Isotachophoresis

Journal of Chromatogaphy Library

-

Volume 6

ISOTACHOPHORESIS Theory, Instrumentation and Applications

JOURNAL O F CHROMATOGRAPHY LIBRARY Volume 1 Chromatography of Antibiotics by G. H. Wagman and M. J. Weinstein Volume 2 Extraction Chromatography edited by T. Braun and G . Ghersini Volume 3 Liquid Column Chromatography. A Survey of Modern Techniques and Applications edited by Z . Deyl, K. Macek and J. Janak Volume 4 Detectors in Gas Chromatography by J. SevEik Volume 5 Instrumental Liquid Chromatography. A Practical Manual on High-Performance Liquid Chromatographic Methods by N. A. Parris Volume 6 Isotachophoresis. Theory, Instrumentation and Applications by F. M. Everaerts, J. L. Beckers and Th. P. E. M. Verheggen Volume 7 Chemical Derivatization in Liquid Chromatography by J. F. Lawrence and R. W. Frei Volume 8 Chromatography of Steroids by E. Heftmann

Journal of Chromatography Library - Volume 6

ISOTACHOPHORESIS Theory, Instrumentation and Applications

Frans M. Everaerts Department of Instrumental Analysis, Eindhoven University of Technology, Eindhoven

Jo L. Beckers Eijkhagen College, Schaesberg

The0 P.E.M. Verheggen Department of Instrumental Analysis, Eindhoven University of Technology, Eindhoven

ELSEVIER SCIENTIFIC PUBLISHING COMPANY AMSTERDAM - OXFORD - NEW YORK 1976

ELSEVIER SCIENTIFIC PUBLISHING COMPANY 335 Jan van Galenstraat P.O.Box 211, Amsterdam, The Netherlands

Distributors for the United States and Canada: ELSEVIER/NORTH-HOLLAND INC. 52,Vanderbilt Avenue New York, N.Y. 10017

Library of Congress Calaloging in Publication Data

Everaerts, Frans M 1941Isotachophoresis : theory, instrumentation, and applications. (Journal of chromatography library ; vo 6) Includes bibliographies and index. 1. Electrophoresis. I. Beckers, Jo L., joint author. 11. Verheggen, Theo P. E. M., joint author. 111. Title. TV. Series. QD79.EaE93 543 ’ .087 7644834 fSBN

0-444-41430-4

Copyright 0 1976 by Elsevier Scientific Publishing Company, Amsterdam All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the publisher, Elsevier Scientific Publishing Company, Jan van Galenstraat 335, Amsterdam Printed in The Netherlands

Dedicated to Pr0f.Dr.h. A.I.M. Keulemans, for providing the possibility of developing this analytical separation technique in his Department of Instrumental Analysis, University of Technology, Eindhoven.

This Page Intentionally Left Blank

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.Historicalreview ........................................................... Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Historical review ......................................................... References ...............................................................

XI11 1 1 1 4

THEORY 2 . Principles of electrophoretic techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary........ ..................................................... 2.1.Introduction .......................................................... 2.2. Principle of zone electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Principle of moving-boundary electrophoresis ................................. 2.4. Principle of isotachophoresis .............................................. 2.4.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2. Simplified model for isotachophoresis: ................................. 2.4.3. Concentration adaptation ........................................... 2.4.4. Some isotachophcrograms ........................................... 2.5. Principle of isoelectric focusing ............................................ 2.6.Discussicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.Conceptofmobility ......................................................... Summary ................................................................ 3.1.Introduction .......................................................... 3.2. Interpretation of electrophoretic migration ................................... 3.3. Ionic mobility and ionic equivalent conductivity ............................... 3.4. Effective ionic mobility .................................................. 3.4.1. Partial dissociation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1.1. Protolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... 3.4.1.2. Complex formation .......................................... 3.4.2. Relaxation and electrophoretic retardation .............................. 3.5. Determination of ionic mobilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1. Relationship between volume and ionic mobility ......................... 3.5.2. Relationship between entropy and ionic mobility ......................... 3.5.3.Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Mathematical model for isotachophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. General equations ...................................................... 4.2.1. Equilibrium equations .............................................. 4.2.2. Electroneutrality equations .......................................... 4.2.3. Mass balances for all ionic species . . . . . . . . . . . 4.2.4. Modified Ohm’s law ...................... 4.2.5. Parameters and equations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Mathematical model for the steady state in isotachophoresis ..................... 4.3.1. Concept of isotachophoretic separation ................................. 4.3.2. Mathematical model of isotachophoresis ................................ 4.3.2.1. Equilibrium equations ....................................... 4.3.2.2. The isotachophoretic condition ................................

