Frans_M_Everaerts_Isotachophoresis_378342.pdf

More documents

Recommendations

Info

CHOICE OF THE pH OF THE LEADING ELECTROLYTE TABLE 5.6 EXPERIMENTALLY DETERMINED pK& VALUES FOR SOME ANIONIC SPECIES IN 95% METHANOL Ionic species PKh Ionic species PKL Acetic acid Adipic acid Azelaic acid Benzoic acid Bu tyric acid Caproic acid Caprylic acid Die thanolamine Formic acid Glutaric acid Hippuric acid Histidine Irnidazole Lauric acid Linoleic acid 7.9 7.55-9.1 7.65-9.0 7.5 8.0 8.0 8.0 9.6 6.45 7.5-9.2 6.95 6.0- 10.15 6.55 7.9 7.9 Maleic acid Malonic acid Monoethanolamine Myristic acid Orotic acid Oxalic acid Palmitic acid F’imelic acid Pyruvic acid Salicylic acid Suberic acid Succinic acid Trie thanolamine Tris Isovaleric acid 4.6-? 5.9-9.7 9.6 8.1 8.8 4.5-8.3 8.0 7.6-8.95 5.9 6.2 7.6-8.95 93 7.25-9.4 7.9 9.05 8.05 principle for his ‘precipitation electrophoresis’. In isotachophoresis in capillary tubes, precipitation is undesirable as it may produce stoppages in the capillary tube. Asecond point to be considered is the use of buffering counter ions with a W absorption power, if a W detector is used. The concenqations of the counter ions and the pH are different in the various zones and, if we use a buffer with a molar extinction coefficient that is influenced by the pN (i.e., if only a particular ionic form of the buffer shows a UV absorbance), the different zones can be detected by the differences in the UV absorption of the buffer ions in these zones. This method of detection can be applied particularly if the sample ions show no or only a slight UV absorption. The third point and the most important function of the buffering counter ionic species is its buffering capacity for the stabilization and regulation of the pH in the different zones, by which effective mobilities of the ionic species are fixed and by which a ‘steady state’ can be maintained. The selection of a suitable pH, in combination with the type of buffer, is considered in the next section. 5.4. CHOICE OF THE pH OF THE LEADING ELECTROLYTE In this section, we shall consider separations according to pK valves because these separations are closely related to the choice of the pH of the leading electrolyte. For separations according to mobilities, the pH is of less importance. Two points are very important here: firstly, the choice of the pH itself, and secondly, the choice of the type of buffering counter ionic species, which defines the pH at which the analysis is to be performed. In general, the pH is chosen in such a way that maximal differences in effective mobilities can be obtained according to eqn. 3.18, but a limitation is that if the pH in a zone differs more than 2 pH units from the pK values of the ionic species in that
94 CHOICE OF ELECTROLYTE SYSTEMS zone, such low effective mobilities are obtained that the potentials required rise above the maximal potential of the stabilized d.c. power supply (this factor is of minor importance for the buffer.) Another limitation is due to the buffering capacity of the counter ions. A maximd buffering capacity is obtained if pK + 1 pK - 1. Hence the buffer will have a low buffering capacity if its pK value differs more than about 2 pH units from the pH in a zone. The pH lies between the pK values of buffer and sample ionic species, so that we cannot use a buffer when its pK value differs more than about 3 pK units from that of the sample ionic species (this is valid when the pK is lower than the pK values of the sample ions). In order to demonstrate this important effect, the relationship between the pK values of ionic species in the sample and the pH values of their zones is shown in Fig.5.1 for a pKB of 6 and a pH of the leading electrolyte, pH, of 5.75. Fig.5.1 shows