Frans_M_Everaerts_Isotachophoresis_378342.pdf

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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.
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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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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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Page 316 and 317: SEPARATION USING CONDUCTIVITY AND W
Page 318 and 319: SEPARATION USING CONDUCTIVITY AND U
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 366 and 367: ENZYMATIC CONVERSION OF GLUCOSE (FR
Page 368 and 369: 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
Page 406 and 407: EXPERIMENTAL 39 1 TABLE A1 THEORETI
Page 408 and 409: DISCUSSION E I I Fig.A2. Graphical
Page 410 and 411: Appendix B Diameter of the narrow-b
Page 412 and 413: 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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