Frans_M_Everaerts_Isotachophoresis_378342.pdf

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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-
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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
Page 312 and 313: SEPARATION USING A THERMOMETRIC DET
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 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
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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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