines (LKB, Bromma, Sweden) so far seem to be compounds that can be applied
both for the dilution of the various zones (carrier function) and €or spacing the various
zones (spacer function), because they consist of numerous amphiprotic compounds. The
ampholines are mixtures of polyamino polycarboxylic acids of general structure
- CH2 -N-( C Hz )x - N-( CH2 )x-NR2
I I
where x = 2 or 3 and R = H or -CHz -CHz -COOH. These compounds are commonly
applied in isoelectric focusing experiments in order to create a stable pH gradient.
In Fig. 13.7, the space function and the carrier function are shown schematically.
As already said, the ampholyte mixtures will give both characteristics to the separation in

SEPARATION OF PROTEINS IN AMPHOLYTE GRADIENTS 325
I I I
Fig.13.7. [llustration of carrier and spacer functions. If an ionic species S is added to a sample
consisting of ions A and B such that mA > ms > mg, and the differences in mobility are sufficient
for a complete separation, S acts as a ‘Spacer’ for ions A and B. If a component C is added to a
sample consisting of ions A and B such that the effective mobility of C is equal to the effective
mobility of B in the operational system chosen, component C acts as a ‘carrier’ for ion B. In specific
instances it is possible for a component to be added such that a mixed zone is formed between the
ions A, B and the component added, although generally an enrichment of A in front and an
enrichment of B at the rear can be expected.
question. One has to bear in mind that although the differences in effective mobility
between the compounds of interest remain constant, the separation capacity decreases
because compounds are added that have effective mobilities between those of the
compounds of interest. The final result of the detection of the various zones, which really
move with equal speed, will be less sharp than under ideal isotachophoretic conditions
because the self-sharpening effect is much lower.
Apart from the addition of, e.g., ampholytes to the leading electrolyte, the terminating
electrolyte can also be doped with a suitable ion with an effective mobility higher than
that of the most mobile protein. However, in some instances the elution effect due to the
substance added may play a dominant role (i.e., isotachophoresis will gradually become
zone electrophoresis). Experiments along these lines will not be discussed in this book,
because they lie far outside its scope.
13.2.2. Experimental
All experiments described in this section were performed in the operational system
specified in Table 13.9. Various operational systems can be used, depending mainly on the
particular proteins to be separated.
Only a general discussion is presented here but we hope it will be sufficient for
scientists interested in the separation of proteins.
Glutamic acid was chosen as the leading ion because it is commercially available in a
very pure form (‘isotachophoretically pure’) and its mobility is sufficiently high in
comparison with that of the most mobile protein at a pH of the leading electrolyte of 7.2.
As already indicated in the analyses discussed in section 13.1, the influence of carbonate
(hydrogen carbonate) on both the qualitative and quantitative results are negligible,

326
TABLE 13.9
AMINO ACIDS, PEF'TIDES AND PROTEINS
OPERATIONAL SYSTEM AT pH 7.2 SUITABLE FOR ANIONIC SEPARATIONS
Solvent: H, 0.
Electric current @A): Ca. 30-50.
Length of narrow-bore tube (cm): Ca. 15.
UV absorption detector wavelength (nm): 256.
Electrolyte
Leading Terminating
Anion Glu- Gly- (adjusted to
a sufficiently
high pH)
Concentration 0.005 M Ca. 0.005M
Counter ion Tris' Tris+
PH 7.2 Ca. 9
Additive 0.05% Polyvinyl None
alcohol (Mowiol)
assuming that the necessary precautions as mentioned in section 13.1 .I are taken. These
precautions must be taken because a less mobile ion is used as the termimator (low
effective mobility) and hence the pH will increase considerably.
In Fig. 13.8, isotachopherograms for several commercially available ampholytes (LKB)
are given. The ampholytes were diluted with double-distilled water (dilution factor 1 :20).
In each instance 0.2 pl of ampholytes with the pZ ranges (b) 3.54, (c) 4-6, (d) 5-8 and
(e) 6-8 were injected, and the leading-terminating electrolyte boundary is shown in (a)
when no sample was introduced. Fig.13.8a also shows the impurities in the electrolytes.
Because the ampholytes were specially developed for use in experiments on isoelectric
focusing, it would be fortuitous if they could be applied directly to experiments based
on isotachophoretic principles. In order to obtain a better gradient between the leading
and terminating electrolytes that would be more suitable for experiments with serum
proteins, various commercially available ampholytes were mixed and several of the mixtures
were found to be suitable. Obviously, if one is interested in the separation of mobile
albumins the most suitable gradient will be completely different from one suitable for
the separation of globulins.
In the remainder of this section we show some isotachopherograms obtained in the
analysis of normal serum and pathological human sera obtained from the St. Josef
Hospital, Eindhoven, The Netherlands. The separations were carried out with the gradient
shown in Fig. 13.9.
The differential trace of the conductivity detector is given in order to show the amount
of substances present, which is not so easy to see if only the linear trace is given. The
gradient, as already mentioned, was determined experimentally by injecting 0.1 pl of a
mixture of ampholytes with different pI ranges in the ratio indicated in Fig.13.9, using
the operational system at pH 7.2 (Table 13.9). It proved to be important to include a larger
proportion of the ampholytes with low p1 ranges.

SEPARATION OF PROTEINS IN AMPHOLYTE GRADIENTS
t
Fig.13.8. Isotachopherogram obtained with the operational system listed in Table 13.9. (a)
Boundary between leading and terminating electrolytes; (b) separation of 0.2 rl (dilution 1:20) of
ampholyte mixture of PI 3.5-4; (c) separation of 0.2 pl (dilution 1:20) of ampholyte mixture of
pI4-6; (d) separation of 0.2 pl (dilution 1:20) of ampholyte mixture of PI 5-8; (e) separation of
0.2 p1 (dilution 1:20) of ampholyte mixture of PI 6-8. A = Increasing UV absorption;R =increasing
resistance; t = time.
In order to compare the results obtained from the analyses of sera, a zone electro-
phoretic separation was also carried out. The experiments were carried out on a porous
cellulose polyacetate strip in veronal buffer, the separated proteins subsequently being
rendered visible with amido black, washed with acetic acid solution and prepared for a
densitometric scan. The results are shown in Fig.13.10 for both normal and pathological
sera. The ratios of the proteins present in these sera are given in Table 13.10.
327

328
-
\
\
30sec
I
R
AMINO ACIDS, PEPTIDES AND PROTEINS
Fig.13.9. Isotachopherogram obtained with the operational system listed in Table 13.9. A 0.1-p1
volume of a mixture of ampholytes (LKB) with a ratio of pI3.5-4: PI 4-6: PI 6-8: water of
1:1.5:0.5:20 was injected. A = Increasing UV absorption;R = increasing resistance; 1 = time.
Figs.13.11 and 13.12 show the separations of the sera in Table 13.10 in a narrow-bore
tube using a conductivity and a UV absorption detector (256 nm). The UV traces are
not shown in Figs.13.1 la and 13.12a as they do not give much useful information, but
if they are of interest Fig.13.8a should be consulted. Fig.13.llb and 13.12b must be
compared with Fig.13.10 in order to obtain a comparison of the isotachophoretic and
zone electrophoretic separations of serum proteins. In order to obtain Figs.13.1 lc and
13.12c, the sera were diluted with the ampholytes by first injecting 0.2 p1 of the serum
to be analyzed into the injection block (Fig.7.5) and then 0.2 p1 of the ampholyte mixture.
This procedure was compared with a procedure in which the sera were diluted before the
analysis, in a small bottle. The reproducibilities of both dilution techniques were identical,
but in the first procedure only a small amount of ampholyte mixture is needed.

SEPARATION OF PROTEINS IN AMPHOLYTE GRADIENTS 329
A
i l l
5 WL
~ .... --+ ..--
Fig.13.10. Separation of a normal serum (A) and a pathological serum (B) by zone electrophoresis.
The analysis was carried out on cellulose polyacetate, the proteins subsequently being coloured with
amido black. The electropherogram was obtained with a Kipp (Delft, The Netherlands) densitometer.
1 = Albumin; 2 = 01, -globulin; 3 = a,-globulin; 4 = p-globulin; 5 = yglobulin. These sera were used in
the analyses in the narrow-bore tubes.
TABLE 13.10
COMPOSITIONS OF NORMAL AND PATHOLOGICAL SERA DETERMINED BY ZONE
ELECTROPHORESIS ON CELLULOSE POLYACETATE STRIPS
This mixture wasused in the analyses presented in Figs.13.11-13.15.
Protein Composition (%)
Normal Pa thological
serum serum
Albumin 45 47
0 1 ~ -Globulin 4 6
~~,-Glob~~lin 12 5
p-Globulin 17 4
y-Globulin
___
22 38
At present it is difficult to draw a conclusion from the results shown in Figs.13.11 and
13.12 because the conditions can vary so easily, resulting in completely different
isotachopherograms.
The isotachopherograms in Figs. 13.1 1 and 13.12, especially the W traces, must be
interpreted in a completely different manner to the traces in Fig.13.10. Although
ampholytes are added to the serum proteins in the analysis shown in Figs.13.11 and 13.12,
a relationship still exists between the amount of a species introduced into the system and
the zone length finally occupied by it, with or without the presence of a supporting

330 AMINO ACIDS, PEPTIDES AND PROTEINS
Fig.13.11. Isotachopherogram of normal serum (Fig.13.10) in an ampholyte gradient (Fig.13.9)
obtained with the operational system listed in Table 13.9. (a) Boundary between leading and
terminating electrolytes; (b) 0.2 pl of normal serum injected; (c) 0.2 MI of normal serum and 0.2 pl
of ampholyte mixture (Fig.13.9) injected. A = Increasing W absorption;R = increasing resistance;
t = time.
electrolyte, assuming that it occupies a position between the boundary of the leading and
terminating zones. Too easy the W traces in Figs.13.11 and 13.12 will be interpreted in a
similar manner to the zone electrophoretic traces in Fig. 13.10. The differential trace from
the conductivity detector is not given because it is rather complex and does not give any
additional information.
The application of a micro-preparative instrument will indicate if the isotachophero-

SEPARATION OF PROTEINS IN AMPHOLYTE GRADIENTS
Fig.13.12. Isotachopherogram of pathological serum (Fig.13.10) in an ampholyte gradient (Fig.13.9)
obtained with the operational system listed in Table 13.9. (a) Boundary between leading and
terminating electrolytes; (b) 0.2 pl of pathological serum injected; (c) 0.2 pl of pathological serum
and 0.2 pl of ampholyte mixture (Fig.13.9) injected. A = Increasing W absorption;R = increasing
resistance; r = time.
grams shown in Fig.13.11 and 13.12 have a practical value, because by applying specific
techniques after the separation more information can be obtained.
The isotachopherograms shown in Fig.13.11 and 13.12 may also be the result of the
reproducible degradation of the various proteins.
Fig.13.13 illustrates the reproducibility of the analysis. In order to show the dif-
ference if another ampholyte mixture is applied, the composition was changed, 0.2 pl of
331

332
AMINO ACIDS, PEPTIDES AND PROTEINS
Fig.13.13. Isotachopherograms of normal serum (Fig.13.10) in an ampholyte gradient obtained with
the operational system listed in Table 13.9. The ampholyte mixture had the following composition:
PI 3.5-4: PI 4-6: PI 6-8: water = 1 : 1 : 0.25 : 20. In both A and B, 0.2 pl of normal serum and 0.2 p1
of ampholyte mixture were injected. A = Increasing UV absorption; R = increasing resistance; t = time.
serum* being diluted with 0.2 pl of the ampholyte mixture. The reproducibility in
Fig.13.13 is acceptable.
Fig. 13.14 demonstrates the effect of a variation in the amount of proteins, in an
identical sample, on the shape of an ampholyte gradient. First 0.1 pl of the ampholyte
mixture was injected and then (a) 0.1 pl, (b) 0.2 p1 and (c) 0.3 pl of normal serum*.
Finally, we present some isotachopherograms that can be compared with those in
Fig. 13.14. These isotachopherograms (Fig. 13.1 5) show that an optimum must always be
sought in the dilution of the serum with the mixture of ampholytes. If too little
ampholyte is added, the sample zones are small and denaturation wil soon occur.
Separate experiments in which a microscope was used to observe the narrow-bore tube
showed that even with human albumin small solid particles are formed (denaturation)
that move between the leading and terminating electrolyte zones, and the particles show
convection. Under isotachophoretic conditions, thermal degradation of the albumin can
soon be expected, as can be understood from Fig.6.7, where the temperatures of zones
*The serum for which an analysis is shown in Fig.13.11 was used.

SEPARATION OF PROTEINS IN AMPHOLYTE GRADIENTS 333
t
i.
Fig.13.14. Isotachopherograms of normal serum (Fig.13.10) in an ampholytegradient obtained with
the operational system listed in Table 13.9. The ampholyte mixture had the following composition:
PI 3.5-4: PI 4-6: PI 6-8: water = 1 : 1 : 0.5: 20. In each instance 0.1 pl of ampholyte mixture was
injected, and (a) 0.1 pl, (b) 0.2 pl and (c) 0.3 p1 of normal serum. A = Increasing UV absorption;
R = increasing resistance; t = time.

334
AMINO ACIDS, PEPTIDES AND PROTEINS
1
Fig.13.15. Isotachopherogram of normal serum (Fig.13.10) in an ampholyte gradient obtained with
the operational system listed in Table 13.9. The ampholyte mixture had the following composition:
pI 3.5-4: pI4-6: PI 6-8: water = I : 1.5: 0.5 : 20. In each instance 0.2 pl of ampholyte mixture was
injected and (a) 0.2 pl, (b) 0.05 pl and (c) 0 p1 of normal serum. A = Increasing UV absorption;
R = increasing resistance; t = time.
in a narrow-bore tube, hanging free in air, are plotted. However, in another system than
applied for the experiments with proteins. In the terminator zone of the operational
system in which the proteins can be separated even higher temperatures can be expected.
If too much ampholyte is added to the serum to dilute the protein zone, the resolution is
decreased.
The isotachopherograms presented in this section show that much more work must be
carried out in this field. The compositicils of the ampholyte mixtures and their

SEPARATION OF SMALL PEPTIDES
reproducibility are very important, because with the ampholyte mixture a constant
gradient, i.e., constant in slope and constant in length, between the leading and
terminating electrolyte zones must be maintained. This is the most important rule in
this typical version of isotachophoretic analysis. More information can be found in ref. 6.
13.3. SEPARATION OF SMALL PEP'IIDES
13.3.1. Introduction
Less attention will be paid to the separation of small peptides, because most of them
can be analyzed both in the system in which amino acids can be separated (Table 13.3)
and in the system in which the analyses with the proteins were performed (Table 13.9).
A single isotachopherogram will be presented.
Fig. 13.16. Isotachopherogram of the analysis of some small peptides obtained with the operational
system listed in Table 13.3. 1 = 5-Bromo-2,4-dihydroxybenzoic acid; 2 = chloride; 3 = glutathione;
4 = glycylglycine; 5 = glycylglycylglycylglycine; 6 = D-leucyl-L-tyrosine; 7 = L-alanine.
335

336 AMINO ACIDS, PEPTIDES AND PROTEINS
13.3.2. Experimental
The operational system used was that specified in Table 13.3. L(+)-Alanine was used as
the terminating electrolyte, adjusted to pH 9.8 by addition of barium hydroxide. The
leading ion, 5-bromo-2,4-dihydroxybenzoic acid (0.004 M), was adjusted to pH 9.05 by
addition of L-lysine. The current was stabilized at 100 MA, and the time of analysis was
approximately 8 min. About 0.01 mole of glutathione (Merck, Darmstadt, G.F.R.),
glycylglycine hydrochloride, glycylglycylglycylglycine and D-leucyl-L-tyrosine (Nutritional
Biochemicals, Cleveland, Ohio, U.S.A.) was injected. The isotachopherogram of the
analysis is shown in Fig.13.16. One should note the chloride*, which is more mobile than
the 5-bromo-2,4-dihydroxybenzoic acid, which has passed the first separation boundary.
Because the chloride is coming from the cathode compartment, it is not a pH disturbance,
which may originate from the semi-permeable membrane, especially as the zone is
reasonably well defined.
It can clearly be seen in the linear traces of both the conductivity detector and the
W absorption detector that the concentration of the leading electrolyte is not changed
after the passage of the mobile chloride ion.
REFERENCES
1 A. Niederwasser and H. Curtius, Z. Klin. Chem. Klin. Biochem., 5 (1969) 4G4.
2 D.H. Spackman, W.H. Stein and S. Moore, Anal. Chem, 30 (1958) 90.
3 F.M. Everaerts and A.J.M. van der Put, J. Chromatogr., 52 (1970) 415.
4 A. Kopwillem, J. Chromatogr., 82 (1973) 407.
5 A.J. de Kok, Graduation Rep., University of Technology, Eindhoven, 1975.
6 F.E.P. Mikkers, Graduation Rep., University of Technology, Eindhoven, 1974.
*The chloride is derived from the sample component glycylglycine hydrochloride.

