Frans_M_Everaerts_Isotachophoresis_378342.pdf

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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 operational 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 concentration 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
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Journal of Chromatogaphy Library -
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Journal of Chromatography Library -
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Dedicated to Pr0f.Dr.h. A.I.M. Keul
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Contents Preface ..................
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CONTENTS IX 6.4.2. The d.c. method
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CONTENTS XI 12.2.2. Applications ..
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It is very well known that charged
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Chapter 1 Historical review SUMMARY
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HISTORICAL REVIEW 3 Independently i
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THEORY
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Chapter 2 Principles of electrophor
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MOVINGBOUNDARY ELECTROPHORESIS a S
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MOVINGBOUNDARY ELECTROPHORESIS 0 iu
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ISOTACHOPHORESIS 13 2.4. PRINCIPLE
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ISOTACHOPHORESIS 15 As the anionic
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ISOTACHOPHORESIS velocities, so tha
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ISOTACHOPHORESIS 19 d.t ’.t I Fig
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ISOTACHOPHORESIS 21 t Fig.2.8. Isot
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ISOELECTRIC FOCUSING 23 The four is
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DISCUSSION 25 Fig.2.11. Survey of t
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Chapter 3 Concept of mobility SUMMA
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IONIC MOBILITY AND IONIC EQUIVALENT
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EFFECTIVE IONIC MOBILITY 31 obtain
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EFFECTIVE IONIC MOBILITY 33 compari
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EFFECTIVE IONIC MOBILITY Fig.3.4. R
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DETERMINATION OF IONIC MOBILITIES 3
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DETERMINATION OF IONIC MOBILITIES 3
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Chapter 4 Mathematical model for is
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GENERAL EQUATIONS 43 anionic specie
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GENERAL EQUATIONS 4.2.1. Equilibriu
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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
Page 134 and 135: THERMOMETRIC RECORDING 6.1.3. Combi
Page 136 and 137: THERMOMETRIC RECORDING Potential gr
Page 138 and 139: THERMOMETRIC RECORDING /2 Fig.6.1.
Page 140 and 141: THERMOMETRIC RECORDING T t l Fig.6.
Page 142 and 143: THERMOMETRIC RECORDING 7 127 It-- I
Page 144 and 145: THERMOMETRIC RECORDING a concentrat
Page 146 and 147: HIGH-FREQUENCY CONDUCTIVITY DETECTI
Page 148 and 149: CONDUCTIVITY DETECTION 133 Fig.6.9.
Page 150 and 151: CONDUCTIVITY DETECTION 135 The d.c.
Page 152 and 153: CONDUCTIVITY DETECTION 137 Hi V 1kR
Page 154 and 155: CONDUCTIVITY DETECTION Fig.6.12. Co
Page 156 and 157: CONDUCTIVITY DETECTION Fig.6.14. Th
Page 158 and 159: CONDUCTIVITY DETECTION 143 6.4.4. T
Page 160 and 161: CONDUCTIVITY DETECTION 145 contact
Page 162 and 163: CONDUCTIVITY DETECTION 147 We can n
Page 164 and 165: CONDUCTIVITY DETECTION 149 N.Y., U.
Page 166 and 167: CONDUCTIVITY DETECTION 151 achieved
Page 168 and 169: UV ABSORPTION METER 153 -_-.- +- R
Page 170 and 171: UV ABSORPTION METER Fig.6.22. Contr
Page 172 and 173: W ABSORPTION METER r +15V -15V Mod
Page 174 and 175: W ABSORPTION METER 159 TABLE 6.4 PR
Page 176 and 177: W ABSORPTION METER 230 240 2% m zm
Page 178 and 179: to Mod (Fig 6.24) oscillator I sync
Page 180 and 181: UV ABSORPTION METER 165 dicular to
Page 182 and 183: Fig.6.31. Isotachopherograms for th
Page 186 and 187: ADDITIVES TO THE ELECTROLYTES 171 6
Page 188 and 189: ADDITIVES TO THE ELECTROLYTES 173 e
Page 190 and 191: ADDITIVES TO THE ELECTROLYTES 175 a
Page 192 and 193: ADDITIVES TO THE ELECTROLYTES 177 o
Page 194 and 195: ADDITIVES TO THE ELECTROLYTES 179 d
Page 196 and 197: ADDITIVES TO THE ELECTROLYTES 181 T
Page 198 and 199: ADDITIVES TO THE ELECTROLYTES Fig.6
Page 200 and 201: ADDITIVES TO THE ELECTROLYTES 185 F
Page 202 and 203: ADDITIVES TO THE ELECTROLYTES 1 2 3
Page 204 and 205: ADDITIVES TO THE ELECTROLYTES 189 n
Page 206 and 207: COATING OF THE MICRO-SENSING ELECTR
Page 208 and 209: DETECTION LIMITS 193 electric drivi
Page 210 and 211: DETECTION LIMITS 195 Fig.6.48. Infl
Page 212 and 213: DETECTION LIMITS 197 TABLE 6.6 SURV
Page 214 and 215: CONCLUSION 199 average length of a
Page 216 and 217: REFERENCES 201 seen. Again it is cl
Page 218 and 219: Chapter 7 Instrumentation SUMMARY T
Page 220 and 221: INJECTION SYSTEMS I! I n Fig.7.1. P
Page 222 and 223: INJECTION SYSTEMS Fig.7.3. Exploded
Page 224 and 225: INJECTION SYSTEMS Fig.7.5. Injectio
Page 226 and 227: COUNTER ELECTRODE COMPARTMENTS 21 1
Page 228 and 229: COUNTER ELECTRODE COMPARTMENTS Even
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Page 232 and 233: 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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