Frans_M_Everaerts_Isotachophoresis_378342.pdf

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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 separation 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
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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
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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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Page 306 and 307: I SEPARATION USING A THERMOCOUPLE A
Page 308 and 309: SEPARATION USING CONDUCTIVITY AND U
Page 310 and 311: Chapter I2 Separation of anionic sp
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Page 316 and 317: SEPARATION USING CONDUCTIVITY AND W
Page 326 and 327: Chapter 13 Amino acids, peptides an
Page 328 and 329: AMINO ACIDS TABLE 13.1 OPERATIONAL
Page 330 and 331: AMINO ACIDS 8- ?- 6- 5- 4- 3- 2- f-
Page 332 and 333: AMINO ACIDS 317 TABLE 13.5 STEP HEI
Page 334 and 335: AMINO ACIDS Fig.13.3. Schematic dia
Page 336 and 337: AMINO ACIDS TABLE 13.6 DETERMINATIO
Page 338 and 339: SEPARATION OF PROTEINS IN AMPHOLYTE
Page 350 and 351: SEPARATION OF SMALL PEPTIDES reprod
Page 352 and 353: Chapter I4 Separation of nucleotide
Page 362 and 363: Chapter 15 Enzymatic reactions SUMM
Page 364 and 365: ENZYMATIC CONVERSION OF GLUCOSE (FR
Page 370 and 371: ENZYMATIC CONVERSION OF PYRUVATE 35
Page 376 and 377: Chapter 16 Separations in non-aqueo
Page 378 and 379: SEPARATION OF ANIONIC SPECIES IN ME
Page 380 and 381: SEPARATION OF CATIONIC SPECIES IN M
Page 388 and 389: EXPERIMENTS IN AQUEOUS METHANOLIC S
Page 390 and 391: Chapter I7 Counter flow of electrol
Page 392 and 393: INTRODUCTION 317 In various operati
Page 394 and 395: EXPERIMENTAL 0% 10 % a b 60 % a b a
Page 396 and 397: EXPERIMENTAL 381 caused by the coun
Page 398 and 399: CONCLUSION 383 c t r ’r; I I"
Page 400 and 401: APPENDICES
Page 402 and 403: Appendix A Simplified model of movi
Page 404 and 405: 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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