Chapter I4
Separation of nucleotides in aqueous systems
SUMMARY
Experiments were carried out in order to separate nucleotides comprising the mono-,
di- and triphosphates of adenosine, cytidine, guanosine and uridine with water as solvent.
The time of analysis is approximately 30-45 min for the thermometric detector and
approximately 15 min for the high-resolution detectors, from the start of the experiment
to the detection of the last zone.
14.1. INTRODUCTION
The nucleotides are amphiprotic substances and at intermediate pH values they are
negatively charged and show a behaviour similar to that of acids. As examples, the
structures of the 5-monophosphates of the nucleotides adenosine, cytidine, guanosine
and uridine are given in Fig. 14.1. This group of substances form the basis of the nucleic
acids and play an important role in carbohydrate, lipid and vitamin metabolisms. The
adenosine and guanosine phosphates are derived from the purine bases adenine and
guanine, and the cytidine and uridine phosphates are derived from the pyrimidine bases
cytosine and uracil.
Exact data on the pK values and mobilities of these nucleotides are not known but it
would be expected that a separation according to pK values would be the most successful.
The pH of the electrolyte system regulates the extent of dissociation of the nucleotides
and is therefore an important factor affecting the effective mobilities.
In the first section some operational systems and data are given for the separation of
nucleotides, using thermometric detection, and in the second section data are given for
separations using a conductivity detector and a UV absorption detector. In this chapter,
the abbreviations A, C, G and U are used for adenosine, cytidine, guanosine and uridine,
respectively, and MP, DP and TP for mono-, di- and triphosphate, respectively.
14.2. SEPARATION USING A THERMOMETRIC DETECTOR
The experiments for the determination of the optimal pH of the operational system at
which the analyses are performed gave a series of operational systems as specified in
Tables 14.1-14.7. These systems were used only with thermometric recording of the
various zones. Later a UV absorption detector became available and this precludes the use
of strongly W-absorbing counter ions. For the experiments in which a thermometric
detector was used, the equipment described in section 7.4.2 was applied.
In Table 14.8, all of the step heights measured for the different systems are given.
They were all obtained with the same thermocouple. In Fig.14.2, the step heights for the
different systems are shown graphically.
331

338 SEPARATION OF NUCLEOTIDES IN AQUEOUS SYSTEMS
U-SLMP G-

SEPARATION USING A THERMOMETRIC DETECTOR 339
TABLE 14.3
OPERATIONAL SYSTEM AT pH 4.2 SUITABLE FOR ANIONIC SEPARATIONS (WAnCl I)
This operational system was used only in experiments in which only a thermometric detector was
available.
Solvent: H, 0.
Electric current @A): Ca. 70.
Electrolyte
Leading Terminating
Anion a- (CH ) CCOO-
Concentration 0.01 N Ca. 0.01 N
Counter ion C, H,WH Tris+
PH 4.2 Ca. .4
Additive None None
I
-- Adenosine
- Cytidine
___--
Guanosine phosphates
Uridine
01 .
3.4 5 6 7 8
PH *
Fig.14.2. Graphical representation of the step heights obtained with a thermometric detector in the
operational systems listed in Tables 14.1-14.7 at 70 PA. 1 = CMP; 2 = AMP; 3 = GMP; 4 = CDP;
5 = UMP; 6 = ADP; 7 = GDP; 8 = ATP; 9 = CTP; 10 = UDP; 11 = GTP; 12 = UTP.

340
SEPARATION OF NUCLEOTIDES IN AQUEOUS SYSTEMS
\
j’!
I,
t
Fig. 14.3. Isotachopherogram of the separation of (A) adenosine phosphates and (B) uridine phosphates
in the operational system listed in Table 14.6. (A): 1 = Chloride; 2 = pyrophosphate; 3 = ATP; 4 = ADP;
5 = AMP; 6 = cacodylate. (B): 1 = Chloride; 2 = UTP; 3 = UDP; 4 = UMP; 5 = cacodylate. A thermo-
couple was used for detection.
TABLE 14.4
OPERATIONAL SYSTEM AT pH 4.6 SUITABLE FOR ANIONIC SEPARATIONS (WAnc1II)
This operational system was used only in experiments in which only a thermometric detector was
available.
Solvent: H, 0.
Electric current bA): Ca. 70.
Electrolyte
Leading Terminating
Anion cl- (CH, )3 CCOO-
Concentration 0.01 N Ca. 0.01 N
Counter ion
PH
C,H,N*H
4.6
Tris+
ca. 4
Additive None None

SEPARATION USING A THERMOMETRIC DETECTOR 341
At higher pH (5-7), the differences in step heights are rather small and it can be
concluded that these systems are less suitable for the separation of complicated mixtures.
TABLE 14.5
OPERATIONAL SYSTEM AT pH 5 SUITABLE FOR ANIONIC SEPARATIONS (WPyrC1)
This operational system was used only in experiments in which only a thermometric detector was
available.
Solvent: H, 0.
Electric current @A): Ca, 70.
Electrolyte
Leading Terminating
Anion cl- (CH,),AsOO-
Concentration 0.01 N Cu. 0.01 N
Counter ion NC5H5+ Tris+
PH 5.0 Ca. 5
Additive None None
TABLE 14.6
OPERATIONAL SYSTEM AT pH 6 SUITABLE FOR ANIONIC SEPARATIONS (WHiscl)
Solvent: H, 0.
Elechc current @A): Ca. 70.
Electrolyte
Leading Terminating
Anion a- (CH, AsOO-
Concentration 0.01 N Ca. 0.01 N
Counter ion His+ Tris+
PH 6 Ca. 6
Additive None None
TABLE 14.7
OPERATIONAL SYSTEM AT pH 7 SUITABLE FOR ANIONIC SEPARATIONS (WIrncl)
Solvent : H, 0.
Electric current @A): Ca. 70.
Electrolyte
Leading Terminating
Anion cl- Benzyl-dl- Asn-
Concentration 0.01 N Ca. 0.01 N
Counter ion Imidazole+ Tris+
PH 7 Ca. 6
Additive None None

342 SEPARATION OF NUCLEOTIDES IN AQUEOUS SYSTEMS
The separation of the mono-, di- and triphosphates of every nucleotide is, however,
possible in each operational system,
Fig.14.3 shows the isotachopherograms for the separations of (A) adenosine phosphates
and (B) uridine phosphates at pH 6; in the former sample pyrophosphate was also present.
The time of separation was about 20 min.
At lower pH, the step heights diverge and larger differences are obtained, because many
of them have pK values in this pH range. These systems are more suitable for the separa-
tion of the nucleotides in complicated mixtures. As an example, the isotachopherogram
of the separation of the nucleotides UTP, UDP, GDP, ADP, UMP, GMP, AMP and CMP is
shown in Fig.14.4. A complete separation could be obtained in 30 min at pH 3.7. Lower
pH values can rarely be used because the low effective mobilities at these pH values
require a higher potential than can be attained and disturbances due to the presence of H’
ions can be expected.
14.3. SEPARATION USING A CONDUCTIVITY DETECTOR (a.c. METHOD) AND A
W ABSORPTION DETECTOR (256 nm)
While in the previous section various operational systems in which the separations of
the nucleotides can be carried out were given, here only one isotachopherogram is given
for comparison. These components are very easy to separate at both low and neutral pH
and a separation according to pK values can easily be performed. Moreover, the nucleotides
TABLE 14.8
STEP HEIGHTS (A QUALITATIVE MEASURE IN THE ISOTACHOPHORETIC ANALYSES) OF
THE NUCLEOTIDES FOR THE DIFFERENT OPERATIONAL SYSTEMS LISTED IN TABLES
14.1- 14.7
The step heights (mm) were determined in the linear trace of the thermocouple signal, and refer to
the temperature of the leading zone at 70 PA and not at 0 FA as in Chapters 11 and 12.
Ionic species WAdCl WaNCl WAnCl I WAnCl I1 WPyrQ WHisCl WImCl
AMP
ADP
ATP
GMP
GDP
GTP
CMP
CDP
CTP
UMP
UDP
UTP
5 36
318
204
388
230
172
740
356
176
3 28
172
476
26 8
184
350
210
160
624
312
188
318
164
120
400
224
150
352
176
120
472
276
168
3 24
168
104
310
170
118
290
152
104
346
192
124
264
136
98
304
164
100
290
140
100
300
-
108
290
186
146
300
192
160
250
184
142
270
178
130
162
108
82
162
112
88
100
68
-
100
78

SEPARATION USING CONDUCTIVITY AND UV DETECTORS 343
have a strong UV absorption, which makes it possible to determine very small amounts
as discussed in Chapters 10* and 6**.
I---
Fig. 14.4. Isotachopherogram of the separation of some nucleotides in the operational system listed
in Table 14.2. 1 = Chloride; 2 = UTP; 3 = UDP; 4 = GDP; 5 = ADP; 6 = UMP; 7 = GMP; 8 = AMP;
9 = CMP; 10 = caproate. A thermocouple was used for detection.
*The UV-absorbing component can be sandwiched between two non-UV-absorbing ions. Due to the
profiles that are always present one can determine very small amounts, even in the picomole region.
**
The UV-absorbing component can be ‘diluted’ with a non-UV-absorbing ion. The component added
has an effective mobility identical with that of the nucleotide of interest. The step height of the
linear trace of the UV detector gives all necessary quantitative information.

344 SEPARATION OF NUCLEOTIDES IN AQUEOUS SYSTEMS
TABLE 14.9
RELATIVE STEP HEIGHTS OF A SERIES OF NUCLEOTIDES IN THE OPERATIONAL SYSTEMS
LISTED IN TABLES 12.5 and 12.6
The accuracy is better than 4%. The values given are to be used only for the identification of the ionic
species in isotachophoretic analyses in the operational systems indicated. The chloride (leading ion)
has a relative step height of 0, while the chlorate has a relative step height of 100. The current was
stabilized at 90 PA for the experiments at pH 3 and at 80 PA for the experiments at pH 4.5. - = No
UV absorption.
Ionic species pH 3.0 pH 4.5
A-2 ',3'-MP
A-3',5'-MP
A3',MP
A-S'-MP
A-2',5'-DP
A-3',5'-DP
A-S'-DP
A-S'-DP-glucose
A-5 '-DP-mannose
A-S'-DP-ribose
A-5'-TP
Chlorate
C-5'-MP
C-5'-DP
(2-5'-TP
Deox y thymidine-MP
Deoxythymidine-DP
Deoxythymidine-TP
G5'-MP
G-S'-DP
GS'-TP
I-S'-MP
I-S'-DP
1-5'-TP
U-S'-MP
U-S'-DP
U-S'-TP
100 h uv 100 h w
hchlorate hchlorate
2989
3178
3132
3610
1484
1469
144 1
1807
1809
1786
728
100
55 10
1628
811
1579
726
4 18
1826
953
621
1621
738
457
1543
6 86
404
99
99
99
99
99
99
99
99
99
99
99
-
94
87
77
99
94
90
99
100
99
99
98
96
98
96
94
1365
1463
1525
1628
799
808
782
977
1038
987
496
100
1872
950
550
1383
664
374
1494
731
444
1438
693
407
1360
668
3 86
100
100
100
100
100
100
99
100
100
99
99
-
98
93
86
99
96
92
100
99
99
100
99
98
100
98
95

SEPARATION USING CONDUCTIVITY AND UV DETECTORS 345
I
4 3 2 1
Fig.14.5. Isotachopherogram of the separation of AMP, ADP and ATP in the operational system at
pH 6 (Table 12.1). An amount of 10 nmole of each component was injected. The current was stabilized at
100 PA. The time of analysis is 8 min; however, this analysis can easily be carried out in 2 min.
1 = Chloride; 2 = ATP; 3 = ADP; 4 = AMP; 5 = MES. A = Increasing W absorption; R = increasing
resistance; t = time.
In Fig.14.5, the separation of AMP, ADP and ATP in the operational system at pH 6
(Table 12.1) is shown. One can refer to Chapter 12 for operational systems that are
suitable for the conductivity detector in combination with the W absorption detector.
In Table 14.9, experimental data obtained with both the conductivity detector
(a.c. method) and the W absorption detector (256 nm) are given.
The equipment used is described in section 7.4.4.

This Page Intentionally Left Blank

Chapter 15
Enzymatic reactions
SUMMARY
The possibility of using isotachophoresis for the determination of non-ionic compounds
via enzymatic reactions has been shown experimentally. The method for determining the
initial velocity of an enzymatic reaction is shown, although it is a disadvantage that the
reaction cannot be followed continuously. Two enzymes, representing two different
classes, were chosen arbitrarily: hexokinase and lactate dehydrogenase. In principle, all
enzymatic reactions can be studied in the operational systems specified in ths chapter.
The time of analysis is approximately 15 min from the start of the experiment to
the detection of the last zone.
1 5.1. INTRODUCTION
Isotachophoresis can be applied in many instances to the study of enzymatic reactions,
because ionic constituents are involved. Both enzymatic conversions and kinetics can be
studied. Because enzymatic conversions can be analyzed, many organic substances that
have no or a low effective mobility, e.g., glucose, fructose and urea, can be determined
quantitatively by the isotachophoretic separation technique. While the spectrophoto-
metric detection of enzymatic reactions sometimes needs a second reaction, isotacho-
phoresis can be carried out with a single reaction. Moreover, the purity of the reaction
constituents can be checked before the reaction; the purity of the starting materials is
very important, especially if activities need to be measured [ 11 .
Two types of reactions are considered in this chapter. The choice was made such that
the two types cover all of the main classes of enzymatic reactions. Firstly, the enzymatic
conversion of glucose into glucose-6-phosphate, followed by the conversion of glucosed-
phosphate into gluconate-6-phosphate is discussed. All enzymatic reactions that make use
of ATP, ADP, AMP, NADP and NADPH can be studied*. Secondly, the enzymatic conver-
sion of pyruvate into lactate is discussed, because in this reaction NAD (NADH) is
involved.
A disadvantage if isotachophoresis is applied to the study of enzymatic reactions is
that the reaction cannot be followed continuously, especially if kinetics are being studied.
The analyses were performed in the equipment as described in section 7.4.4.
*Abbreviations used: ADP = adenosine-5’-diphosphate; ATP = adenosine-5’-triphasphate;
LDH = lactate dehydrogenase; MES = morpholinoethanesulphonic acid; NAD = nicotinarnide-adenine
dinucleotide (oxidized); NADH = nicotinamide-adenine dinucleotidc (reduced); NADPH = nicotin-
amide-adenine dinucleo?ide phosphate (reduced); NADP = nicotinamide-adenine dinucleotide
phosphate (oxidized).
341

348 ENZYMATIC REACTIONS
1 5.2. ENZYMATIC CONVERSION OF GLUCOSE (FRUCTOSE) INTO GLUCOSE-6-
PHOSPHATE (FRUCTOSE-6-PHOSPHATE) WITH HEXOKINASE FROM YEAST
Many papers have dealt with the enzymatic conversion of glucose and fructose into
glucose-6-phosphate and fructose-6-phosphate by hexokinase (e.g., refs. 2-4). Apart
from ATP and the enzyme hexokinase, the reaction can be performed only if sufficient
Mgz+ or Ca2+ is present. Singly charged ions mostly inhibit the reaction [5] . Mg2+ forms
a suitable complex with ATP, but it is beyond the scope of this book to go into too much
detail concerning the complexity of the enzyme reaction itself.
Kinetics and conversions are commonly studied via analyses of the substrate and
product concentrations, especially the changes that occur during the reaction in the
former instance. This is often effected by utilizing a physical property of one of the
reaction constituents: the UV absorption at an appropriate wavelength. This measure-
ment gives all necessary qualitative and quantitative information and is very sensitive. The
reaction discussed briefly in this section, however, can be studied only if it is followed
by a second reaction because ATP and ADP have almost identical UV spectra and
glucose-6-phosphate and glucose have negligible UV absorption. The overall reaction can
be expressed as follows:
hexokinase
Glucose + ATP glucosed-phosphate + ADP
G6PDH
Glucose-6-phosphate + NAPD 6-phosphogluconate + NADPH
(15.1)
(15.2)
The difference in UV absorption between the ions NADP and NADPH at 340 nm gives
information about the conversion of glucose given in eqn. 15.1.
In principle, by means of isotachophoresis all ionic constituents can be determined,
both qualitatively and quantitatively. Suitable operational systems are listed in Tables 15.1
and 15.2. The conditions listed in Table 15.1 are used for measurements at ‘hgh’ concen-
TABLE 15.1
OPERATIONAL SYSTEM AT pH 3.8 SUITABLE FOR ANIONIC SEPARATIONS
Solvent: H, 0.
Electric current @A): Ca. 50-70.
Purification: The buffer was purified by recrystallization in water-ethanol, the crystals
being washed with acetone. The terminator was purified by recrystallization.
Electrolyte
Leading Terminating
Anion c1- p NH, -C, H, -COO-
Concentration 0.01 N Ca. 0.01 N
Counter ion pAla+ H+
PH 3.8 r4
Additive 0.05% Polyvinyl None
alcohol (Mowiol)

ENZYMATIC CONVERSION OF GLUCOSE (FRUCTOSE)
TABLE 15.2
OPERATIONAL SYSTEM AT pH 5 SUITABLE FOR ANIONIC SEPARATIONS
Solvent : H, 0.
Electric current (PA): Ca. 50-70.
Purification: e-Aminocaproic acid was purified by recrystallization.
Electrolyte
Leading Terminating
Anion
Concentration
c1-
0.005 N
Glu-
Ca. 0.005 N
Counter ion HOOC-C,H, -CH, N+H Tris’
PH 5 ca. 5
Additive 0.05% Polyvinyl None
alcohol (Mowiol)
trations of the reaction constituents, while those listed in Table 15.2 make analyses at
‘low’ concentrations of the reaction constituents possible.
Fig. 15.1 shows the analyses of all of the anionic constituents given in eqns. 15.1 and
15.2 after the conversion. In Fig. 15.2, the anionic constituents before the conversion are
shown. The impurities present in the biochemically pure chemicals were not further
identified, as’they did not disturb or obscure the final analytical results. Fig.15.1 and
15.2 show that it is possible to study the reactions given in eqns. 15.1 and 15.2
simultaneously or separately.
For kinetic measurements via isotachophoresis the reaction must be stopped at a
chosen time, in order to analyze the composition of the reaction mixture. The enzymatic
reaction is stopped by injecting a small amount (50 pl) of the reaction mixture into a
small glass tube (melting-point tube) that is wrapped with a Kanthal spring. The tube is
placed in a high-frequency field for cu. 8 sec, when the temperature of the spring reaches
the Curie point and the temperature of the contents of the tube reaches about 80°C. The
enzyme is denatured at this temperature, and a reproducible analysis of the reaction
mixture was achieved. Other techniques for stopping the enzymatic reaction were less
reproducible, e.g., adding inhibitors to the reaction mixture, rapidly changing the
‘optimal’ pH or filtering off the enzyme over a protein filter.
In Table 15.3, six solutions are given with which the conversion of glucose was followed.
The concentration of Mg2+ was chosen to be twice that of ATP so as to ensure that the
ATP was present as MgATP’ . All solutions were buffered to pH 8 by addition of Tris.
The enzyme suspension used in the experiments described in this section was prepared
by mixing in a bottle 1 p1 of yeast hexokinase (10 mgfml; 140 U/mg), 20 pl of glucosed-
phosphate dehydrogenase (1 mg/ml; 140 U/mg) and 29 pl of a 2 mg/ml solution of
serum albumin in double-distilled water. Although the reaction in eqn. 15.2 was not
followed, glucose-6-phosphate dehydrogenase was added in order to compare results
under reaction conditions that were as similar as possible.
The experiments showed that the results obtained in instances when only hexokinase
was added to the reaction mixture were similar to those when glucose-6-phosphate was
consumed by glucose-6-phosphate dehydrogenase and converted into 6-phosphogluconate.
349

350
7
2
ENZYMATIC REACTIONS
Fig.lS.l. Isotachopherogram of the separation of a reaction mixture in which the enzyme hexokinase
(from yeast) and the enzyme G6 PDH are involved. 1 = Chloride; 2 = ATP; 3 = 6-PG; 4 = NADPH;
5 = ADP; 6 = NADP; 7 = glutamate. A = Increasing UV absorption, R = increasing resistance; t = time.