clearly that the buffer has a low buffering capacity if its pKB differs more than about 3 pH units from the pK of the ionic species. In general, a buffer should be chosen with a pKB less than 3 pH units lower than the pK value of the anionic species to be separated. After considering the limitations concerning demands for the buffering ions at a chosen pH, the next problem is to choose the pH, value, and two approaches are possible. If no data such as pK values and mobilities are available, the experimental method must be used. All step heights of the substances concerned have to be measured at different pH, values and for several electrolyte systems, after which a pH, value can be chosen for the optimal separation (maximal differences in step heights). A combination of systems can be applied. If all data are known, theoretically all effective mobilities can be calculated and from the results obtained the pH, that shows maximal differences in effective mobilities can be decided. In order to demonstrate those two approaches to separations according to pK values, eleven anionic species that cannot be separated according to mobilities in the system histidine/histidine hydrochloride at a pH, of 6.02 (see Table 12.1) were selected. The effective mobilities were computed with a computer program for five electrolyte systems at different pH, values and all step heights were measured for the systems. In Table 5.7, the conditions are given for the five electrolyte systems and Table 5.8 gives the calculated effective mobilities (theoretical method) and measured step heights (experimental method). In Fig.5.2 the step heights are plotted for the different systems. The results indicate that the differences in effective mobilities (and step heights) are much greater at lower pH values of the leading electrolyte, which permits better separations to be achieved. Some separations have been carried out. Fig.5.3A shows the electropherogram for the separation of a mixture of trichloroacetate, P-chloropropionate, benzoate, crotonate, paminobenzoate and trimethyl acetate at a pH, of 6.02. The terminator was glutamate, and the leading ion was chloride. No complete separation could be achieved. In Fig.5.3B, the separation is shown for the same mixture in system E (see Table 5.7) at a pH, of4. Trimethyl acetate was used as the terminator and a complete separation could be easily achieved. A thermometric detector (copper-constantan thermocouple) was used.
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 14 and 15:
It is very well known that charged
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
Page 64 and 65: GENERAL EQUATIONS 49 Uthzone (U-llt
Page 66 and 67: GENERAL EQUATIONS 4.2.4. Modified O
Page 68 and 69: GENERAL EQUATIONS TABLE 4.2 REDUCED
Page 70 and 71: MATHEMATICAL MODEL FOR THE STEADY S
Page 84 and 85: VALIDITY OF THE ISOTACHOPHORETIC MO
Page 92 and 93: CHECK OF THE ISOTACHOPHORETIC MODEL
Page 94 and 95: CHECK OF THE ISOTACHOPHORETIC MODEL
Page 96 and 97: REFERENCES 81 Fig.4.17. Relationshi
Page 98 and 99: Chapter 5 Choice of electrolyte sys
Page 100 and 101: CHOICE OF THE SOLVENT 85 In general
Page 102 and 103: CHOICE OF THE SOLVENT TABLE 5.2 THE
Page 104 and 105: CHOICE OF THE SOLVENT pH and pKvalu
Page 106 and 107: CHOICE OF THE SOLVENT 91 TABLE 5.4
Page 110 and 111: CHOICE OF THE pH OF THE LEADING ELE
Page 112 and 113: CHOICE OF THE TERMINATING AND LEADI
Page 114 and 115: ADDITIONS TO THE ELECTROLYTE SOLUTI
Page 116 and 117: EXAMPLES Choice of solvent. Can wat
Page 118 and 119: EXAMPLES 103 solvent, hoping that a
Page 120 and 121: EXAMPLES TABLE 5.11 STEP HEIGHTS OF
Page 122 and 123: EXAMPLES TABLE 5.12 STEP HEIGHTS OF
Page 124 and 125: Fig.5.9. Isotachopherograms of the
