Frans_M_Everaerts_Isotachophoresis_378342.pdf

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EXAMPLES 103 solvent, hoping that a different solvation will affect the ionic mobilities in a favourable way. As the ions concerned do not show any proton interaction, we must seek a suitable solvent in the class of amphiprotic solvents, preferably with a high dielectric constant. In that case, we can try methanol. The ionic mobilities of the alkali metals in methanol are given in Table 5.4. It can be seen that the differences in the ionic mobilities are much more favourable in methanol, so that we can use methanol for the separation of the alkali metals. The second problem is the use of a leadtng ion. In nearly all solvents, the alkali metals have higher ionic mobilities than other positive ions except H’. Therefore, we should choose H’ as the leading ion (in methanol there are some positive ions with a higher effective mobility than CS', e.g., the tetramethylammonium ion). As the terminating ionic species many positive ions can be used; in this example we tried Cu2+ ions. In general, it is preferable to use a buffering counter ion, but for the separation af the alkali metals the pH is not important and, because no disturbances can be expected, we chose a non-buffering ion (chloride) as the counter ionic species. We carried out some experiments in order to check this system, The step heights measured with a thermocouple are given in Table 5.9. As the leading electrolyte we used a solution of 0.OlNhydrochloride in methanol (95%) (MHCl) and as the terminator a solution of 0.1N coppel(I1) chloride in methanol. Firstly, all step heights of the alkali metals were measured and then a separation of a mixture of the alkali metals was carried out. The isotachopherogram for this separation is shown in Fig.5.5. A rapid and complete separation was easily obtained. In Chapter 16, more data about cationic separations with both water and methanol as solvents are given. Example D. In Chapter 14 the separation of some nucleotides is considered; in this example, we shall discuss how to choose a suitable electrolyte system for the separation of nucleotide diphosphates. As the solvent we use water as it dissolves all ionic species easily. As not all pK values and mobilities of the diphosphates are known, we have to use the experimental method in order to find a suitable electrolyte system. We have chosen some electrolyte systems with pH, values of 3.4,3.7,4.2,4.6, 6.0 and 7.0 (these values were chosen because several nucleotides have pK values between 2 and 5). As the diphosphates will be separated as negative ions at the chosen pH values, we use chloride as the leading ion and positively charged ionic species as the buffering counter ionic species. It can be seen that the pH, values of the systems do not differ much from the pK values. The conditions for the chosen electrolyte systems are given in Table 5.10 and the measured step heights are given in Table 5.1 1 and are shown graphically in Fig.5.6. It can be seen from Fig.5.6 that maximal differences in effective mobilities are obtained at lower pH values. For the separation of a mixture of diphosphates, we chose a pH, of 3.7. The isotachopherogram of this separation is shown in Fig.5.7. The detection was performed with a thermometric detector (copper-constantan thermocouple). Example E. Fatty acids, especially the higher fatty acids, are slightly soluble in water, so that water cannot be used as the solvent for their separation. Methanol is a better
104 7> I: CHOICE OF ELECTROLYTE SYSTEMS Fig.5.5. Isotachopherogram for the separation of alkali metals in methanol (for operational system, see Table 16.4). The detection was performed with a thermometric detector. T= increasing temperature; t = time: The electric current was stabilized at 70 PA. 1 = H+; 2 = Cs+; 3 = Rb'; 4 = K+; 5 = Na+; 6 = LT; 7 = Cu2+. TABLE 5.10 DIFFERENT ELECTROLYTE SYSTEMS FOR THE SEPARATION OF NUCLEOTIDES For operational systems, see section 14.2 No. System pH Leading electrolyte Electric current Terminator o( A) I WAdQ 3.4 0.01 NHCl + adenosine 70 Caproic acid I1 WaNCl 3.7 0.01 NHCl + a-naphthylamine 70 Caproic acid 111 WAnCI(1) 4.2 0.01 N HCl + aniline 70 Pivalic acid IV WAnCl(I1) 4.6 0.01 NHCl +aniline 70 F'ivalic acid v WHiScl 6.0 0.01 NHCl + histidine 70 Cacodylic acid VI WImCl 7.0 0.01 NHCl+ imidazole 70 Benz yl-dl-asparigine
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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
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 108 and 109: CHOICE OF THE pH OF THE LEADING ELE
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 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
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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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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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