ENZYMATIC CONVERSION OF GLUCOSE (FRUCTOSE)
1
I
A
3
I’
Fig.15.2. Isotachopherogram of the reaction mixture before the enzymatic conversion. 1 = Chloride;
2 = ATP; 3 = NADP; 4 = glutamate. Various impurities from the chemicals are present, but these do
not interfere with the separation. A = Increasing UV absorption; R = increasing resistance; r = time.
Fig. 15.3 shows an isotachopherogram of the reaction mixture after only hexokinase
had been added.
The enzymatic reaction was performed with the six solutions specified in Table 15.3,
using 1.5 ml in the reaction flask each time. This flask was thermostated in a metal block
at 25"C, a heat-sink compound being applied between the flask and the metal block for
351

352 ENZYMATIC REACTIONS
TABLE 15.3
COMPOSITION OF THE SOLUTIONS APPLIED FOR THE DETERMINATION OF THE INITIAL
REACTION VELOCITY
4 chemicals were purchased from Boehringer (Mannheim, G.F. R.)
Solution Concentration (mmole/l)
No.
Glucose ATP MgC1, ADP
1.00
2.00
2.00
2.00
1 .oo
0.50
3.29
3.39
0.97
0.54
0.56
0.53
6.00
6.00
2.00
1 .oo
1 .oo
1 .00
0.10
0.09
0.08
0.07
0.07
0.03
good thermal contact. To the solution was added 1 p1 of the mixed enzyme suspension
mentioned above. Under the conditions chosen, a maximum of 0.028 U of hexokinase is
present. During the first 20 min, several samples were taken and the reaction stopped by
thermal denaturation of the enzyme, as described above. After the denaturation, the
melting-point tubes were quickly cooled and kept in an ice-bath until required for analysis.
The analyses were carried out in the operational system specified in Table 15.2, about
5 pl of sample being analyzed each time. The results are given in Table 15.4.
The concentrations of ATP, ADP and glucose-6-phosphate are in acceptable agreement.
The measurements at high concentrations of ATP were less reproducible; possibly at
these concentrations the degradation of ATP plays an important role.
The initial reaction velocity, determined graphically from the values in Table 15.4, are
given in Table 15.5.
If smaller amounts of glucose need to be studied, then the ‘mixed-zone’ method must
be applied. The ATP or ADP must be diluted in its zone with a suitable non-UV-absorbing
component. Therefore an anion must be sought that has, in the operational system chosen,
an effective mobility identical with that of ATP. The quantitative information can now
be obtained via a calibration graph, from the linear trace in the UV (see Fig.6.33). For
ADP, hydroxybutyric acid can be applied at a pH of about 4, although analyses at pH > 6
show that the influence of the pH of the zone is less important.
Another six solutions were prepared for the determination of the enzymatic conver-
sions and are listed in Table 15.6. The analyses were carried out in the operational system
specified in Table 15.1,0.5 pl being injected into the system each time. To 900 pl of the
solution specified in Table 15.6,30 p1 of yeast hexokinase (10 mg/ml; 140 U/mg) were
added. The solution was maintained at room temperature, the pH of the reaction mixture
being adjusted to 8 by addition of Tris. The results of the conversions are shown
graphically in Figs.15.4 and 15.5 for glucose and fructose, respectively. All enzymatic
reactions in which ATP, ADP, AMP, NADP, NADPH are involved can be studied by
isotachophoresis in the operational system given [6] .

ENZYMATIC CONVERSION OF GLUCOSE (FRUCTOSE)
- 6
t
4
Fig.15.3. Isotachopherogram of the reaction mixture (Fig.15.2) after the addition of the enzyme
hexokinase. 1 = Chloride; 2 = ATP; 3 = ADP; 4 = NADP; 5 = G6P; 6 = glutamate. A = Increasing
UV absorption; R = increasing resistance; t = time.
353

354 ENZYMATIC REACTIONS
TABLE 15.4
COMPOSITIONS OF THE SOLUTION LISTED IN TABLE 15.3 AFTER THE ADDITION OF THE
ENZYME HEXOKINASE.
~
Solution Reaction time
No. (min)
1 0
2
4
7
11
15
2
3
0
2
4
7
9.5
11
15
0
2
4
6
15
20
~~ ~~ ~ ~~
Concentration Solution Reaction time Concentration
(mmole/ 1) No. (min) (mmole/l)
ATP ADP G6P ATP ADP G6P
3.29 0.10 - 4 0
3.30 0.12 0.04 2
3.35 0.14 0.05 4
3.22 0.17 0.13 6
3.11 0.19 0.17 10
3.16 0.25 0.20 16
3.39 0.09 - 5 0
3.25 0.10 0.03 2
3.24 0.10 0.05 4
3.28 0.17 0.07 6
3.13 0.19 0.12 10
3.26 0.21 0.10 15
3.21 0.28 0.17 21
0.54 0.07 -
0.51 0.07 -
0.52 0.11 -
0.49 0.12 0.05
0.46 0.14 0.09
0.42 0.20 0.13
0.56 0.07 -
0.52 0.09 -
0.49 0.10 -
0.49 0.13 0.02
0.48 0.15 0.08
0.47 0.20 0.08
0.39 0.27 0.10
0.97
0.92
0.94
0.08
0.09
0.12
-
0.01
0.02
6 0
2
4
0.58
0.52
0.48
0.03 -
0.06 -
0.09 -
0.94 0.12 0.05 8
0.47 0.10 0.05
0.88 0.17 0.10 10
0.45 0.10 0.04
0.85 0.21 0.12
~ - _ _
TABLE 15.5
INITIAL REACTION VELOCITY OF THE SOLUTIONS LISTED IN TABLE 15.3 DETERMINED
GRAPHICALLY FROM THE DATA IN TABLE 15.4
Solution
No.
"conversion
(lo* mcle!l- min:
1
"conversion
(10' 1 * min/mole)
1 1.00 1.00
2 1.18 0.85
3 0.6 1.67
4 0.8 1.25
5 0.85 1.17
6 0.87 1.16

ENZYMATIC CONVERSION OF PYRUVATE 355
TABLE 15.6
COMPOSITION OF THE SOLUTIONS APPLIED FOR THE DETERMINATION OF THE EXTENT
OF CONVERSION
Solution Concentration (mmole/l)
No.
.-
ATP MgSO, Glucose Fructose
1.5
1.5
1.5
1.5
1.5
1.5
2
1.45
0.5
-
TABLE 15.7
OPERATIONAL SYSTEM AT pH 4.7 SUITABLE FOR ANIONIC SEPARATIONS
Solvent: H, 0.
Electric current (PA): Ca. 70-100.
Purification: MES is purified by recrystallization three times from water-ethanol, the
crystals being washed with acetone. e-Aminocaproic acid is purified by
recrystallization.
Electrolyte
Leading Terminating
Anion c1- MES-
Concentration 0.01 N Ca. 0.01 N
Counter ion* HOOC-C, Ha CH, N'H Tris+
and His+
PH 4.5 (c-Aminocaproic Ca. 4.5
acid); 4.7 (His)
Additive 0.05% Polyvinyl None
aIcohol (Mowiol)
*The leading electrolyte is prepared as follows: first, 0.01 N hydrochloric acid; secondly, eaminocaproic
acid is added till a pH value of 4.5 has been reached; and finally, histidine is added till a pH value of
4.7 has been reached.
15.3. ENZYMATIC CONVERSION OF PYRUVATE INTO LACTATE WITH LACTATE
DEHYDROGENASE FROM PIG HEART
This type of enzymatic reaction was chosen because NAD (NADH) is involved. The
reaction is:
LDH
Pyruvate + NADH+ + H+ NAD + lactate* (15.3)
A reaction mixture was prepared by mixing 25 ml of buffer solution (0.01 Nhydrochloric
*At pH 7.5 lactate is formed; at pH 8.9 pyruvate is formed.
-
2
1
0.5

356
E
ENZYMATIC REACTIONS
Fig.15.4. Relationship between the lengths of the zones of the various reaction constituents, as found
in the isotachopherograms, and the amount of glucose converted. c = concentration of glucose (mmole/l).
acid adjusted to pH 7.6 by addition of Tris), 195.3 mg of NADH
. - and
.. 109 mg of pyruvic
acid in a reaction flask. In this instance also the enzymatic reaction was stopped at chosen
time intervals for kinetic measurements. The same procedure is followed as described in
section 15.2. Each time 0.3 pl was injected and analyzed in the operational system

ENZYMATIC CONVERSION OF PYRUVATE 357
0. 5 I 1.5 2
c*
ADP
F6P
ATP
Fig.15.5. Relationship between the lengths of the zones of the various reaction constituents, as found
in the isotachopherograms, and the amount of fructose converted. c = Concentration of fructose
(mmole/ 1).
specified in Table 15.7.
The example in Fig.lS.6 shows two isotachopherograms that illustrate the reaction
mixture before and after the conversion (80 min). The composition of the reaction
mixture as a function of time is given in Fig.15.7 and the data are listed in Table 15.8.
From Fig.15.7, it can be seen that lactate and NAD fit a straight Iine exactly, while
the pyruvate and NADH lines are curved of the beginning, possibly due to the impurities

35 8 ENZYMATIC REACTIONS
I
c-
1
t-
t
Fig.15.6. Isotachopherograms showing the enzymatic conversion of pyruvic acid into lactic acid by
LDH (pig heart). Left, reaction mixture after the conversion; right, reaction mixture before the
conversion. 1 = Chloride; 2 = sulphate; 3 = pyruvate; 4 = lactate; 5 = NADH; 6 = NAD; 7 = MES.
A = Increasing UV absorption; R = increasing resistance; f = time.

ENZYMATIC CONVERSION OF PYRUVATE 359
I0 20 30 40 50 ca 70
- time
Fig.15.7. Zone lengths of the various reaction constituents as a function of the reaction time in an
enzymatic reaction in which LDH is involved.
TABLE 15.8
CHANGE IN COMPOSITION OF A REACTION MIXTURE CAUSED BY THE ENZYME LACTATE
DEHY DROGENASE
Reaction time Zone length (mm)
(min)
Pyr uvate Lactate NADH NAD
0
10
30
40
60
70
80
90
100
120
27.4
27.3
24.3
23.3
22.0
21.7
20.9
21.1
25.0
20.4
1.4
2.7
4.2
4.9
6.5
7.3
7.9
7.1
2.9
8.7
~-____
17.6
16.2
11.3
9.5
6.1
5.0
3.7
5 .O
14.1
2.1
that are always present in the chemicals.
For this series of experiments, a mass balance was made for all reaction constituents.
In order to determine the amounts of pyruvate and NADH in nanomoles that fit a zone
length of 1 mm, the values obtained at t = 0, the reaction mixture before the conversion,
were applied, as these amounts are known exactly at this time. For lactate and NAD, a
calibration graph was constructed, which is quicker* than using the calibration constant
*Moreover, accurate data are not available for the determination of the calibration constant.
-
2.3
4.5
5.8
8.3
9.3
10.2
9.0
2.5
11.1

360 ENZYMATIC REACTIONS
TABLE 15.9
MASS BALANCE OF: THE REACTION CONSTITUENTS
Reaction time Amount (nmole)
(min)
-~___
Pyruvate Lactate NADH NAD
40 - 148.4 134.8 - 127.2 128.2
60 - 195.5 196.4 - 180.6 183.4
70 -206.3 227.2 - 197.8 205.5
80 -235.3 250.3 -218.2 225.4
method (Chapter 10) because only two componentsneed to be quantitatively determined.
The results of these experiments are given in Table 15.9.
Table 15.9 shows that an acceptable agreement is obtained, although it is not clear
why the values for pyruvate and lactate on the one hand and for NADH and NAD on
the other are so similar. The reason may lie in impurities in the standards used for the
calibration, and especially impurities present in NAD and NADH. All enzymatic reactions
in which NAD or NADH are involved can be analyzed by isotachophoresis [7].
REFERENCES
1 A.J. Berry, A.J.M. Lot and G.1:. Grannis, Clin. Chem., 19 (1973) 1255.
2 H.J. Fromm and V. Zewe,J. Biol. Chern., 237 (1962) 3027.
3 H.J. Frornm, J. Biol. Chem., 239 (1964) 3045.
4 P. Ottolenghi, C.R. Trav. Lab. Carlsberg, 34 (1964) 237.
5 N.C. Melchior and J.B. Melchior, J. Biol. Chern., 231 (1958) 609.
6 J. Hoenkamp, Graduation Rep., University of Technology, Eindhoven, 1975.
7 T. Willemsen, Graduation Rep., University of Technology, Eindhoven, 1974.

Chapter 16
Separations in non-aqueous systems
SUMMARY
Experimental data are mainly presented for isotachophoretic separations with methanol
as solvent. Data are given only for experiments in which a thermometric detector was used
because, especially for the conductivity detector, the sharpness is poor, possibly owing
to electroendosmosis, if concentrated methanol solutions are applied.
The time of analysis is approximately 30-45 min for the thermometric detector and
approximately 15 min for the high-resolution detectors from the beginning of the
experiment to the detection of the last zone.
Some isotachopherograms are shown of a standard mixture of ions obtained with a
conductivity detector and a UV absorption detector when methanol-water mixtures were
applied.
16.1. INTRODUCTION
As already mentioned in Chapter 5, solvents other than water can be used for isotachophoretic
analyses. In this chapter, some examples of other solvents are considered,
especially methanol and also a mixture of methanol and water.
In section 16.2 results are given for separations of anionic species using a thermometric
detector and in section 16.3 the separation of cationic species in methanol is considered.
The results obtained when a conductivity detector (a.c. method) and a UV absorption
detector were applied are discussed in section 16.4. No results are given for methanol
(95%) as solvent with these detectors because the resolution is bad, possibly owing to
electroendosmosis. No surfactant, e.g., polyvinyl alcohol as used in the aqueous experiments,
could be found that would suppress the electroendosmosis. The isotachopherograms
lack sharpness with both detectors, although the UV detector can be applied in
numerous experiments.
As will be shown, the influence of other dielectric constants and differences in solva-
tion, resulting in different pK values and mobilities, allows numerous possibilities
compared with isotachophoretic experiments in aqueous solutions.
The 95% methanol (technical grade) used for the separations described in sections
16.2 and 16.3 were purified by running it through a column filled with a mixed-bed ion
exchanger (Merck V). The equipment used for the experiments with the thermometric
detector is discussed in section 7.4.2, while for the experiments discussed in section 16.4
the equipment with high-resolution detectors described in section 7.4.4. was used.
361

362 SEPARATIONS IN NON-AQUEOUS SYSTEMS
16.2. SEPARATION OF ANIONIC SPECIES IN METHANOL USING A THERMO-
METRIC DETECTOR
For experiments with anionic species in methanol, the pH, and pK values of the
substances involved in the separation are very important. Some pK values are presented
in Chapter 5 and from these data we chose as the leading electrolyte a mixture of Tris
and hydrochloric acid in methanol at pHC 9, the concentration of chloride ions being
0.01 N(Tab1e 16.1). This means that a combination of a ‘separation based on pK values’
and a ‘separation based on mobilities’ was used, as most acids have pKz values of 8-9.
The step heights measured in this system are given in Table 16.2.
Simultaneous separations are possible in this system if the differences in step heights
are about 7% (relative to 0 PA). As discussed in Chapter 12, some fatty acids have been
measured in aqueous systems, but many of them have similar effective mobilities and some
are not sufficiently soluble for them to be separated. Their solubility in methanol is much
better and also the differences in mobility seem to be greater. A separation of fatty acids
in methanol has already been shown in Fig.5.8.
In the separation of some dicarboxylic acids, it is remarkable that often different step
heights were obtained when they were measured after various times. In particular, for
fresh and old solutions of oxalic acid different step heights were obtained. It was found
that fresh solutions of oxalic acid gave a step height of 300 mm, whereas a 2-day-old
solution gave a step height of 112 mm. Between these times, isotachopherograms were
obtained showing two step heights, one at 112 and one at 300 mm, where the zone lengths
were different according to the time of preparation. These steps were stable, i.e., when a
mixture of oxalic acid and another substance, with a step height between the two for
oxalic acid, was introduced the electropherogram showed three peaks in accordance with
those of oxalic acid and the other substance.
In Fig.16.1A the isotachopherogram of dl-malic acid is shown, in Fig.16.lB that
of oxalic acid (two steps) and in Fig.16.1C that of a mixture of dl-malic acid and oxalic
TABLE 16.1
OPERATIONAL SYSTEM AT pH* 9 SUITABLE FOR ANIONIC SEPARATIONS
Solvent: CH OH.
Electric current (PA): Ca. 50-70.
Purification: Methanol (95%, technical grade) was purified by running it through a column
filled with a mixed-bed ion exchanger (Merck V).
Electrolyte
Leading Terminating
Anion
Concentration
c1-
0.01 N
E.g., (CH,),AsOO-,
C, H ,, COO-
Ca. 0.01 N
Counter ion Tris’ Tris’
PH* 9 Ca. 8
Additive None None