Page 126 and 127: EXAMPLES II 4 3 Fig.5.11. Isotachop
Page 128 and 129: REFERENCES 113 Example I. As exampl
Page 130 and 131: INSTRUMENTATION
Page 132 and 133: 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 184 and 185:
UV ABSORPTION METER 169 50 Fig.6.33
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
Page 230 and 231:
COUNTER ELECTRODE COMPARTMENTS 21 5
Page 232 and 233:
EQUIPMENT the large bore, a more di
Page 234 and 235:
EQUIPMENT 219 belonging to three cl
Page 236 and 237:
EQUIPMENT 221 signal of the thermoc
Page 238 and 239:
EQUIF'MENT 223 No effect on the res
Page 240 and 241:
EQUIPMENT 225 Fig.7.14. Electrophor
Page 242 and 243:
EQUIPMENT Fig.7.16. Photograph of t
Page 244 and 245:
EQUIPMENT 229 mounted equiplanar, w
Page 246 and 247:
COUNTER FLOW OF ELECTROLYTE 231 ins
Page 248 and 249:
COUNTER FLOW OF ELECTROLYTE p High
Page 250 and 251:
COUNTER FLOW OF ELECTROLYTE Fig.7.2
Page 252 and 253:
COUNTER FLOW OF ELECTROLYTE 231 aro
Page 254 and 255:
COUNTER FLOW OF ELECTROLYTE p high
Page 256 and 257:
COUNTER FLOW OF ELECTROLYTE 24 1 7.
Page 258 and 259:
COUNTER FLOW OF ELECTROLYTE I I 31
Page 260 and 261:
COUNTER FLOW OF ELECTROLYTE max. 15
Page 262 and 263:
APPLICATIONS
Page 264 and 265:
Chapter 8 Introduction SUMMARY In t
Page 266 and 267:
INTRODUCTION 25 1 the various ionic
Page 268 and 269:
Chapter 9 Practical aspects SUMMARY
Page 270 and 271:
DISTURBANCES CAUSED BY H+ AND OH 25
Page 272 and 273:
Page 274 and 275:
DISTURBANCES CAUSED BY H+ AND OH- 2
Page 276 and 277:
Page 278 and 279:
DISTURBANCES DUE TO CO, 263 Electro
Page 280 and 281:
ENFORCED ISOTACHOPHORESIS 265 t T T
Page 282 and 283:
WATER AS TERMINATOR 267 function of
Page 284 and 285:
PURIFICATION OF THE TERMINATOR 7 4
Page 286 and 287:
REFERENCES 271 AS an example, the c
Page 288 and 289:
Chapter 10 Quantitative aspects SUM
Page 290 and 291:
THERMOMETRIC MEASUREMENTS 215 vj c
Page 292 and 293:
THERMOMETRIC MEASUREMENTS TABLE 10.
Page 294 and 295:
CONDUCTIMETRIC MEASUREMENTS factor
Page 296 and 297:
CONCLUSION 28 1 Tables 10.3-10.5 wa
Page 298 and 299:
Chapter 11 Separation of cationic s
Page 300 and 301:
SEPARATION USING A THERMOCOUPLE AS
Page 302 and 303:
Page 304 and 305:
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 314 and 315:
Page 316 and 317:
SEPARATION USING CONDUCTIVITY AND W
Page 318 and 319:
Page 320 and 321:
Page 322 and 323:
Page 324 and 325:
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 340 and 341:
Page 342 and 343:
Page 344 and 345:
Page 346 and 347:
Page 348 and 349:
Page 350 and 351:
SEPARATION OF SMALL PEPTIDES reprod
Page 352 and 353:
Chapter I4 Separation of nucleotide
Page 354 and 355:
Page 356 and 357:
Page 358 and 359:
Page 360 and 361:
Page 362 and 363:
Chapter 15 Enzymatic reactions SUMM
Page 364 and 365:
ENZYMATIC CONVERSION OF GLUCOSE (FR
Page 366 and 367:
Page 368 and 369:
Page 370 and 371:
ENZYMATIC CONVERSION OF PYRUVATE 35
Page 372 and 373:
Page 374 and 375:
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 382 and 383:
Page 384 and 385:
Page 386 and 387:
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.
Page 414 and 415:
LITERATURE 399 B.P. Konstantinov an
Page 416 and 417:
LITERATURE 401 1971 L. Arlinger, Is
Page 418 and 419:
LITERATURE 403 1973 L. Arlinger and
Page 420 and 421:
LITERATURE 405 A. Chrambach and J.S
Page 422 and 423:
LITERATURE 407 M. Coxon and M.J. Bi
Page 424 and 425:
Symbols and abbreviations SYMBOLS A
Page 426 and 427:
SYMBOLS AND ABBREVIATIONS SUPERSCRI
Page 428 and 429:
Subject index A Absorption meter, U
Page 430 and 431:
SUBJECT INDEX 41 5 Detection limit
Page 432 and 433:
SUBJECT INDEX 41 7 --_ , separation
show all

Frans_M_Everaerts_Isotachophoresis_378342.pdf

Create successful ePaper yourself

Delete template?

Save as template?