Frans_M_Everaerts_Isotachophoresis_378342.pdf

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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” equipment 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
Page 2 and 3: Journal of Chromatogaphy Library -
Page 4 and 5: Journal of Chromatography Library -
Page 6 and 7: Dedicated to Pr0f.Dr.h. A.I.M. Keul
Page 8 and 9: Contents Preface ..................
Page 10 and 11: CONTENTS IX 6.4.2. The d.c. method
Page 12 and 13: CONTENTS XI 12.2.2. Applications ..
Page 16 and 17: Chapter 1 Historical review SUMMARY
Page 18 and 19: HISTORICAL REVIEW 3 Independently i
Page 20 and 21: THEORY
Page 22 and 23: Chapter 2 Principles of electrophor
Page 24 and 25: MOVINGBOUNDARY ELECTROPHORESIS a S
Page 26 and 27: MOVINGBOUNDARY ELECTROPHORESIS 0 iu
Page 28 and 29: ISOTACHOPHORESIS 13 2.4. PRINCIPLE
Page 30 and 31: ISOTACHOPHORESIS 15 As the anionic
Page 32 and 33: ISOTACHOPHORESIS velocities, so tha
Page 34 and 35: ISOTACHOPHORESIS 19 d.t ’.t I Fig
Page 36 and 37: ISOTACHOPHORESIS 21 t Fig.2.8. Isot
Page 38 and 39: ISOELECTRIC FOCUSING 23 The four is
Page 40 and 41: DISCUSSION 25 Fig.2.11. Survey of t
Page 42 and 43: Chapter 3 Concept of mobility SUMMA
Page 44 and 45: IONIC MOBILITY AND IONIC EQUIVALENT
Page 46 and 47: EFFECTIVE IONIC MOBILITY 31 obtain
Page 48 and 49: EFFECTIVE IONIC MOBILITY 33 compari
Page 50 and 51: EFFECTIVE IONIC MOBILITY Fig.3.4. R
Page 52 and 53: DETERMINATION OF IONIC MOBILITIES 3
Page 54 and 55: DETERMINATION OF IONIC MOBILITIES 3
Page 56 and 57: Chapter 4 Mathematical model for is
Page 58 and 59: GENERAL EQUATIONS 43 anionic specie
Page 60 and 61: GENERAL EQUATIONS 4.2.1. Equilibriu
Page 62 and 63: GENERAL EQUATIONS 41 i Similar equa
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GENERAL EQUATIONS 49 Uthzone (U-llt
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GENERAL EQUATIONS 4.2.4. Modified O
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GENERAL EQUATIONS TABLE 4.2 REDUCED
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MATHEMATICAL MODEL FOR THE STEADY S
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VALIDITY OF THE ISOTACHOPHORETIC MO
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CHECK OF THE ISOTACHOPHORETIC MODEL
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CHECK OF THE ISOTACHOPHORETIC MODEL
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REFERENCES 81 Fig.4.17. Relationshi
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Chapter 5 Choice of electrolyte sys
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CHOICE OF THE SOLVENT 85 In general
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CHOICE OF THE SOLVENT TABLE 5.2 THE
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CHOICE OF THE SOLVENT pH and pKvalu
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CHOICE OF THE SOLVENT 91 TABLE 5.4
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CHOICE OF THE pH OF THE LEADING ELE
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CHOICE OF THE pH OF THE LEADING ELE
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CHOICE OF THE TERMINATING AND LEADI
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ADDITIONS TO THE ELECTROLYTE SOLUTI
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EXAMPLES Choice of solvent. Can wat
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EXAMPLES 103 solvent, hoping that a
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EXAMPLES TABLE 5.11 STEP HEIGHTS OF
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EXAMPLES TABLE 5.12 STEP HEIGHTS OF
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Fig.5.9. Isotachopherograms of the
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EXAMPLES II 4 3 Fig.5.11. Isotachop
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REFERENCES 113 Example I. As exampl
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INSTRUMENTATION
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Chapter 6 Detection systems SUMMARY
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THERMOMETRIC RECORDING 6.1.3. Combi
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THERMOMETRIC RECORDING Potential gr
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THERMOMETRIC RECORDING /2 Fig.6.1.
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THERMOMETRIC RECORDING T t l Fig.6.
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THERMOMETRIC RECORDING 7 127 It-- I
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THERMOMETRIC RECORDING a concentrat
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HIGH-FREQUENCY CONDUCTIVITY DETECTI
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CONDUCTIVITY DETECTION 133 Fig.6.9.
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CONDUCTIVITY DETECTION 135 The d.c.
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CONDUCTIVITY DETECTION 137 Hi V 1kR
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CONDUCTIVITY DETECTION Fig.6.12. Co
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CONDUCTIVITY DETECTION Fig.6.14. Th
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CONDUCTIVITY DETECTION 143 6.4.4. T
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CONDUCTIVITY DETECTION 145 contact
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CONDUCTIVITY DETECTION 147 We can n
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CONDUCTIVITY DETECTION 149 N.Y., U.