SEPARATION OF ANIONIC SPECIES IN METHANOL 363
TABLE 16.2
QUALITATIVE INFORMATION (STEP HEIGHTS) FOR SOME ANIONS IN THE OPERATIONAL
SYSTEM LISTED IN TABLE 16.1
The step heights H refer to the step height of the leading zone (173 rnrn is the step height from 0 to 70 pA
for the zone of the leading electrolyte).
Ionic species H(mrn) Ionic species Hhm)
Acetic acid
Acetic acid, phenyl
Acetic acid, trichloro
Adipic acid
Azelaic acid
Benzoic acid
Benzoic acid, o-amino
Benzoic acid, p-amino
Benzoic acid, rn’-amino
Benzoic acid, 5-bromo3,4-dihydroxy
Benzoic acid, 2,4 dihydroxy
Butyric acid
Cacodylic acid
Capric acid
Caproic acid
Caprylic acid
Crotonic acid
Hydrofluoric acid
Formic acid
Hippuric acid
Lactic acid
Lauric acid
*Double step.
88
240
76
260
212
216
304
412
308
264
224
176
800
380
296
336
180
148
37
256
232
408
Linoleic acid
Maleic acid
Malic acid, dl
Malonic acid
Mandelic acid, dl
Myristic acid
Oleic acid
Oxalic acid
Palmitic acid
Pelargonic acid
Pimelic acid
Pyruvic acid
Salicylic acid
Salicylic acid, acetyl
Salicylic acid, sulpho
Stearic acid
Suberic acid
Succinic acid
Sulphanylic acid
Sulphonic acid, 2-naphthalene
Valeric acid
508
176 t 344*
244
120 + 188*
210
440
5 04
112 t 300*
480
360
264
96 + 298*
112
108 t 220*
108
508
280
224
200
162
2 74
acid. The isotachopherograms were measured I day after the preparation of the sample
solutions.
pK measurements on oxalic acid showed the disappearance of one pK step during the
time involved. A fresh solution gave two pK values, while a 2-day-old solution gave only
one. A large proportion of the methanol in the solutions of oxalic acid in methanol was
evaporated off and, after the subsequent addition of water, the resulting solution was also
measured in an aqueous operational system. Here the products of the old methanolic
solution gave a higher step height than normal, while the product from the fresh
methanolic solution gave the normal step height of oxalic acid in water. Old solutions
of oxalic acid in methanol gave, after several hours in water, two step heights, but after
about 1 day they gave only one step height (the normal step height of oxalic acid). It can
be concluded from these experiments that oxalic acid undergoes spontaneous conversion
into its monoester in methanolic solutions. Other dicarboxylic acids showed similar
effects, but on a smaller scale.
Dicarboxylic acids such as dihydroxymaleic acid showed a large number of step heights
and it is clear that the analyses of such substances will be difficult. In Fig.16.2, the

i
3
T
364 SEPARATIONS IN NON-AQUEOUS SYSTEMS
- - c_
t t t
Fig.16.1. Isotachopherograms of the separation of dl-malic acid (A), oxalic acid (B) and a mixture of
oxalic and dl-malic acids C. A: 1 = Chloride; 2 = dl-malate; and 3 = cacodylate. B: 1 = Chloride; 2,
3 = components that belong to the oxalate [the oxalate (2) and possibly an ester (3)] ; 4 = cacodylate.
C: 1 = Chloride; 2 = oxalate; 3 = malate; 4 = the ester of oxalate and methanol (?); 5 = cacodylate.
isotachopherogram of pure dihydroxymaleic acid is given. The terminator was cacodylic
acid.
The substances listed in Table 16.2 are almost all organic acids; in general, inorganic
acids were sparingly soluble in methanol. Because of its lower dielectric constant,
complex formation occurs to a much greater extent in methanol than in water, and also
the greater effect of the activity coefficients and the decreasing effect on the mobility
make methanol unsuitable for isotachophoretic experiments with inorganic ionic species.
For the halides, however, which have almost identical effective mobilities in water and
cannot be separated, some experiments were carried out in methanol. As with alkali
metals (see section 16.3.1), the halides have greater differences in mobilities in methanol
and can easily be separated. In Table 16.3, the absolute ionic mobilities and measured
step heights in water and methanol are given. The leading electrolyte in water was
0.01 N hydrobromic acid and in methanol 0.01 N hydriodic acid. In Fig.16.3, the
separation of the halides is shown, with sodium dihydrogen orthophosphate as terminator.
In the sample used to obtain Fig.16.3, formate was added in order to have the possibility
of comparing the step heights with those in Table 16.2.
16.3. SEPARATION OF CATIONIC SPECIES IN METHANOL USING A THERMO-
METRIC DETECTOR
Some cationic species were measured in three different systems, viz., the unbuffered
system MHCl, listed in Table 16.4, and the buffered systems MKAC and MTMAAC listed
in the Tables 16.5 and 16.6 respectively.

SEPARATION OF CATIONIC SPECIES IN METHANOL
-3
Fig. 16.2. Isotachopherogram of dihydroxymaleic acid in methanol. The leading ion is chloride and
the terminating ion is cacodylate. Numerous ‘impurities’ are obtained. 1 = Chloride; 2 = dihydroxy-
maleate; 3 = cacodylate.
16.3.1. The operational system MHCl
The step heights of the cations in the methanolic systems are given in Table 16.7. The
differences in step heights required for a complete separation must be about 8-10 mni.
In comparison with the aqueous systems, especially for monovalent cations, separations
can be achieved much more successfully in methanol. Trivalent cations are difficult to
separate as their isotachopherograms show very wide, sometimes double, steps because
365

366 SEPARATIONS IN NON-AQUEOUS SYSTEMS
TABLE 16.3
MOBILITIES AND STEP HEIGHTS OF HALIDES IN WATER AND METHANOL
The step heights refer to 0 PA. These values indicate the possibility of effecting separations.
Anion Water Methanol
Br- 81.3
I- 79.8
CI- 79.0
F- 56.6
m-105 (cm2/V*sec) h (mm) m - lo5 (cmz/V - sec) h (mm)
105 58.6
106 66.1
107 54.1
135 42.2
HCOO- 56.6 136.5 51.7 176
6-
Fig.16.3. Isotachopherogram of the separation of h.alides in the operational system listed in Table 16.1.
Formic acid is included for comparison. 1 = I-; 2 = Br-; 3 = C1-; 4 = CHOO-; 5 = F-; 6 = H, PO, *-.
clusters can be formed. For this reason, only the separations of monovalent and divalent
cations were investigated. Fig. 16.4 shows which cations can be separated simultaneously
and, in Fig.16.5, the isotachopherogram for the separation of alkali metals is given, the
153
138
164
196

SEPARATION OF CATIONIC SPECIES IN METHANOL 367
TABLE 16.4
OPERATIONAL SYSTEM (UNBUFFERED) SUITABLE FOR CATIONIC SEPARATIONS (MHCI)
Solvent: CH,OH.
Electric current (MA): Ca. 50.
Purification: Methanol (95%, technical grade) was purified by running it through a column
filled with a mixed-bed ion exchanger (Merck V).
Electrolyte
Leading Terniinating
+
Cation
Concentration
Counter ion
H+
0.01 N
c1-
CdZ
C'. 0.01 N
c1-
PH - -
Additive None None
TABLE 16.5
OPERATIONAL SYSTEM AT pH 7.4 SUITABLE FOR CATIONIC SEPARATIONS (MKAC)
Solvent: CH,OH.
Electric current (MA): Ca. 50.
Purification: Methanol (9576, technical grade) was purified by running it through a column
filled with a mixed-bed ion exchanger (Merck V).
Electrolyte
Leading Terminating
Cation K+ Cd"
Concentration 0.01 N Ca. 0.01 N
Counter ion CH, COO- c1-
PH 7.4 -
Additive None None
leading ion being W and the terminating ion Cuz+. In Fig.16.6, the isotachopherogram is
given for the separation of a mixture of (CH3)41V, (C2H5)4N+, NH;, K', Na', Ca2+, Li+,
Co2+, Mn2+ and Cu2+, the leading ion being H’ and the terminating ion Cd2+.
16.3.2. The operational system MKAC
In this system, the leading electrolyte is potassium acetate in methanol adjusted to a
pH of 7.4 by adding acetic acid. The pH is measured with a glass electrode and a calomel
electrodel filled with an aqueous saturated solution of potassium chloride, as a reference
electrode; The terminator used is cadmium chloride in methanol. There are large differ-
ences in clornparison with the step heights of the cations in the system MHC1, particularly

368 SEPARATIONS IN NON-AQUEOUS SYSTEMS
TABLE 16.6
OPERATIONAL SYSTEM AT pH 6.9 SUITABLE FOR CATIONIC SEPARATIONS (MTMAAC)
Solvent : CH OH.
Electric current (PA): Ca. 50.
Purification: Methanol (95%, technical grade) was purified by running it through a column
filled with a mixed-bed ion exchanger (Merck V).
Electrolyte
Leading Tcrminating
Cation (CHJ4 N+ Cd”
Concentration 0.01 N Ca. 0.01 N
Counter ion CH, COO- cl-
PH 6.9 -
Additive None None
TABLE 16.7
QUALITATIVE INFORMATION (STEP HEIGHTS) OF SOME CATIONIC SPECIES IN THE
OPERATIONAL SYSTEMS LISTED IN TABLES 16.4-16.6
The step heights (mm) refer to 0 wA.
Cation H (mm) Cation H (mm)
H+
K+
Na+
Llt
Rb+
cs+
Ag+
NK
Tris+
TI
(CH3),+
(C, H, ).,+
(C, H, ),+
Guanidine+
Succinyl
choline’
Imidazole’
MHCl MKAC MTMAAC
124 -
195 195
222 230
25 7 270
180 -
168 -
- 1031
179 -
292 321
- 218
154 151
170 177
265 260
192 203
209 191
176 599
*n.s.m. = not sufficiently mobile.
-
198
230
260
188
173
193
317
150
186
250
198
184
NiZ+
MgZ+
Zn2+
PbZ+
BaZ*
Caz+
CdZi
co2+
cuz+
Mn2+
Fez+
Fe3+
A13+
Cr3+
Ce3+
La3+
MHCl MKAC MTMAAC
26 2
240
n.s.m.*
n.s.m.
232
24 1
628
272
383
296
390
340
256
290
310
330
510
397
821
946
335
425
1025
497
n.s.m.
483
n.s.m.
n.s.m.
n.s.m.
n.s.m.
n.s.m.
n.s.m.
560
436
960
1080
350
456
540
-
5 20
-

SEPARATION OF CATIONIC SPECIES IN METHANOL
Fig.16.4. Simultaneous separation of some cations in the operational system MHCl (Table 16.4.).
Cations in a circle or in a circle connected to another circle by a line cannot be separated. Abbrevia-
tions: Guan = guanidine; Im = imidazole; S.C. = succinyl choline; Tba = (C,H,),N; Tea = (C,H,),N;
Tma = (CH,),N.
Pig.16.5. Isotachopherogram of the separation of the alkali metals in the operational system listed in
Table 16.4 1 = H+; 2 = CS+; 3 = Rb+; 4 = K+; 5 = Na+; 6 = Li'; 7 = Cua+.
369

370
1,
\
t
C
SEPARATIONS IN NON-AQUEOUS SYSTEMS
Fig.16.6. Isotachopherogram of the separation of some cations in the operational system listed in
Table 16.5. 1 = H+; 2 = (CH,),N+; 3 = (C,H,),N+; 4 = NH:; 5 = K’; 6 = Na'; 7 = Ca2+; 8 = Li';
9 = Co"; 10 = MnZ+; 11 = Cu2+; 12 = Cd*+.
for divalent cations. The trivalent cations have such a low effective mobility that they do
not migrate in an appropriate way.
Fig.16.7 shows which cations are simultaneously separated in this system and Fig.16.8
shows the isotachopherogram of the separation of some cations.
The most important metals in blood can easily be separated in this operational system.
Qualitative separations are more difficult because large differences in concentrations exist

SEPARATION OF CATIONIC SPECIES IN METHANOL
Fig.16.7. Simultaneous separation of some cations in the operational system listed in Table 16.5.
Cations in the same circle cannot be separated. Abbreviations as in Fig.16.4.
-
r
Fig.16.8. Isotachophoretic separation of a mixture of anions carried out in the operational system
listed in Table 16.5. A thermometric detector was used. 1 = K+; 2 = guanidine+; 3 = Na'; 4 = Li+;
5 = Baz+. 6 = MgZ+. 7 = CaZ+; 8 = NiZ+; 9 = Zn2+,
371

312
Fig. 1 6.9.
Fig. 16.10.
,J I !
B
70% CH3OH
B
30% .CH30H
SEPARATIONS IN NON-AQUEOUS SYSTEMS
r-
A
0% CH30H
A
:

EXPERIMENTS IN AQUEOUS METHANOLIC SYSTEMS 313
between the various ionic species and the resolution of the thermometric detector, as
discussed in Chapters 6 and 10, is low.
16.3.3. The operational system MTMAAC
In the preceding system, the leading ion is K’ and cationic species with mobilities
less than that of K' cannot be determined. Because many ions are more mobile than K’,
we carried out some experiments with the leading electrolyte tetramethylammonium
acetate, the tetramethylammonium ion being the most mobile cationic species used in
our experiments. In Table 16.7 it can be seen that most step heights agree with those in
the system MKAC. All divalent cations are slightly slower, possibly owing to the higher pH.
16.4. EXPERIMENTS IN AQUEOUS METHANOLIC SYSTEMS USING A CONDUCTI-
METRIC DETECTOR (a.c. METHOD) AND UV ABSORPTION DETECTOR (256 nm)
More detailed research must be carried out with methanol-water mixtures as solvents
before conclusive results can be given; it is always difficult to recommend specific propor-
tions of these two solvent components because they depend mainly on the substances to
be analyzed. Therefore, only four experiments will be briefly discussed here, carried out
in 100% water and 9: 1,4: 1 and 7:3 water-methanol mixtures. The methanol (95%,
technical grade) was purified by running it through a mixed-bed ion exchanger (Merck V).
Double-distilled water was applied. The experiments were carried out in the operational
system at pH 5.0 (Table 11.3), except for the solvent. The current has been stabilized at
70 PA, the amplifications of the conductivity detector (a.c. method) and the UV absorp-
tion detector were not changed and the speed of the recorder paper was 6 cm/min for all
analyses.
Fig.16.9 shows the results of the analyses for 100% water and 9:1 water-methanol,
and Fig.16.10 shows those for 4:1 and 7:3 water-methanol.
From the isotachopherograms shown, it is clear that the effective mobilities of the
various constituents of the sample are influenced in different ways. Moreover, it can be
seen that the resolution of both detectors is sufficient.
Fig.16.9. Isotachophoretic separation of a standard mixture of cations (Fig.ll.7) carried out in the
operational system at pH 5 (Table 11.3) in (A) water and (B) 9: 1 water-methanol. 1 = K'; 2 = Ba2+;
3 = Na+; 4 = (CH,),N*; 5 = PbZ+; 6 = Girard reagent D+; 7 = Tris'; 8 = histidine'; 9 = creatinine+;
10 = benzidine'; 11 = e-aminocaproic acid+; 12 = y-aminobutyric acid'. The amplifications of the
detectors were not changed. A = Increasing UV absorption; R = increasing resistance; t = time.
Fig.16.10. Isotachophoretic separation of a standard mixture of cations (Figs.11.7 and 16.9) in
water-methanol systems (A, 4:l; B, 7:3) carried out in the operational system at pH 5 (Table 11.3).
A = Increasing UV absorption; R = increasing resistance; t = time.

This Page Intentionally Left Blank

Chapter I7
Counter flow of electrolyte
SUMMARY
It has been found that a counter flow of electrolyte can be used if the concentration
differences between the ionic species of the sample are large. If the effective mobilities of
the ionic species of the sample are too small, no counter flow of electrolyte can be chosen
that will give a complete separation in the available length of narrowbore tube. More
attention must be paid to all types of disturbances to the boundary profiles.
17.1. INTRODUCTION
As already discussed in Chapter 7, a counter flow of electrolyte can be applied in
isotachophoretic analysis in order to increase the effective length available for the separation
of a given sample into its constituent ionic species. In zone electrophoresis, of course,
a counter flow of electrolyte cannot be applied. In moving-boundary experiments, only
the first zone can be stopped, which may be advantageous if one is interested only in the
ion following,the leading ion, or if two ionic species are to be separated.
Two main reasons can be given why counter flow of electrolyte is wanted [ 1,2] :
(1) the difference in concentration of the various ions of the sample is too great to
achieve a complete separation (Fig.4.5);
(2) the differences in the effective mobilities of the various ions of the sample are too
small (for the length of narrow-bore tube chosen) [3-91.
It is logical that the first reason is chosen when studying the disturbances to the various
zone profiles, because a sufficiently large difference in effective mobility is assumed to be
present between the various ions for a complete separation to be expected. The second
reason places more stringent demands on the resolution of the technique. A counter
flow of electrolyte can be applied succesfully only if the disturbances to the profile of
the zone boundary are suppressed sufficiently. The suggestion that the length of the
narrow-bore tube simply needs to be increased for an improved separation is also of
doubtful validity if the influence of electroendosmosis is not sufficiently well understood
and the disturbances are not suppressed; more experiments need to be performed in order
to elucidate these phenomena.
In this section we shall consider the effect of a counter flow of electrolyte, given as a
percentage of the electrophoretic migration, on disturbances to the profiles. We shall
define a 100% counter flaw of electrolyte to be such that the zone boundaries no longer
move. Hence the electrophoretic migration is equal to the hydrodynamic counter flow of
electrolyte.
The regulation of the counter flow of electrolyte via signals derived from a detector, at
which the zones are stopped, was found to be the most stable, although the regulation as
given in section 7.5.5. was found to be a good alternative because an extra high-resolution
315

316 COUNTER FLOW OF ELECTROLYTE
detector is not needed. This regulation, however, places demands on the electrolytes of
the operational system, which must be of high purity and also of carefully selected
concentration. If the terminating electrolyte is impure, an impurity may influence the
final recording if its effective mobility is greater than that of the terminating ion (see
eqn. 7.4.). Both the pH and the concentration of the terminating electrolyte may have
an influence on the regulation of the counter flow of electrolyte. At the position
where the terminator is present in the narrow bore (1 mm) of the injection system (see
Fig.7.5.), a well defined potential gradient is obtained as a result of the electrophoretic
driving current chosen and the concentration of the electrolyte present (Ohm’s law). In
isotachophoretic experiments without a counter flow of electrolyte, the concentration
of the terminator has hardly an influence, assuming that it is present in such a concentra-
tion that it is able to adapt to the isotachophoretic concentration, which is determined
by the concentration of the leading electrolyte chosen (ratio of the anion and cation
concentrations). If a counter flow of electrolyte is now applied that is regulated by the
total potential gradient between the electrodes, the total resistance over the bore (1 mm)
filled with terminating electrolyte changes, owing to the counter flow of electrolyte applied,
if the composition of the terminating electrolyte is not already that which it would
finally attain as a result of the isotachophoretic conditions.
If the concentration of the terminating electrolyte is too low, then in a long run the
potential gradient will decrease, because the final conductivity* is higher. This results in a
movement of the sample zones in the direction of the detector. If the Concentration of
the terminating electrolyte is too high, then the sample zones will be pushed back,
although this effect is much smaller and generally has a less important effect on the final
results.
If a later experiment with a counter flow of electrolyte is to be carried out, it is
preferable to rinse the reservoir fdled with terminating electrolyte and to re-fill it with
a fresh solution, although this is not always necessary in experiments without a counter
flow of electrolyte**. Moreover, it was found experimentally to be preferable to fill the
reservoir at least three-quarters full with the terminating electrolyte, otherwise impurities
can penetrate into the narrow-bore tube more easily. If impurities are present in the
leading electrolyte, they may influence the final recording (see eqn. 7.3).
Particular attention should be paid to the electrolyte present in the counter electrode
compartment at the side of the semi-permeable membrane where the electrode is
mounted. In experiments without a counter flow of electrolyte, double-distilled water is
suitable because the membrane is not polluted by the counter ion chosen. Especially if
the equipment is used for various operational systems, the mutual effect of the various
counter ions is minimal. If experiments with a counter flow of electrolyte are considered,
the pH shift due to the membrane and the electrode reaction may influence the final
result more quickly. If double-distilled water is still preferred, this compartment must
be rinsed continuously during the analysis. If leading electrolyte is present, this compart-
ment must still be rinsed and re-filled after each experiment, assuming that the time of
analysis is not excessive, otherwise it must also be supplied continuously with fresh
electrolyte.
*The buffer ions are flushed into the canals filled with terminating ions.
**If experiments are carried out at a low concentration (0.01 M), it is preferable to start each experiment
again with a fresh solution of the terminating electrolyte.

INTRODUCTION 317
In various operational systems, the reaction products formed by the electrode
reaction* may influence and obscure the final result. As a result, the concentration
and/or composition of the leading electrolyte changes during the time the counter flow
of electrolyte is applied. Consequently, the concentration of the sample zones changes
(qualitative information) and the length of the narrow-bore tube occupied by these
various zones also changes (quantitative information), which is in contradiction to the
experiments shown in Figs.12.7 and 13.16 (see also Chapter 9). The shift in the step height
often found in the trace of the linear signal of the conductivity detector (ax. method) or the
potential gradient detector (d.c. method) can be caused by changes in the concentration
and composition of the leading electrolyte**, and also by impurities that may possibly be
present in the leading electrolyte and/or the terminating electrolyte. In the last instances,
the zones are particularly obscure, depending on the effective mobility of the various
impurities, as can be seen from eqns. 7.3 and 7.4.
If the counter flow of electrolyte occurs too soon, i.e., if sample zones are present that
have not reached the isotachophoretic concentration (note that ‘mixed’ zones also have
the isotachophoretic concentration), problems can be expected because the sample can
be flushed (partly) into the terminating electrolyte present in the narrow bore (1 mm)
or even in the reservoir filled with the terminating electrolyte. Problems can particularly
be expected if the sample is injected at too high a concentration, which leads to a high
conductivity at the position where the sample is injected. The various ions in this region
have a much lower velocity than would be expected in the narrow-bore tube according to
the isotachophoretic conditions.
Fig.17.1 summarizes the situation at the moment when the counter flow of electrolyte
must be applied, and illustrates the ‘dilution’ effect and the ‘concentration’ effect of
isotachophoresis.
In Fig.l7.la, the sample AB is introduced, already sandwiched between the leading
electrolyte (L) and the terminating electrolyte (T). In the steady state, the zone length of
A+B is less than the length of narrow-bore tube they originally occupied. Therefore,
(1 7.1 )***
v,> VL,
In this case, the counter flow of electrolyte can be applied at the beginning of the
experiment.
In Fig. 17.1 b, the ‘dilution’ effect is shown. Now,
(17.2)***
If in this instance the counter flow of electro1:rte is applied at the beginning of the experi-
ment, the sample is flushed back, resulting in a disturbance.
*E.g., in experiments carried out in the operational system at pH 6 (Table 12.1), where histidine is
used as the buffering counter ion, a reddish component is formed.
**This may also occur in experiments without a counter flow of electrolyte if the electrolyte (doubledistilled
water) in the compartment at the side of the membrane where the electrode is mounted is
not replenished regularly (see Chapter 9).
m
V;C and Vp are the velocities of the front B/T. Visot is the velocity of the front L/A.

378 COUNTER FLOW OF ELECTROLYTE
11
I , I
I
I I
1 1
B
6
1 A
I
,
I
,
v,
I
Fig.17.1. Diagram to illustrate the 'concentration' (a) and 'dilution' (b) principles of isotachophoretic
analyses.
17.2. EXPERIMENTAL
If the disturbance caused by the counter flow ofelectroIyte is to be studied, a
scanning detector is needed or dyes must be applied. We studied the disturbance by
using the dyes amaranth red, bromophenol blue and fluorescein. Acetate and glutamate
were found to be suitable for spacing the dyes. The separations were carried out in the
operational system at pH 6 (Table 12.1) and the results are shown in Fig.17.2 and 17.3.
The photographs in Fig.17.2 were obtained in a fluoroethylene polymer (FEP)
narrow-bore tube of I.D. approximately 0.5 mm, while the isotachopherograms in
Fig.17.3 were obtained with a conductivity detector (a.c. method) that had a probe made
almost completely of Perspex (see Chapter 6) with I.D. 0.4 mm. It can be seen that the
optimal sharpness of zones is obtained if a small counter flow of electrolyte is applied.
This optimal counter flow depends on, amongst other things, the viscosity of the
electrolyte, the diameter of the bore, the temperature inside the bore and the material
of which the narrow bore is made. This is why the recording of the zones by the a.c.
method (Fig.17.3) indicates another optimum for the sharpness of the zone boundaries,
~ ~ ~.
~
Fig.17.2. Results of experiments carried out in the operational system at pH 6 (Table 12.1) to show
the disturbance of the profiles by a counter flow of electrolyte (indicated in percentages). In (a) a
free solution was applied, while in (b) an electrolyte in which the viscosity was increased by addition of
2% of hydroxyethylcellulose (purified by shaking it with a mixed bed ion exchanger) was used; the
viscosity of the solution was approximately 100 cP. 1 = Chloride; 2 = amaranth red; 3 = acetate;
4 = bromophenol blue; 5 = glutamate; 6 = fluorecein; 7 = MES. It can clearly be seen that the zone
boundaries are first sharpened, and then are disturbed. The disturbance is a function of the viscosity.
In the two photographs in the bottom right-hand corner, two examples are given of the disturbance
of the zune boundary as a function of the effective mobilities of the consecutive zones: (a) shows the
boundary amaranth red/MES and (b) shows the boundary amaranth red/glutamate (loo%* counter
flow of electrolyte).

EXPERIMENTAL
0% 10 %
a b
60 %
a b
a b
70 %
a b
20 %
a b
80 %
a b
30 %
a b
90 %
a b
40%
a b
100 %
a b
50 %
a b
319
loo%*
a b

380 COUNTER FLOW OF ELECTROLYTE
d
C
b
a
I / 6 5
- f
Fig.17.3. Isotachophoretic separation of the mixture of components indicated in Fig.17.2, recorded
with a conductivity detector (a.c. method) when a counter flow of electrolyte was applied. As shown
in Fig.17.2, the zone boundaries become sharper if a small counter flow of electrolyte occurs.
R = Increasing resistance; f = time. 1 = Chloride; 2 = amaranth red; 3 = acetate; 4 = bromophenol blue;
5 = glutamate; 6 = fluorescein; 7 = MES.
tR

EXPERIMENTAL 381
caused by the counter flow of electrolyte, than the photographic registration shown in
Fig.17.2. It should be noted that the zones shown in the isotachopherogram in Fig.17.3d
move more slowly than those in Fig.17.3a, that the speed of the recorder paper was the
same in both analyses, and that nevertheless the zones shown in Fig.17.3d are sharper.
Moreover, Fig.17.3d shows that an impurity is present that is difficult to see in Fig.17.3a.
This shows once more the risk involved when zone profiles are used for the quantitative
determination of small amounts of components (see section 10.5).
Both Figs.17.2 and 17.3 show that the thickness of the electrodes is sufficient, if the
disturbances of the zone boundaries are not suppressed, assuming that the electrode
reactions do not play an important role. Moreover, the suppression of the boundary
disturbance by a counter flow of electrolyte can only be used for specific boundaries,
because the consecutive boundaries all have their own curvature. From the photographs in
Fig.17.2 (both the experiments in which 2% of hydroxyethylcellulose was added and
those carried out in the free solution), the profile disturbances were photographically
enlarged. The results are given in Fig.1 7.4: at higher rates of counter flow of electrolyte,
the disturbance proved to be no longer a function of the viscosity.
Another series of experiments was carried out in order to investigate the disturbance
of the zone boundary profile when the difference in effective mobilities is small.
Fig.17.4. Disturbance of the zone profiles by a counter flow of electrolyte. (a) Experiments in a free
solution; (b) experiments with 2% of hydroxyethylcellulose added to the leading electrolyte (viscosity
= 100 cP). V, = electrophoretic velocity; V, = hydrodynamic velocity (counterflow of electrolyte).

382 COUNTER FLOW OF ELECTROLYTE
Amaranth red was used as the sample in the operational system at pH 6 (Table 12.1).
Various terminators with different effective mobilities were applied. The results are shown
graphically in Fig.17.5.
These experiments indicated that the disturbance is greater;the smaller are the differ-
ences in effective mobilities. In Fig.17.2 (loo%*), two examples of these boundary
disturbances are shown: (a) the boundary when MES was applied as the terminator and
(b) the boundary when glutamate was applied as the terminator. Experiments with
acetate as the terminator showed that the amaranth red is flushed back into the
terminating reservoir. From these series of experiments, we can conclude that a ‘slow’
terminator is needed in order to prevent too much sample from being flushed back.
Fig.17.5. Disturbance of the zone boundary as a function of the effective mobility of the following
zone. ah = Difference in effective mobility; L = length of the disturbance of the zone boundaries
(mm). This figure can be compared with Fig.17.2 (loo%*). In these experiments hMES = 139;
hglutamate = 77.5; hacetate = 17.5; hamarant,, red = 0; hence Ah refers to the step height of the
amaranth red zone.
Fig.17.6. Isotachopherogram of a standard mixture of anions (Fig.12.5) without (A) and with (B) a
counter flow of electrolyte, showing that several mixed zones disappear. The experiments were carried
out in the operational system at pH 6 (Table 12.1). The differences in heights of the peaks (impurities)
in the linear trace of the W detector should be noted. These zones are marked with asterisks. They
are enriched during the time that the counter flow of electrolyte is applied (see section 10.5). A slight
difference between the recordings from the W absorption detector and the conductivity detector is
always obtained because the diameters of the probe and the PTFE narrow-bore tube are not the same.
Moreover, differences between the traces of the conductivity and the UV absorption detectors can be
expected in (A), because the W absorption detector is mounted closer to the injection point. The
zone of carbonate, marked with a large asterisk, increases with time (see Chapter 9). A = Increasing UV
absorption; R = increasing resistance; t = time.

CONCLUSION 383
c
t
r
’r; I
I"

3 84
17.3. CONCLUSION
COUNTER FLOW OF ELECTROLYTE
From section 17.2, we found that the disturbances are not as small in experiments in
narrow-bore tubes as was emphasized from the results of earlier experiments. If boundaries
were found to be less sharp, both the regulation and the construction of the equipment
were always found to be the cause. We now know that for enrichment of substances, a
very mobile leading ion and a much less mobile terminating ion is needed.
Fig.17.6 shows two analyses (with and without a counter flow of electrolyte) in order
to illustrate when the optimal effect of a counter flow of electrolyte can be expected.
The experiments were performed in the operational system at pH 6 (Table 12.1), the test
mixture of anions (Fig.12.5) being injected. AU experimental conditions were the same
except for the time for analysis: in (B) a counter flow of electrolyte was applied, which
doubled the time of analysis (from 15 to 30 min). A complete separation could be
achieved. The experiments were carried out with a membrane pump (section 7.5.5) in the
equipment described in section 7.4.4.
REFERENCES
1 F.M. Everaerts, J. Vacik, Th. P.E.M. Verheggen and J. Zuska, J. Chromatogr., 49 (1970) 262.
2 F.M. Everaerts, J. Vacik, Th.P.E.M. Verheggen and J. Zuska,J. Chromatogr., 60 (1971) 397.
3 J.W. Westhaver, J. Res. Nut. Bur. Stand., 38 (1947) 137.
4 S.L. Madorsky and S. Straus, J. Res. Nut. Bur. Stand., 38 (1947) 169.
5 S.L. Madorsky and A.K. Brewer., US. Put., 2,645,610, 1953; C.A., 47 (1953) 10374a.
6 B.P. Kostantinov and V.B. Fiks., Rum. J. Phys. Chem., 38 (1964) 1038.
7 B.P. Kostantinov and E.A. Bakulin, Russ. J. Phys. Chem., 39 (1965) 315.
8 W. Preetz, Talanta, 13 (1966) 1649.
9 W. Preetz and H.L. Pfeifer, Tulanta, 14 (1967) 143.
10 J. van de Venne, Graduation Rep., University of Technology, Eindhoven, 1975.

APPENDICES

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Appendix A
Simplified model of moving-boundary electrophoresis for the
measurement of effective mobilities
A. 1. INTRODUCTION
If the separation in isotachophoresis is complete, only one ionic species of the sample
is present in each sample zone and the parameters of each zone are related to those of the
preceding zone. Calculations of the pH, concentrations of the ionic species in the zone
and other parameters are possible and a mathematical model for the buffered systems
is given in Chapter 4. If the separation is not complete, Le., if mixed zones are present
and/or the influence of the background ions is too great, the conditions for real isotachophoresis
are lost and the model described is no longer valid. In particular, the influence
of the background will dominate in unbuffered systems. Sometimes the separation
procedure can be better understood by using a model similar to that for the movingboundary
technique.
Several workers [ 1-61 have given mathematical models for the moving-boundary system,
but it is very difficult to work with an exact model and some simplifications have to be
made. All zones do not contain one ionic species of the sample, but the number of ionic
species in the zones increases to the rear side. Only the first zone, following the leading
electrolyte zone, contains one ionic species of the sample. All zones have correlations
with both their preceding and following zones, which explains the difficulties involved
in computations (see Chapter 4). A simpler model was used by Brouwer and Postema [7],
who described a model for the separation procedure during isotachophoresis, which in
principle is moving-boundary electrophoresis. Concentration effects, the influence of pH
and the differences in temperature were neglected. Although this is not a general model,
it can be used for unbuffered systems of monovalent, fully ionized ionic species for the
calculation of effective mobilities. In this Appendix, we describe a model comparable to
that of Brouwer and Postema [7]. With the equations presented, a computer program
was developed and the calculations carried out with it are compared with the results of
experiments. Further, we describe the procedure for the determination of effective
mobilities of strong electrolytes using the moving-boundary principle. Also, the effective
mobilities of, for example, weak fatty acids can be determined working at high pH where
they are fully ionized, for it is assumed that the influence of pH or pK values can be
neglected.
A.2. MODEL OF MOVINGBOUNDARY ELECTROPHORESIS
When carrying out experiments on moving-boundary electrophoresis, the capillary
tube can be filled with an electrolyte of a strong acid if the separation of, for example,
cations is desired. The cation present has a mobility that is higher than that of any other
cation of the sample. The sample is situated at one end of the capillary tube, in the anode
compartment. For the derivation of the equations, the following assumptions are made:
387

388 SIMPLIFIED MODEL OF MOVINGBOUNDARY ELECTROPHORESIS
fully ionized cations and anions are considered; the contribution of the background ions
to the conductance of a zone is negligible; the influence of differences in pH and concen-
trations are neglected; the electric current is stabilized; diffusion, hydrodynamic flow
and electroendosmosis are neglected; and the solution initially present in the capillary
tube and anode compartment has a well known, constant composition. The equations that
need to be considered are the electroneutrality equations, the modified Ohm’s law and
the mass balances for all catioaic species.
A.2.1. Electroneutrality equations
If the influence of the background ions can be neglected and when all ionic species
are fully ionized, the concentration of the counter ions will always be identical with the
concentrations of the cations present in a zone, if monovalent ions are considered.
A.2.2. Modified Ohm’s law
if the influence of the presence of the hydrogen and hydroxyl ions is neglected.
A.23. Mass balances for all cationic species
In the stationary state, the amount of each ionic species passing a separation boundary
is equal to the amount reaching the separation boundary. For each ionic species and all
separation boundaries we can write (see also section 4.2.3):
CA,.,V-I(EU-lmAr-vV)= ‘Ar,lJ (EVmAr-vV)
Substituting for uu:
*The subscript U refers to the Uth zone, which contains Uionic species of the sample.