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CONDUCTIVITY DETECTION 151 achieved
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UV ABSORPTION METER 153 -_-.- +- R
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UV ABSORPTION METER Fig.6.22. Contr
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W ABSORPTION METER r +15V -15V Mod
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W ABSORPTION METER 159 TABLE 6.4 PR
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W ABSORPTION METER 230 240 2% m zm
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to Mod (Fig 6.24) oscillator I sync
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UV ABSORPTION METER 165 dicular to
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Fig.6.31. Isotachopherograms for th
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UV ABSORPTION METER 169 50 Fig.6.33
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ADDITIVES TO THE ELECTROLYTES 171 6
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ADDITIVES TO THE ELECTROLYTES 173 e
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ADDITIVES TO THE ELECTROLYTES 175 a
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ADDITIVES TO THE ELECTROLYTES 177 o
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ADDITIVES TO THE ELECTROLYTES 179 d
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ADDITIVES TO THE ELECTROLYTES 181 T
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ADDITIVES TO THE ELECTROLYTES Fig.6
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ADDITIVES TO THE ELECTROLYTES 185 F
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ADDITIVES TO THE ELECTROLYTES 1 2 3
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ADDITIVES TO THE ELECTROLYTES 189 n
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COATING OF THE MICRO-SENSING ELECTR
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DETECTION LIMITS 193 electric drivi
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DETECTION LIMITS 195 Fig.6.48. Infl
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DETECTION LIMITS 197 TABLE 6.6 SURV
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CONCLUSION 199 average length of a
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REFERENCES 201 seen. Again it is cl
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Chapter 7 Instrumentation SUMMARY T
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INJECTION SYSTEMS I! I n Fig.7.1. P
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INJECTION SYSTEMS Fig.7.3. Exploded
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INJECTION SYSTEMS Fig.7.5. Injectio
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COUNTER ELECTRODE COMPARTMENTS 21 1
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COUNTER ELECTRODE COMPARTMENTS Even
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COUNTER ELECTRODE COMPARTMENTS 21 5
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EQUIPMENT the large bore, a more di
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EQUIPMENT 219 belonging to three cl
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EQUIPMENT 221 signal of the thermoc
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EQUIF'MENT 223 No effect on the res
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EQUIPMENT 225 Fig.7.14. Electrophor
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EQUIPMENT Fig.7.16. Photograph of t
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EQUIPMENT 229 mounted equiplanar, w
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COUNTER FLOW OF ELECTROLYTE 231 ins
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COUNTER FLOW OF ELECTROLYTE p High
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COUNTER FLOW OF ELECTROLYTE Fig.7.2
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COUNTER FLOW OF ELECTROLYTE 231 aro
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COUNTER FLOW OF ELECTROLYTE p high
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COUNTER FLOW OF ELECTROLYTE 24 1 7.
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COUNTER FLOW OF ELECTROLYTE I I 31
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COUNTER FLOW OF ELECTROLYTE max. 15
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APPLICATIONS
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Chapter 8 Introduction SUMMARY In t
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INTRODUCTION 25 1 the various ionic
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Chapter 9 Practical aspects SUMMARY
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DISTURBANCES CAUSED BY H+ AND OH 25
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DISTURBANCES CAUSED BY H+ AND OH- 2
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DISTURBANCES DUE TO CO, 263 Electro
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ENFORCED ISOTACHOPHORESIS 265 t T T
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WATER AS TERMINATOR 267 function of
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PURIFICATION OF THE TERMINATOR 7 4
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REFERENCES 271 AS an example, the c
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Chapter 10 Quantitative aspects SUM
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THERMOMETRIC MEASUREMENTS 215 vj c
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THERMOMETRIC MEASUREMENTS TABLE 10.
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CONDUCTIMETRIC MEASUREMENTS factor
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CONCLUSION 28 1 Tables 10.3-10.5 wa
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Chapter 11 Separation of cationic s
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SEPARATION USING A THERMOCOUPLE AS
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I SEPARATION USING A THERMOCOUPLE A
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SEPARATION USING CONDUCTIVITY AND U
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Chapter I2 Separation of anionic sp
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SEPARATION USING A THERMOMETRIC DET
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SEPARATION USING CONDUCTIVITY AND W
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Chapter 13 Amino acids, peptides an
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AMINO ACIDS TABLE 13.1 OPERATIONAL
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AMINO ACIDS 8- ?- 6- 5- 4- 3- 2- f-
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AMINO ACIDS 317 TABLE 13.5 STEP HEI
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AMINO ACIDS Fig.13.3. Schematic dia
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AMINO ACIDS TABLE 13.6 DETERMINATIO
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SEPARATION OF PROTEINS IN AMPHOLYTE
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SEPARATION OF SMALL PEPTIDES reprod
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Chapter I4 Separation of nucleotide
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Chapter 15 Enzymatic reactions SUMM
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ENZYMATIC CONVERSION OF GLUCOSE (FR
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ENZYMATIC CONVERSION OF PYRUVATE 35
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Chapter 16 Separations in non-aqueo
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SEPARATION OF ANIONIC SPECIES IN ME
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SEPARATION OF CATIONIC SPECIES IN M
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EXPERIMENTS IN AQUEOUS METHANOLIC S
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Chapter I7 Counter flow of electrol
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INTRODUCTION 317 In various operati
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EXPERIMENTAL 0% 10 % a b 60 % a b a
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EXPERIMENTAL 381 caused by the coun
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CONCLUSION 383 c t r ’r; I I"
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APPENDICES
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Appendix A Simplified model of movi
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PROCEDURE OF COMPUTATION A.3. PROCE
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EXPERIMENTAL 39 1 TABLE A1 THEORETI
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DISCUSSION E I I Fig.A2. Graphical
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Appendix B Diameter of the narrow-b
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Appendix C Literature 1897-1966 F.
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LITERATURE 399 B.P. Konstantinov an
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LITERATURE 401 1971 L. Arlinger, Is
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LITERATURE 403 1973 L. Arlinger and
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LITERATURE 405 A. Chrambach and J.S
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LITERATURE 407 M. Coxon and M.J. Bi
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Symbols and abbreviations SYMBOLS A
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SYMBOLS AND ABBREVIATIONS SUPERSCRI
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Subject index A Absorption meter, U
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SUBJECT INDEX 41 5 Detection limit
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SUBJECT INDEX 41 7 --_ , separation
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