PROCEDURE OF COMPUTATION
A.3. PROCEDURE OF COMPUTATION
For a separation boundary between the zones U- 1 and U, eqn. A1 gives
u- 1 U
~u-i * 2 (mA,+mB)cA,,U-i =E,* C (mAr+mB)cAr,U (A71
r= 1 r=l
Combining eqns. A7 and A6 gives
JI
In fact, this is a modification of the ‘Dole polynomals’ [ 1,8] and solutions for the
equations are valid if
If the composition of the leading electrolyte and the sample solution are known, all
parameters can be computed with the equations given above. The velocity of the concen-
tration boundaries can be neglected.
In the first instance, the concentrations of the ionic species in the last sample zone
are taken to be equal to the original concentrations in the sample. Although this assump-
tion is not correct, the ratio between the concentrations in the sample remains constant
in the zones, according to eqn. A2, which gives
(It is assumed that the velocity of the concentration boundary can be neglected.)
Using eqns. AS and A9, flu-] ,u can be calculated if all mobilities are known. With
flu- ,u and eqn. A6, all concentrations of the zone U- 1 can be calculated, and thus all
concentrations and values can be calculated for all zones.
The concentration of the ionic species in the first sample zone can be calculated in
two ways, either with the equations given above or using the isotachophoretic condition
as described in Chapter 4. In the first computation, we chose arbitrarily as concentrations
for the ionic species in the last sample zone those concentrations present in the original
sample solution, and all quantities could be obtained. If the parameters of the first zone
obtained in this way did not agree with those obtained by the isotachophoretic method,
we re-computed from the first to the last zone with the quantities obtained with the
isotachophoretic condition, using the 0 values from the moving-boundary procedure. By
this calculation, new concentrations for the sample zones can be proposed and new
/3 values can be calculated. This procedure of ‘iteration’ must be repeated until the
P values and concentrations fit.
389

390 SIMPLIFIED MODEL OF MOVINGBOUNDARY ELECTROPHORESIS
A.4. EXPERIMENTAL
With the equations presented above, a computer program was developed and experiments
were carried out in order to check this model. To this end, all concentrations
should be determined in all zones. However, as this is difficult, another possibility is to
measure the velocities of the zones by means of a detector.
Each zone has a specific constant velocity, vu = EumAU; for practical reasons, we use
relative velocities instead of absolute velocities* :
If the distance between the point of injection and the point of detection is P, the time
needed for a particular ionic species to be detected will be
or
P = VUtU
The correlation between vu and vL will be
P= VUtU = V LtL
and hence
v = VU/VL = tL/tU
U
The times of detection can be measured from the time of the starting point of the
analysis up to the time of the appearance of the zone of a particular ionic species. Because
the velocity of the leading electrolyte is equal to the velocity of the first sample zone
(isotachophoretic condition), we use the relationship
v' =tL/tU=tJt"
U
In this way, the measured ratio tl/tU from the electropherograms can be used to check
the computed ratio vu/vL (vh).
In order to check the model, some experiments were carried out. The values of tl ItU
were measured from the electropherograms for different mixtures of P, Na+, Li+, (CH3)4NC
and (CzHS)4M. The leading electrolyte was 0.01 Nhydrochloric acid and the current was
stabilized at 70 PA. Experimental and theoretical values are given in Table Al.
In Fig.Al these values are represented graphically (the broken lines represent the
experimental values). The experimental values agree very well with the calculated values
and it can be concluded that this model is suitable in many instances.
Because the relative time of detection for a mixture of two ions of known concentrations
is constant in a given system and depends only on the mobilities, it can be used
for the determination of the effective mobility of an ion. In order to demonstrate this
*The relative velocity of a moving-boundary zone is related to the isotachophoretic velocity of the
leading zone (isotachophoretic condition).

EXPERIMENTAL 39 1
TABLE A1
THEORETICAL AND EXPERIMENTAL VALUES OF THE RELATIVE TIME OF DETECTION
FOR SOME CATIONS IN A MOVING-BOUNDARY ELECTROPHORETIC SYSTEM
System Value K' Na' (CH,),N' Li' (C,H5)4N'
A Concentration (N)
fL/tv, theoretical
tL/fu, measured
B Concentration (N)
tL/tv, theoretical
fL/tU, measured
C Concentration (N)
fL/tu, theoretical
f L/tU, measured
D Concentration (N)
fL/f u, theoretical
rL/t u, measured
E Concentration (N)
tL/t u, theoretical
fL/tU, measured
0.01
1 .ooo
1.00
0.02
1.000
1.00
0.02
1 .ooo
1 .oo
0.02
1.000
1.00
0.02
1.000
1.00
0.01 0.01
0.904 0.863
0.90 0.85
0.01 0.0 1
0.848 0.805
0.84 0.79
0.02 0.02
0.889 0.845
0.88 0.83
0.01 0.01
0.872 0.837
0.87 0.83
0.01 0.02
0.877 0.845
0.87 0.84
0.01 0.01
0.793 0.717
0.79 0.70
0.01 0.01
0.736 0.664
0.73 0.65
0.01 0.01
0.753 0.671
0.75 0.66
0.02 0.02
0.793 0.723
0.78 0.71
0.01 0.02
0.767 0.708
0.76 0.70
effect, the theoretical values of all relative detection times as a function of the mobility
of a cation are shown in Fig.A2. The concentrations of the cations varied from 0.01 N
to infinite dilution and the leading electrolyte was 0.01 N hydrochloric acid.
From the experimental values for the relative time of detection given in Fig.A2, the
mobility can be derived. Measurements were carried out with samples of Na', (CH3)4N+
and (CzH5)4N+ and the results are shown in Table A2. It can be seen that the theoretical
and measured values agree.
In Fig.A2 a linear relationship is obtained for a zero concentration of a cation mixed
with 0.01 N potassium chloride in a sample. This corresponds with the theory, because
TABLE A2
THEORETICAL AND EXPERIMENTAL MOBILITIES OF SOME CATIONS
Ion Concentration Ion
(N) W)
Theoretical Measured
K+ 0.01 Na' 0.01 0.8375 50.5 51.25
0.01 0.005 0.7930 51.5
K' 0.01 (CH,),N+ 0.0 1 0.7900 45.0 45.7
0.01 0.005 0.7200 45.5
K' 0.01 (C,H,),N+ 0.01 0.6770 30.0 32.2
0.01 0.005 0.6000 33.2

392 SIMPLIFIED MODEL OF MOVINGBOUNDARY ELECTROPHORESIS
.t r
2 3 4 5 1 2 3 4 5 1
- -
I
1 2 3 4 5 1 2 3 4
-I
5
Fig.Al. Graphical representation of the theoretical and experimental values for the times of detection
for some cations in a moving-boundary system (see Table Al.). r = ratio I , ItU. s = ionic species:
1 = K'; 2 = Na+; 3 = (CH,),N'; 4 = Li+; 5 = (C,H,),N+. A, B,C, D and E refer to A, B,C, D and E in
Table Al.
now elution phenomena prevail (a uniform voltage gradient is present over the whole of
the capillary tube and consequently the relative times of detection show a Iinear relation-
ship with the mobility).
AS. DISCUSSION
As shown in section A.4, a theoretical relationship between the relative time of detec-
tion and mobility can be used for the determination of effective mobilities, although of
course an experimentally obtained relationship between mobility and relative times of
detection can also be used.
Sometimes, disturbances during isotachophoretic analyses can be understood better
by using a moving-boundary model instead of an isotachophoretic model, and an
example was given in section 9.2.1.2. In Fig.9.2, the relationship between the pH of the
terminator and the ratio tl ItU (vb) is given for theoretical and experimental values. This
is, in fact, the separation of a mixture of two cations, viz., a mixture of H' and K: and
it will be clear that different pH values (different concentrations of H’) will cause
different relative velocities of the K' zones.
Moving boundary electrophoresis can hardly be used as an analytical technique. Of
course, separations can be carried out in this way and Fig.A3 shows an electropherogram

DISCUSSION
E I
I
Fig.A2. Graphical representation of the calculated relative times of detection as a function of the
mobilities for different concentrations of the cations, mixed with 0.01 N KCl, after the leading
electrolyte (0.01 N HCl). rn = mobility (10-5.cm2/V-sec).
- I
Fig.A3. Separation of a mixture of cations in moving-boundary electrophoresis. All initial concentrations
were 0.01 M. The current was stabilized at 70 PA. The leading electrolyte was 0.01 M HC1 in
methanol (95%, w/w). 1 = H+; 2 = (CH,),N+; 3 = (CH,),N+ + NH;; 4 = (CH,)," + NH,' + K+;
5 = (CH,),N+ + NH,' + K' + Na'; 6 = (CH,),N' + NH,' + K' + Na' + Caz+; 7 = (CH,),N' + NH:
+ K' + Na+ + Caz+ + Li'; 8 = (CH,),N' + NH,' + K' + Na+ + Ca2' + Li+ + CozC; 9 = (CH,),N' +
NH,' + K' + Na' + Ca ’+ + Lit + Coz+ + MnZ+; 10 = (CH,),N' + NH,* + K+ + Na' + Ca" + Li' + Coz+
+ Mnzt + Cuz+.
393

394 SIMPLIFIED MODEL OF MOVINGBOUNDARY ELECTROPHORESIS
for the separation of a mixture of (CH3)41V+, NH;, K’, Na”, Ca2’, Li’, Co , Mn” and
Cu2+ in narrow-bore tubes, with H+ as the leading ion and methanol as solvent. The separation
is reasonable but an interpretation will be very difficult if the sample is unknown, as
both the retention times (time of appearance of a ‘moving-boundary’ zone) and step
heights depend on both the mobilities and the concentrations of the ionic species in the
sample. Moving-boundary electrophoresis can thus hardly be used, unless the mixture has
a simple composition. For simple routine analyses, however, this method can probably be
used, because much simpler apparatus (compared with ‘isotachophoretic’ equipment) can
be constructed.
REFERENCES
1 V.P. Dole, J. Amer. Chem. SOC., 67 (1945) 1119.
2 R.A. Alberty, J. Amer. Chem. SOC., 72 (1950) 2361.
3 J.C. Nichol, J. Amer. Chem. SOC., 72 (1950) 2367.
4 J.C. Nichol, E.B. Dismukes and R.A. Alberty,J. Amer. Chem. SOC., 80 (1958) 2160.
5 E.B. Dismukes and R.A. Alberty, J. Amer. Chem. Soc., 76 (1954) 191.
6 D. Tondeur and J.A. Dodds., J. Chim. Ph,ys., 3 (1972) 441.
7 G. Brouwer and G.A. Postema, J. Electrochem. SOC., 117 (1970) 7 and 874.
8 M. Bier, Electrophoresis, Vol. 1, Academic Press, New York, 1959.

Appendix B
Diameter of the narrow-bore tube, applied for separation
In Chapters 6 and 7, equipment is described in whch a narrow-bore tube with an I.D.
of 0.45 mm was used. The current density in the equipment discussed was about
500 pA/mm2. From experiments conducted in 1970, it was well known that, if glass
narrow-bore tubes with 1.D.s of 0.2 and 0.1 mm were used for isotachophoretic experi-
ments, very small temperature differences could be measured between the various zones
with the micro-sensing thermocouples (section 6.2). Moreover, the increase in electro-
endosmotic flow even made analyses with dyes, as described in Chapter 17 (Fig.17.2),
impossible. No additives, e.g., Mowiol (polyvinyl alcohol) were applied at that time.
As soon as the equipment and detectors described in Chapters 6 and 7 had been
developed and tested, some research was carried out so that the I.D. of the narrow-bore
tube could be decreased. Three main reasons for desiring this reduction can be given:
(a) With a smaller I.D. of the narrow-bore tube, the total detectable amount of the
ionic species to be separated can be decreased, because the length of the zones increase on
decreasing the diameter of the narrow-bore tube.
(b) If comparable current densities are applied, the temperature differences between
the successive zones is less if the narrow bore-tube has a smaller 1.D. A smaller profile of
the zone boundary is thus obtained, especially between zones with ionic species that have
very low effective mobilities.
(c) If higher current densities can be permitted, the time of analysis will decrease.
A conductimeter (Fig.6.10) was therefore constructed, with the electrodes (10 pm
Pt-Ir) glued in a manner similar to that discussed for the probe (Fig.6.16). In ths
instance, the linear conductimeter, as discussed in section 6.4.4, can still be used. The I.D.
of the probe was made to be 0.2 mm. A PTFE narrow-bore tube, with an I.D. of 0.2 mm
and an O.D. of 0.45 mm, was mounted between the injection block (Fig.7.5) and the
conductivity probe. A similar narrow-bore tube was mounted between the probe and the
counter electrode compartment (Fig.7.9). The slit of the W absorption detector
(Fig.6.30) was also adapted. Although the diameter of the narrow-bore tube is much
smaller, the UV absorption detector can still be used because the wall thickness of the
narrow-bore tube is much smaller. It is well known that PTFE has a great W absorption.
Experimentally, it was found that the electroendosmotic flow could be decreased by
addition of, e.g., Mowiol (polyvinyl alcohol). The current density applied could be
increased up to at least 1500 pA/mm2. The sharpness of the zones increased by decreasing
the diameter of the narrow-bore tube, partly owing to the small differences in temperature
between the adjacent zones.
The main advantage of decreasing the diameter is the small heat production. Even
between the leading electrolyte and terminating electrolyte the increment is small,
compared with experiments in whch a narrow-bore tube of I.D. 0.45 mm was used. This
effect can easily be seen if terminating ions that have a very low effective mobility are
applied and the electric current is switched off. Because the conductivity is a function of
temperature, the shift in the linear signal of the conductivity detector (a.c. method) gives
395

396 DIAMETER OF THE NARROW-BORE TUBE
an impression of the temperature of the electrolytes inside the probe. With the probe with
an I.D. of 0.2 mm, the shifi is 0.4% of the total signal for ACES in the operational system
listed in Table 12.1 if a current density of 500 pA/mm* is applied. With the probe with
an I.D. of 0.45 mm, the shift is 2% under similar conditions. In addition, the drift in the
signal (warming up of the probe) after the terminator has passed tile micro-sensing
electrodes, again at a current density of 500 pA/mm*, can be neglected for probes with a
small J.D.
Whether or not I.D. can be decreased further depends on, among other factors, the
possibility of suppressing the electroendosmotic flow, stable regulation of the current-
stabilized power supply (I < 10 pA) and the technology involved in making probes with
such a small I.D.

Appendix C
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391

398 LITERATURE
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400 LITERATURE
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LITERATURE 401
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University of Technology, Eindhoven, 1972.
Th.P.E.M. Verheggen, E.C. van Ballegooijen, C.H. Massen and F.M. Everaerts, Detection
electrodes for electrophoresis, J. Chromatogr., 64 (1972) 185.
A. Vestermark and B. Sjodin, Isotachophoresis used alone or in two-dimensional combina-
tion with zone electrophoresis for the small-scale isolation of labelled ribulose-1 ,5-
diphosphate, J. Chromatogr., 71 (1972) 588.
A. Vestermark and B. Sjodin, Isotachophoresis in two-dimensional combination with
zone electrophoresis for the concentration and separation of glucose metabolites,
J Chromatogr., 73 (1 9 72) 2 1 1.

LITERATURE 403
1973
L. Arlinger and H. Lundin, UV-Detection of both absorbing and non-absorbing ions in
analytical isotachophoresis, Protides Biol. Fluids, Boc. Colloq., 21 (1973) 667.
J.L. Beckers, Isotachophoresis. Some fundamental aspects, Thesis, University of Tech-
nology, Eindhoven, J.H. Pasmans, 's-Gravenhage, 1973
J.L. Beckers, F.M. Everaerts and W.J.M. Houterrnans, The qualitative separation of
fatty acids by isotachophoresis, J. Chromatogr., 76 (1973) 277.
B.A. Bilal, Zur Untersuchung von Gleichgewichten instabiler Komplexe und die
Bestimmung der Beweglichkeiten der einzelnen Spezies mittels Gegenstrom-Ionen-
wanderung, 2. Naturforsch. A, 28 (1973) 1226.
I. Clemmensen, Three new E-antigenic fibrinogen fractions found in a commercial
plasmin preparation, Sci. Tools, 20 (1973) 7.
I. Clemmensen and P.J. Svendsen, Isolation of the plasmin resistance E-antigenic
fibrinogen breakdown product by isotachophoresis, Sci. Tools, 20 (1973) 5.
F.M. Everaerts, Isotachophoresa, Chem. Listy, 67 (1973) 9.
F.M. Everaerts, J.L. Beckers and Th.P.E.M. Verheggen, Some theoretical and practical
aspects of isotachophoretical analysis, Ann. N. Y. Acad. Sci., 209 (1973) 419.
F.M. Everaerts, A.J. Mulder and Th.P.E.M. Verheggen, Isotachophoresis: Analytical tool
in electrophoresis, Amer. Lab., (1973) 37; Znt. Lab., (1973) 43.
J.S. Fawcett, Continuous flow isoelectric focusing and isotachophoresis, Ann. N. Y. Acad.
Sci., 209 (1973) 112.
A. Griffth, N. Catsimpoolas and J. Kenney, Analytical gel isotachophoresis of proteins
with carrier ampholyte spacers, Ann. N. Y. Acad. Sci., 209 (1973) 457.
T. Haruki and J. Akiyama, New potential gradient detection system for isotachophoresis,
Anal. Lett., 6 (1973) 985.
J.O.N. Hinckley, Longitudinal temperature gradients in transphoresis and isotachophoresis
in relation to detection, Biochem. SOC. Trans., 1 (1973) 574.
T. M. Jovin, Multiphasic zone electrophoresis. 1. Steady-state moving-boundary systems
formed by different electrolyte combinations, Biochemistry, 12 (1973) 87 1.
T. M. Jovin, Multiphasic zone electrophoresis. 11. Design of integrated discontinuous
systems for analytical and preparative fractionation, Biochemistry, 12 (1973) 879.
T. M. Jovin, Multiphasic zone electrophoresis. 111. Further analysis and new forms of
discontinuous buffer systems, Biochemistry, 12 (1973) 890.
L. JuSka, Contribution to the isotachophoretic theory, Graduation Report, Charles
University, Prague, 1973.
A. Kopwillem, Analytical isotachophoresis in capillary tubes used for the separation
of ions involved in the enzymatic transformation of glucose to 6-phosphogluconate,
Acta Chem. Scand., 27 (1973) 2426.
A. Kopwillem, Analytical isotachophoresis in capillary tubes. Transformation of pyruvate
to succinate by calf heart mitochondria1 enzymes, J. Chrornatogr., 82 (1973) 407.
A. Kopwillem, F. Chillemi, A.B. Bosisio-Righetti and P. Righetti, Analytical isotacho-
phoresis and gel electrofocusing of synthetic peptides, Protides Biol. Fluids, Proc.
Colloq., 21 (1973) 657.
O.V. Oshurkova, N.S. Lyadov and LA. Koshurkina, Separation of multicomponent

4 04 LITERATURE
electrolytes in halogenide solutions by the moving boundary method, Zh. Prikl.
Khim. (Leningrad), 46 (1973) 776.
D. Peel, Isotachophoresis (displacement electrophoresis), in E. Reid (Editor),
Methodological Developments in Biochemistry, Longman, London, 1973, Ch. 21, p. 205.
F.R. Rorsman and J.M. Castagnino, Isotachophoresis. Fundamentals and application,
Bioquim. Clin., 7 (1973) 183.
R.J. Routs, The choice of electrolyte conditions for isotachophoretic separations, Ann.
N. Y. Acad. Sci., 209 (1 973) 445.
Z. Ryslav?, Isotachophoretic experiments with counterflow of electrolyte, Thesis,
Charles University, Prague, 1973.
B. Sjodin and A. Vestermark, The enzymatic formation of a compound with the expected
properties of carboxylated ribulose 1 ,5-diphosphate, Biochim. Biophys. Acta, 297
(1973) 165.
P.J. Svendsen, On the procedure of preparative isotachophoresis, Sci. Tools, 20 (1973) 1.
P.J. Svendsen, Isotachophoretic separation of electrically charged sample components,
Swed. Pat. 357, 891 (Cl. B. Olk) July 16, 1973, Appl. 4779/70.
V. Taglia and M. Lederer, Isotachophoresis on paper. I. Investigation of general conditions
and separation of some inorganic anions, J. Chromatogr., 77 (1973) 467.
V. Taglia, Isotachophoresis on paper. 11. The separation of Ag(I), Tl(I), HgZ2+ and Pb(II),
J. Chromatogr., 79 (1973) 380.
A. Vestermark, Determination of pH differences occurring during isotachophoresis with
different systems of leading and terminating electrolytes, Ann. N. Y. Acad. Sci., 209
(1973) 470.
1974
L. Arlinger, Andy tical isotachophoresis in capillary tubes. Separation of human
hemoglobin, Protides Biol. Fluids, Proc. Colloq., 22 (1974) 691.
L. Arlinger, Analytical isotachophoresis. Principle of separation and detection, Protides
Biol. Fluids, Proc. Colloq., 22 (1974) 66 1.
L. Arlinger, Analytical isotachophoresis. Resolution, detection limits and separation
capacity in capillary columns, J. Chromatogr., 91 (1974) 785.
L. Arlinger, Spectrophotometric detection of zone boundaries formed, for example, in
isotachophoretic separation, Ger. Offen Pat. 2,401,620 (Cl. G. Oh) July 25, 1974;
Swed. Appl. 492-1173.
M. Bier, J.O.N. Hinckley, A.J.K. Smolka and R.S. Snyder, Potential use of isotachophoresis
in space,Protides Biol. Fluids, Proc. Colloq., 22 (1974) 673.
P. BoEek, M. Deml and J. Janik, Quantitation in isotachophoresis. The concept of
relative correction factors, J. Chromatogr., 91 (1974) 829.
T.C. Bdg-Hansen, P.J. Svendsen, O.J. Bjerrum C.S. Nielsen and J. Ramlau, Preparative
isotachophoresis of membrane proteins in solubilizing and dissociating media,
Protides Biol. Fluids, Proc. Colloq., 22 (1974) 679.
C.H. Brogren and P.J. Svendsen, Preparative isotachophoresis combined with biospecific
interaction and neuraminidase treatment in purification of human serum cholinesterase,
Protides Biol. Fluids, Proc. Colloq., 22 (1974) 685.

LITERATURE 405
A. Chrambach and J.S. Skyler, The application of steady-state stacking to macromolecular
fractionation by polyacrylamide gel electrophoresis, Protides Biol. Fluids, Proc. Colloq.,
22 (1974) 701.
M. Coxon and M.J. Binder, Isotachophoresis (displacement electrophoresis, transphoresis)
theory, structure of the ionic species interface, J. Chromatogr., 95 (1974) 133.
M. Coxon and M.J. Binder, Radial temperature distribution in isotachophoresis columns
of circular cross-section, J. Chromatogr., 101 (1974) 1.
N.R. Curvetto, N.A. Balmaceda and G.A. Orioli, Isotachophoresis and isoelectric focusing
of soil humic substances in polyacrylamide gel, J. Chromatogr., 93 (1974) 248.
M. Demjanenko, Detection of zone boundaries with electrodes not in direct contact
with the electrolytes inside the capillary, Graduation Report; Thesis, Charles University,
Prague, 1974.
J.P.D. Dunn and R.B. Kemp, Isotachophoretic studies of adenosine phosphates and
divdent cations of perfused mouse liver cells, Protides Biol. Fluids, Roc. Colloq.,
22 (1974) 727.
F.M. Everaerts, Isotachophoresis. Quantitative aspects of the separation of mixtures of
anions, J. Chromatogr., 91 (1974) 823.
F.M. Everaerts, P. Prod and Th.P.E.M. Verheggen, Conductometric detection during
isotachophoresis, Protides Biol. Huids, Proc. Colloq., 22 (1 974) 721.
F.M. Everaerts and P.J. Rommers, Isotachophoresis. Phenomena that occur when conductometric
detection is applied, J. Chromatogr., 91 (1974) 809.
F.M. Everaerts, and Th.P.E.M. Verheggen, Isotachophoresis. Applications in the biochemical
field,J. Chromatogr., 91 (1974) 837.
P. Faigl, Some physical-chemical aspects of conductrometric detection in capillary
isotachophoresis, Graduation Report, Charles University, Prague, 1974.
A.L. Griffith and N. Catsimpoolas, Analytical gel isotachophoresis with ampholine
spacers, in R.C. Allen and H.R. Maurer (Editors), Electrophoretic Isoelectric Focusing
Polyaclylamide Gel (Proc. Small. Conf., 1972), 1974,~. 158.
H. Hatano, Chromatography and its related fields. Systematization of chromatography,
Kagaku No Ryoiki, 28 (1974) 145.
M. Hess, L. Davies and D. Allen, Basic structure of mouse histocompatibility antigens.
Eur. J. Biochem., 41 (1974) 1.
J.O.N. Hinckley, Transphoresis and isotachophoresis. Automatable fast analysis of
electrolytes, proteins and cells with suppression of gravitational effects, Clin. Chem.,
20 (1974) 973.
S. HjertCn, Free displacement electrophoresis (isotachophoresis), Protides Biol. Fluids,
Proc. Colloq., 22 (1974) 669.
A. Kopwillem, Purification control of synthetic peptides by means of analytical
isotachophoresis, Protides Biol. Fluids, Proc. Colloq., 22 (1974) 71 5.
A. Kopwillem, H. Lundin, A.B. Bosisio-Righetti and P. Righetti, Analytical isotacho-
phoresis in capillary tubes: Preliminary study of phenylketonuric sera, Protides
Biol. Fluids, Proc. Colloq., 22 (1974) 737.
F.E.P. Mikkers, The isotachophoretic separation of proteins and the development of
‘double-isotachophoretic’ system for discontinuous electrophoresis, Graduation
Report, University of Technology, Eindhoven, 1974.

406 LITERATURE
A.J. Mulder and J. Zuska, Isotachophoresis. Conductivity measurement and signal
handling, J. Chromatogr., 9 1 (1 974) 8 19.
T.W. Nee, Theory of isotachophoresis (displacement electrophoresis, transphoresis),
J. Chromatogr., 93 (1974) 7.
Z. Prusik, Free-flow electromigration separations, J. Chromatogr., 9 1 (1974) 867.
M.Y. Rosseneu, V. Blaton, H. Caster, H. Peeters and A. Kopwillem, Isotachophoresis of
human APO-HDL polypeptides, Protides Biol. Fluids, Proc. Colloq., 22 (1974) 697.
B. Sjodin, A. Kopwillem and J. Karlsson, Isotachophoresis: A new technique for determination
of tissue metabolite concentrations, Protides Biol. Fluids, Proc. Colloq.,
22 (1974) 733.
B.F. Sunden, Automatic sample futation in counterflow isotachophoresis, Ger. Offen
Pat. 2,363,195 (C1.B. Olk,G.Oln) June 27, 1974; Swed. Appl. 16, 594/72.
K. Uyttendaele, M. de Groote, H. Peeters and F. Alexander, Detection of traces of
proteins by isotachophoresis, Protides Biol. Fluids, Proc. Colloq., 22 (1974) 743.
J. Vacik and J. Zuska, Capillary isotachophoresis with electrolyte counter-flow. Temperature
and concentration profiles of the zone boundary, J. Chromatogr., 9 1 (1974)
795.
B. Vocisek, Some remarks about W-detection in capillary isotachophoresis, Graduation
Report, Charles University, Prague, 1974.
A.J. Willemsen, Enzymatic reactions followed via isotachophoresis, Graduation Report,
University of Technology, Eindhoven, 1974.
1975
L. Arlinger, Apparatus for isotachophoretic separation, Ger. Offen Pat. 2,454,105,
May 15, 1975; Swed. Appl. 73 15,417 (14 Nov. 1973).
L. Arlinger, Analytical isotachophoresis in capillary tubes: Analysis of proteins in spacer
gradients, in P.G. Righetti (Editor), Progress in Isoelectric Focusing and Isotacho-
phoresis, North-Holland, Amsterdam, Oxford and Elsevier, New York, 1975, p. 33 1.
G. Baumann and A. Chrambach, Gram preparative isotachophoresis of proteins,
Fed. Proc., Fed. Amer. SOC. Exp. Biol., 34 (1975) 685.
P. BoEek, M. Deml and J. Janik, Instrumentation for high-speed isotachophoresis,
J. Chromatogr., 106 (1975) 283.
T.C. BQg-Hansen, P.J. Svendsen and O.J. Bjerrum, On the biospecific interaction of Con A
and glycoproteins in preparative isotachophoresis, in P.G. kghetti (Editor), Progress
in Isoelectric Focusing and Isotachophoresis, North-Holland, Amsterdam, Oxford
and Elsevier, New York, 1975, p. 347.
C.-H. Brogren, P.J. Svendsen and T.C. Bdg-Hansen, On the purification of proteins by
neuraminidase treatment and preparative isotachophoresis, in P.G. Righetti (Editor),
Progress in Isoelectric Focusing and Isotachophoresis, North-Holland, Amsterdam,
Oxford and Elsevier, New York, 1975, p. 359.
J.F. Brown and J.O.N. Hinckley, Electrophoretic thermal theory. 11. Steady-state radial
temperature gradients in circular section columns, J. Chromatogr., 109 (1975) 218.
J.F. Brown and J.O.N. Hinckley, Electrophoretic thermal theory. 111. Steady-state
temperature gradients in rectangular section columns,J. Chromatogr., 109 (1975) 225.

LITERATURE 407
M. Coxon and M.J. Binder, Transverse temperature distributions in isotachophoresis
columns of rectangular cross-section, J. Chromatogr., 107 (1975) 43.
A.C.G. de Kok, The analysis of amino acids via isotachophoresis, Graduation Report,
University of Technology, Eindhoven, 1975.
M. Deml. P. BoCek and J. Jan&, High-speed isotachophoresis: Current supply and detection
system. J. Chromatogr., 109 (1975) 49.
F.M. Everaerts and Th.P.E.M. Verheggen, Analytical isotachophoresis: some practical
and funadmental aspects, in P.G. Righetti (Editor), Progress in Isoelectric Focusing
and Isotachophoresis, North-Holland, Amsterdam, Oxford and Elsevier, New York,
1975, p. 309.
B. Gas, A mathematical model for isotachophoretic separation processes, Graduation
Report, Charles University, Prague, 1975.
J.O.N. Hinckley, Electrophoretic thermal theory. I. Temperature gradients and theii.
effects. J. Chromatogr., 109 (1975) 209.
J.C.M. Hoenkamp, The enzymatic phosphorylation, studied via isotachophoresis,
Graduation Report, University of Technology, Eindhoven, 1975.
A. Kopwillem, Analytical isotachophoresis, Fed. Proc., Fed. Amer, SOC. Exp. Biol.,
34 (1975) 685.
A. Kopwillem, U. Moberg, G. Westin-Sjodahl, R. Lundin and H. Sievertsson, Analytical
isotachophoresis in the analysis of synthetic peptides, Anal. Biochem., 67 (1975) 166.
M. Kovai, Isotachophoresis. Separation and analysis of some organic acids, Graduation
Report, Comenius University, Bratislava, 1975.
A.J.P. Martin and F. Hampson, The analytical isotachophoresis of insulin, in P.G. Righetti
(Editor), Progress in Isoelectric Focusing und Isotachophoresis, North-Holland,
Amsterdam, Oxford and Elsevier, New York, 1975, p. 327.
R. Mollby, S.G. Hjalmarsson and T. Wadstrom, Separation of Escherichia co2i heat-labile
entero toxin by preparative isotachophoresis, Febs Lett., 56 (1975) 30.
G.T. Moore, Theory of isotachophoresis. Development of concentration boundaries,
J. Chromatogr., 106 (1975) 1.
Z. RySlav?, J. Vacik and J. Zuska, Temperature profiles in capillary isotachophoresis,
J. Chromatogr., 114 (1975) 315.
F. Schonhofer and F. Grass, Beitrage zur Theorie der elektrophoretischen Ionen fokussie-
rung anorganischer Ionen mit schwachen Komplexbildnern, J. Chromatogr., 110
(1975) 265.
A.J.K. Smolka and M. Bier, Isotachophoresis of living cells, Fed. Proc., Fed. Arner. SOC.
Exp. Biol., 34 (1975) 685.
S. Stankoviansky, P. bmanec and D. Kaniansky, Conductivity detection of zones in
isotachophoresis with a high-frequency bridge, J. Chromatogr., 106 (1 975) 13 1.
M. Svoboda, The combination of conductrometric detection and UV-absorption detection
in capillary isotachophoresis, Graduation Reporf, Charles University, Prague, 1975.
M. Svoboda, Some theoretical and practical aspects of photometric theory and spectro-
photometric detection in capillary isotachophoresis, Zkesis, Charles University, Prague,
1975.
K. Uyttendaele, V. Blaton, F. Alexander, H. Peeters, M.de Groote, N. Vinaimont-
Vandecasteele and J. Chevalier, Agarose isotachophoresis of human sweat, in P.G.

408 LITERATURE
Righetti (Editor), Progress in Isoelectric Focusing and Isotachophoresis, North-Holland,
Amsterdam, Oxford and Elsevier, New York, 1975, p. 341.
J.L.M. van de Venne, Some aspects of isotachophoretic analyses, Graduation Report,
University of Technology, Eindhoven, 1975.
J. Vozkova, Determination of dissociation constants via capillary isotachophoresis,
Graduation Report, Charles University, Prague, 1975.
A.J. Willemsen, Application of isotachophoresis in enzymology, J. Chromatogr., 105
(1975)405.
1976
J. Akiyama and T. Mizuno, Sensitivity of newly designed potential gradient detector
for isotachophoresis, J. Chromatogr., 119 (1976) 605.
L. Arlinger, Preparative capillary isotachophoresis. Principle and some applications,
J. Chromatogr., 119 (1976) 9.
P. BoEek, K. Lekova and J. Jan& Separation of some typical Krebs cycle acids by highspeed
isotachophoresis, J. Chromatogr., 117 (1976) 97.
F.M. Everaerts, M. Geurts, F.E.P. Mikkers and Th.P.E.M. Verheggen, Analytical isotachophoresis,
J. Chromatogr., 119 (1976) 129.
A. Kopwillem, W.G. Merriman, R.M. Cuddeback, A.J.K. Smolka and M. Bier, Serum
protein fractionation by isotachophoresis using amino acid spacers, J. Chromatogr.,
118 (1976) 35.
H. Miyazaki and K. Katoh, Isotachophoretic analysis of peptides,J. Chromatogr., 119
(1976) 369.
M. Svoboda and J. Vacik, Capillary isotachophoresis with ultraviolet detection. Some
quantitative aspects, J. Chrornatogr., 119 (1976) 539.

Symbols and abbreviations
SYMBOLS
A
A
a
B
b
C
C
'act
C*
DL7
D
E
e
F
fc
G
H
h
I
i
MI
N
n
ni
0
P
Q
4
R
r
S
S
T
increasing UV absorption;
empirical constant for a given series of ions of the same charge
ionic species A
activity
buffer ionic species B, ionic species B;
empirical constant for a given series of ions of the same charge
distance of closest approach (A)
capacity (F)
concentration (molell)
actual concentration of the ionic species in the narrow-bore tube (molell)
equivalent concentration (g-equiv./l)
dielectric constant of the solvent
dielectric constant of a solution
electric field strength (V/cm);
electromotive force (V)
charge on an electron (C)
Faraday constant (C/g-equiv.);
force (N)
friction factor
constant
step height (qualitative information) (generally mm)
step height (qualitative information) (generally mm)
electric current (d.c.) (A);
ionic strength (g-equiv./ml)
electric current (a.c.) (A)
equilibrium constant (in general, K is written for Ka)
calibration constant
gas constant per mole (erg/"K)
constants
length of a zone (cm)
length of a zone (sec)
migration distance of ion A (cm)
mobility (cm' /V * s);
molality (mole/l)
molecular weight of the solvent
Avogadro's number
number of pKa values of a molecule
number of ions i per ml
cross-section of the narrow-bore tube (cm’)
distance between point of injection and point of detection (cm)
total amount of an ionic species (mole);
volume transport (ml/sec)
charge on an ion (e.s.e.; C)
gas constant (erg/"K);
electric resistance (sym. increasing resistance) (a)
radius (A)
entropy
electrolyte constant;
migration way
absolute temperature (OK);
increasing temperature
409

410 SYMBOLS AND ABBREVIATIONS
1
V
v
oi
a*
P
Y
f
1)
K
A
A*
x
x*
IJe
w
SUBSCRIPTS
A
B
C
corn
H
i
ind
isot
i
L
OH-
0
r
ref
rel.
ret.
time (sec);
transport number
d.c. voltage drop (V);
increasing voltage;
molecular volume (A')
velocity (cm/sec);
a.c. voltage drop
volume injected (pl)
function of K h
function of K b
maximum number of positive charges for an ionic species;
valency of an ion
extent of dissociation;
constant
real extent of dissociation;
constant
ratio of two electric field strengths
activity coefficient;
correction factor according to Onsager
potential (V)
viscosity (g/cm - sec)
function of the concentration
molar conductance (cm2 /a * mole)
equivalent conductance (cm2 /a - equiv.)
electric conductivity of a zone [(Q crn)-']
equivalent conductance of an ionic species (cm'/s2 - equiv.)
magnetic permeability
frequency (Hz)
ionic species A
ionic species B
concentration
computed
ionic species H'
a number indicating the step of dissociation;
summation index
indicator electrode
isotachophoretic
a number indicating the step of dissociation;
liquid junction
leading ion/zone
ionic species OH-
at zero concentration
type of ionic species
reference electrode;
reference value
relaxation
retardation

SYMBOLS AND ABBREVIATIONS
SUPERSCRIPTS
EXAMPLES
'A,,lJ,z-i
('H, CJ)’
ABBREVIATIONS
AMP, ADP, ATP
CMP, CDP, CTP
E'EP
GMP, GDP, GTP
G6P
G6PDH
GABA
Guan
HNP
I. C.
Im
LDh
MES
MKAC
NADP
NAD
6 PG
PTI:E
S.C.
Tba
Tea
Tma
Tris
TPX
UMP, UDP, UTP
WKAC
standard solution
terminating ion/zone
Uth zone
Vth zone
sample solution
maximum possible charge
to the ith degree
relative
total
maximum number of positive charges of an ionic species
refers to equivalents instead of molar quantities;
refers to quantities in a certain solution
concentration of the ionic species A, with z-i positive charges in the Uth zone
the concentration of H' in the Uth zone to the ith degree
adenosine mono-, di- and triphosphate
cytidine mono-, di- and triphosphate
fluoroethylene polymer
guanosine mono-, di- and triphosphate.
glu co se-6 -ph ospha t e
glucose-6-phosphate dehydrogenase
y-aminobutyric acid
guanidine
half-neutralization point
integrated circuit
Imidazole
lactate dehydrogenase
morpholinoethanesulphonic acid
an example of an operational system, listed in a table, with methanol as the solvent
nicotinamide-adenine dinucleotide phosphate
nicotinamide dinucleotide
6-phosphogluconic acid
polytetrafluoroethylene
succinyl choke
tetrabutylammonium
tetraethylammonium
tetramethylammonium
trishydroxymethylarninornethane
methylpentene polymers
uridine mono-, di- and triphosphate
an example of an operational system, listed in a table, with water as the solvent
41 1

This Page Intentionally Left Blank

Subject index
A
Absorption meter, UV 153-170
a.c. conductivity detector, differentiator 154
Acidic media 87
Acidic solvents 86
a.c. method, calibration of conductimeter 15 1
___ , circuit suitable for determination of the
conductivity 146, 148
___ , linearity of two conductimeters 152
a.c. method of resistance determination 135, 143
Activity coefficients 76
Adaptation in concentrations 44
Adapted zones 18
Additives 180-190
_-_ , effect on electroendosmotic flow
171-173
--- , effect on micro-sensing electrodes
174- 180
--_ , influence.on isotachophoretic separation
183
Additives to the electrolytes 171-190
Alkali metals, determination 102
Alkali metals in methanol 104
Aluminium block, isotachophoretic equipment
with thermostating via 221-224
Amino acids, peptides and proteins 31 Iff
Amino acids, qualitative information 316, 317,
322
Amphiprotic media 87
Ampholyte gradients, separation of proteins
322-335
Analytical isotachophoresis, survey of detectors
used 122
Anionic species, separation in aqueous solutions
using a conductivity detector (a.c. method)
and a UV absorption detector (256 nm)
300-310
--- , separation in aqueous solutions using a
thermometric detector 295-300
--_ , separation in methanol using a
thermometric detector 362-364
Anions, qualitative information 297, 298,
307-310,363
Aqueous methanolic systems, separations using a
conductimetric detector (a.c. method) and UV
absorption detector (256 nm) 373
Aqueous solutions, separation of anionic species
using a conductivity detector (a.c. method)
and a UV absorption detector (256 nm)
300-310
_-_ , separation of anionic species using a
thermometric detector 295-300
___ , separation of cationic species using a
conductivity detector (a.c. method) and a UV
absorption detector (256 nm) 293, 294
___ , separation of cationic species using a
thermocouple as detector 283-293
Aqueous systems, separation of nucleotides using
a conductivity detector (a.c. method) and a
UV absorption detector (256 nm) 342-345
___ , separation of nucleotides using a
thermometric detector 337ff
Axial temperature differences 75, 76
B
Background electrolyte 7
Basic media 87
Basic solvents 86
Beharrliche Funktion 2, 41
Boundary, concentration 44
___ , separation 44
-__ , ___ ,velocity 48
Buffering capacity 94, 249
C
Calibration constant 273,275,280, 281
Calibration of concentrations 99
Calibration of conductimeter, ax. method 151
Calibration of d.c.-a.c. converter 141
Carrier functions 325
Carriers 99, 100
Cationic species, separation in aqueous solutions
using a conductivity detector (a.c. method)
and a UV absorption detector (256 nm)
293,294
___ , separation in aqueous solutions using a
thermocouple as detector 283-293
___ , separation in methanol using a
thermometric detector 364-373
413

414 SUBJECT INDEX
Cations, qualitative information 287, 292, 293,
368
Choice of buffering counter ionic species 92,93
Choice of electrolyte system 83ff
Choice of electrolyte system, scheme 101
Choice of pH of the leading electrolyte 93-96
Choice of solvent 84-92
Choice of terminating and leading ionic species
96-99
Circuit for counter flow regulation via the
membrane pump 242, 245
Circuit for d.c. method 137
Circuit suitable for determination of the
conductivity, a.c. method 146, 148
Circuit for differentiating thermometric signals
123
Circuit for on-off regulation of the pumping
mechanism during counter-flow 239
Circuit for potential gradient detector 137
Circuit for regulation of the counter-flow of
electrolyte via level regulation 233
Circuit for thermostating 222
Circuit of UV source 157
Coating of micro-sensing electrodes 191-193
--- ,effect 192, 194
Combination of systems 11 1
Compartment, counter electrode 21 1-217
--- -__ , cylindrical 213-215
~
-_- --_ , with flat membrane 215-217
Complex formation 33-35
Computation procedure for isotachophoresis 62
Computation procedure in moving-boundary
electrophoresis 389
Computer program (steady-state in
isotachophoresis) 74
Concept of mobility 27ff
Concentration adaption 18, 19
Concentration boundary 44
‘Concentration’ principle of isotachophoretic
analyses 378
Concentrations, calibration 99
Conductance, equivalent 29,36, 86
Conductimeters, calibration, a.c. method 151
-_- , two, linearity, a.c. method 152
Conductimetric detector (a.c. method) and UV
absorption detector (256 nm), separations in
aqueous methanolic systems 373
Conductivity, ionic equivalent 29-31
Conductivity detection 133 -1 52
-_- , high-frequency 130-133
--- , __- , construction 131-133
Conductivity detector 133
Conductivity detector (a.c. method) and UV
absorption detector (256 nm), separation of
anionic species in aqueous solutions 300-310
Conductivity detector (a.c. method) and UV
absorption detector (256 nm), separation of
cationic species in aqueous solutions 293,294
Conductivity detector (a.c. method) and UV
absorption detector (256 nm), separation of
nucleotides in aqueous systems 342-345
Conductivity probe 136
Conductivity probe with equiplanar-mounted
measuring electrodes 144
Conductivity probe with equiplanar-mounted
sensing electrodes 143-152
Construction, high-frequency conductivity
detection 131 -1 33
Construction of thermocouples 119-1 25
Conversion, enzymatic 348-360
Conversion of data 270, 271
Corrosion inhibitors 184
Coulomb’s law 85
Counter electrode compartment 211-217
__- , cylindrical 213-215
-__ ,with flat membrane 215-217
Counter flow, 100% 375
Counter flow of electrolyte 230-245, 375ff
-_- , influence of impurities 231
--- , via level regulation, circuit 233
Counter flow regulated by the current-stabilized
power supply 241-245
Counter flow regulation, via the membrane pump,
circuit 242,245
Counter flow with direct control on the pumping
mechanism via the power supply 237,238
Counter flow with level regulation 231-233
Counter flow with light-dependent resistor
regulation 23 3 -23 7
Counter flow with no regulation 238-240
Counter flow with on-off regulation of the
pumping mechanism, circuit 239
Counter ionic species, buffering, choice 92,93
Current, leak 188
Current density, influence on the
isotachopherogram 195
Current stabilized power supply 229
Cylindrical counter electrode compartment
213-215
D
Data, conversion 270, 271
d.c.-a.c. converter 140-142
__- , calibration 141
d.c. method, circuit 137
d.c. method of resistance determination 135
De-gassing of electrolytes 252

SUBJECT INDEX 41 5
Detection limit 129, 278
--_ , high-resolution detectors 193-199
___ , improvements 195
Detectors, specific 11 8
--_ , synchronous, UV detection 163
--_ , universal 1 18
Detectors used in analytical isotachophoresis,
survey 122
Determination of conductivity circuit suitable,
ax.-method 146, 148
Determination of effective mobilities 392
Determination of trace amounts 168
Diameter of the narrow-bore tube 395ff
Differential signal peaks 20
Differentiating thermometric signals, circuit 123
Differentiator for the a.c. conductivity detector
154
Diffusion, influence on the zone boundaries
74,75
‘Dilution’ principle of isotachophoretic analyses
378
‘Dilution’ technique 352
--_ , UV detector 169
Disc electrophoresis 17, 265-267
Disc electrophoretic system 67
Dissociation, partial 3 1-35
Disturbances caused by hydrogen and hydroxyl
ions 253-263
Disturbances due to the presence of carbon
dioxide 263, 264
Disturbances due to the presence of hydrogen
and hydroxyl ions in buffered systems
260-263
Disturbances from the leading zone 257-260
‘Dole polynomals’, modification 389
E
Electrode, activated 187
-_ _ , bipolar sensing 176
--_ , charge-transfer 175, 176
_-- , ‘ideally’ polarized 175, 176
_-- , metallic 174
-__ , micro-sensing, coating 19 1 - 193
_-_ , --_ , effect of coating 192, 194
--- , (partially) passivated 186
--_ , passivated 185
--_ , polarized 174
--_ , reversible 174
Electrode reactions 256
Electroendosmosis 171, 173
Electroendosmotic flow, effect of additives
171-173
Electrolyte, background 7
___ , de-gassing of 252
--- , leading 13
__- , supporting 7
___ , terminating 13
Electrolyte systems, choice 83ff
-__ --- ,scheme 101
Electroneutrality, principle 15,60
Electroneutrality equations 47, 48, 388
Electrophoresis, disc 17, 265-267
__- , moving-boundary 9-12, 387ff
--- , -__ , procedure of computation 389
__- , stacking 18
-_- ,zone 7
Electrophoretic system, disc 67
Enforced isotachophoresis 264-267
Enzymatic conversion 348-360
Enzymatic reactions 347ff
Equilibrium equations 45-47,58
Equipment 217-229
Equivalent weight 29
F
Fatty acids 103
_-- , separation in a methanolic system 108
Friction factor 27, 38
Friction force 27
Funktion, beharrliche 2, 41
Galvanic separation 150
Gas bubbles 252, 253
H
Halides, qualitative information 366
Heights, step 20
Henderson-Hasselbalch equation 33
High-frequency conductivity detection 130-133
__- , construction 131-133
High-resolution detectors, detection limits
193-199
, isotachophoretic equipment with 224-229

416 SUBJECT INDEX
I
Identification 20
--_ , reference materials 99
Impurities, influence on counter-flow of
electrolyte 23 1
--_ , marking a zone boundary 165
--_
, zones of 96
Indirect UV method 166
Inhibitors, corrosion 184
Injection block 208-211
--- , simplified 211, 212
Injection systems 203-21 1
--_ , four-way tap 204, 205
--_ , six-way valve 205-208
Ionic atmosphere 28
Isoelectric focusing 23, 24
Isoelectric point 23
Isotachopherograms 20-22
--- , influence of current density 195
Isotachophoresis, computation procedure 62
--- , enforced 264-267
, mathematical model 41ff, 58-62
--_ , principle 13-23
-_- , steady state 55-69
--- , _-_ , computer program 74
Isotachophoretic analysis, resolution 1 89
Isotachophoretic condition 14, 15, 58, 260
Isotachophoretic equipment 11 7
Is0 tachophore tic equipment with high-resolution
detectors 224-229
Isotachophoretic equipment with thermostating
via aluminium block 221-224
Isotachophoretic equipment with water-jacket
219-221
Isotachophoretic model, check 76-81
Isotachophoretically separated system 13, 57
Isotachophoretic separations, concept 5 5 -5 8
--- , influence of additives 183
--_ , influence of surfactants 189
Isotachophoretic separations in non-aqueous
systems 361ff
Iteration procedure 62-69
L
Law of independent migration 31
Leading electrolyte 13
-_- , pH, choice 93-96
Leading ionic species, choice 96-99
Leading zone, enlarged 255
Leak current 188
Length of a zone 18
Linearity of two conductimeters, a.c. method
152
M
Mass balance of the buffer 59, 60
Mass balances 48-50, 388
Measurement of effective mobilities 387ff
Mechanical construction of the UV source 158
Membrane pump 24 1-245
-__ , circuit for counter flow regulation via 242,
24 5
‘Memory’ effect 182
Methanol, alkali metals in 104
_-- , separation of anionic species using a
thermometric detector 362-364
--_ , separation of cationic species using a
thermometric detector 364-373
Methanol as solvent 87-92
Methanolic system, separation of some fatty acids
108
Micro-sensing electrodes, coatinq 191-193
--_ , effect of additives 174-1 80
--- , effect of coating 192, 194
--_ , polarization 176
Migration, independent, law 3 1
Migration paths 50
Mixed zones 9,13
Mobility, absolute ionic 30, 31
--_ , concept 27ff
-__ ,ionic 29-31
--- _-- ,effective 31-37,49, 83, 86
- - - - - - - - - , determination 392
- - - - - - , - - - , measurement 3 87 ff
--- , --- , relationship with entropy 39
--- , __- , relationship with volume 37, 38
-__ , separations according to 83, 98, 100
Mobility at infinite dilution 30
Moving-boundary electrophoresis 9-12, 387ff
-__ , procedure of computation 389
Moving-boundary procedure 13
N
Narrow-bore tube; diameter 395ff
Non-aqueous systems, isotachophoretic
separations 361ff
Nucleotides, qualitative information 342, 344

SUBJECT INDEX 41 7
--_ , separation 103
-__ , separation in aqueous systems using a
conductivity detector (a.c. method) and a UV
absorption detector (256 nm) 342-345
_ _ , separation ~ in aqueous systems using a
thermometric detector 337ff
0
Ohm’s law 17
--_ , modified 51, 61,62, 388
Operational system 249
Overpotential 176
Overshoot 177, 178
P
Parabolic profile 199
Peptides, amino acids and proteins 311 ff
___ , small, separation 335, 336
pH, determination 90
--_ , operational definition 89
pK values, determination 89-92
___ , separations according to 83, 94, 98, 100
Polarization of micro-sensing electrode 176
Potential gradient detector 135
__- , circuit 137
Potentials, acidity 86
Power supply, current stabilized 229
Procedure of computation in moving-boundary
electrophoresis 389
Profile of a zone boundary 172
Proteins, amino acids and peptides 311ff
- _
-__ , separation in ampholyte gradients
322-335
Protolysis 33
Q
Qualitative information 21-23
Qualitative information of amino acids 316, 317,
322
Qualitative information of anions 297, 298,
307-310, 363
Qualitative information of cations 287, 292, 293,
36 8
Qualitative information of halides 366
Qualitative information of nucleotides 342, 344
Quantitative aspects 273
Quantitative determination 21, 23
Quantitative information 21-23
R
Radial temperature differences 75, 76
Reference materials for identification 99
Regulating function 42
Relaxation 36, 37
Relaxation effect 28
Reproducibility 275, 279
Resistance determination, a.c. method 135, 143
--_ , d.c. method 135
Resolution of isotachophoretic analysis 189
Retardation, electrophoretic 28, 36, 37
R~values 8
S
Self-conductance 85
Self-correcting effect 11
Self-correction 15
Separation, partial 9
___ , time needed 56
Separation boundary 44
___ , velocity 4 8
Separation of anTonic species in aqueous solutions
using a conductivity detector (ax. method)
and a UV absorption detector (256 nm)
300-310
Separation of anionic species in aqueous solutions
using a thermometric detector 295-300
Separation of anionic species in methanol using a
thermometric detector 362-364
Separation of cationic species in aqueous
solutions using a conductivity detector (ax.
method) and a UV absorption detector
(256 nm) 293,294
Separation of cationic species in aqueous
solutions using a thermocouple as detector
283-293
Separation of cationic species in methanol using a
thermometric detector 364-373
Separation of fatty acids in a methanolic system
108
Separation of nucleotides 103
Separation of nucleotides in aqueous systems
using a thermometric detector 337ff

41 8 SUBJECT INDEX
Separation of nucleotides in aqueous systems
using a conductivity detector (a.c. method)
and a UV absorption detector (256 nm)
342-345
Separation of peptides 335, 336
Separation of proteins in ampholyte gradients
322-335
Separations according to mobilities 83, 98, 100
Separations according to pK values 83, 94, 98,
100
Separations in aqueous methanolic systems using
a conductimetric detector (a.c. method) and
UV absorption detector (256 nm) 373
Solvents, choice 84-92
--- , methanol 87-92
--_ , acidic 86
--- ,basic 86
--_ , classes 87
Spacer functions 325
Spacers 99,100
Stabilizers 99
Stacking electrophoresis 18
Stationary state 48
Steady state 13,57
Steady-state in isotachophoresis, computer
program 74
Supporting electrolyte 7
Surface-active chemicals 99
Surface-active compounds used in
isotachophoretic analyses 181
Surfactants, influence on the isotachophoretic
separation 189
Synchronous detector, UV detection 163
Systems, combination 11 1
T
Tailing 8
Tap, four-way 204,205
Terminating electrolyte 13
Terminating ionic species, choice 96-99
Thermocouple, construction 119-1 25
--- , differential 121, 124
Thermocouple as detector, separation of cationic
species in aqueous solutions 283-293
Thermometric detector, separation of anionic
species in aqueous solutions 295-300
--- , separation of anionic species in methanol
362-364
--_ , separation of cationic species in methanol
364-373
--_ , separation of nucleotides in aqueous
systems 337ff
Thermometric recording 119-1 30
Thermometric signals, differentiating, circuit 123
Thermostating, circuit 222
Trace amounts, determination 168
Transport number 30
Trouble-shooting 249, 250
U
UV absorption detector (256 nm) and a
conductimetric detector (a.c. method),
separations in aqueous methanolic systems
373
UV absorption detector and conductivity
detector (a.c. method), separation of anionic
species in aqueous solutions 300-310
UV absorption detector (256 nm) and
ccnductivity detector (a.c. method),
separation of cationic species in aqueous
solutions 293, 294
UV absorption detector (256 nm) and
conductivity detector (a.c. method),
separation of nucleotides in aqueous systems
342-345
UV absorption meter 153-170
W cell 164, 165
UV detector, dilution technique 169
W detector combination with modulated UV
source 162
UV detector combination with non-modulated
UV 160
W method, indirect 166
UV source, circuit 157
_-- , construction 155-159
_-_ , mechanical construction 158
V
Valve, six-way 205-208
W
Water-jacket, isotachophoretic equipment with
219
Z
Zone boundary, influence of diffusion 74
-__ , profile 172
Zone electrophoresis 7
Zone length 18
Zones, mixed 9, 13
_-_
, adapted 18
Zones of impurities 96