I 7 7 I 9 13 13 15 18 20 23 24 21 21 21 21

29 31 32 33 33 36 31 31 39 40 40 41 41 41 43 45 41 48 51 51

55 55 58 58 58

VIII

CONTENTS

4.3.2.3. Mass balance of the buffer .................................... 4.3.2.4. Principle of electroneutrality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2.5. Modified Ohm’s law .... 4.3.3. Computer program for calculation of the steady state ...................... 4.3.3.1. Computation procedure ...................................... 4.3.3.2. Iteration procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3.3. Discussion ..................................... 4.4. Validity of the isotachophoretic model ...................................... 4.4.1. Introduction ..................................................... 4.4.2. Influence of diffusion on the zone boundaries ........................... 4.4.3. Influence of axial and radial temperature differences ...................... 4.4.4. Influence of activity coefficients ..................................... 4.5. Check of the isotachophoretic model ....................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

59 60 61 62 62 62 69 69 69 74 75 76 76 81

.................................................

83 83 83 84 84 87 87 89 92 93 96 99 99 99 99 99 100 100 113

5 . Choice of electrolyte systems

Summary ................................................................ 5.1.Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.1. General remarks .................................................. 5.2. Choice of the solvent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1. Methanol as a solvent .............................................. 5.2.1.1. Comparative behaviour with water .............................. 5.2.1.2. Determination of pK values in methanolic solutions ................ 5.3. Choice of the buffering counter ionic species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Choice of the pH of the leading electrolyte ................................... 5.5. Choice of the terminating and leading ionic species ............................. 5.6. Additions to the electrolyte solutions ............................... 5.6.1. Stabilizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.2. Surface-active chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6.3. Reference materiall~foridentificationandi~e~ifi~atio~~~ calibration of concentrations . . . . . . . 5.6.4. Spacers and carriers .................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7.Discussion ............................................................ 5.8.Examples ............................................................ References . ..........................................................

INSTRUMENTATION

6 . Detection systems

................................................

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 .1. Universal detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.2. Specific detectors ....................................... 6.1.3. Combinations of universal and specific detectors ......................... 6.2. Thermometric recording . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

..................................................

6.2.3. Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.4. Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.5. Conclusion ............................... ................... ................... 6.3. High-frequency conductivity detection . . . . . . . . . . . . . . 6.3.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.2.Construction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Conductivity detection . . . . . . . . . . . ............................... 6.4.1. Introduction . . . .................... ........................

117 117 117 118 118 119 119 119 119 125 126 129 130 130 131 133 133

CONTENTS 6.4.2. The d.c. method of resistance determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.3. The d.c.-a.c. converter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.4. The a.c. method of resistance determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.5. Conductivity probe with equiplanar-mounted sensing electrodes . . . . . . . . . . . . . 6.5. UV absorption meter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.2. Construction of the UV source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.3. UV detector in combination with a non-modulated UV source . . . . . . . . . . . . . . . 6.5.4. UV detector in combination with a modulated UV source . . . . . . . . . . . . . . . . . . 6.5.5.UVcell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5.6. Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6. Additives to the electrolytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6.2. Effect of additives on the electroendosmotic flow ......................... 6.6.3. Effect of additives on the micro-sensing electrodes ........................ 6.6.4.Additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7. Coating of the micro-sensing electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7.2. Experimental . . . . . . . . . . . . . .................................. 6.8. Detection limits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.8.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.8.2.Experimental .................................................... 6.9.Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 . Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Injection systems ...................... ............................. 7.2.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2.Four-way tap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.3. Six-way valve . . . . . . . . . . . . .................................... 7.2.4. Injection block . . . . . . . . . . . . . . . . . . .............................. 7.2.5. Simplified injection block . . .................................... 7.3. Counter electrode compartments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2. Cylindrical counter electrode compartment ............................. 7.3.3. Counter electrode compartment with flat membrane ....................... 7.4.Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.2. Narrow-bore tube surrounded with a water-jacket ......................... 7.4.3. Narrow-bore tube thermostated with an aluminium block . . . . . . . . . . . . . . . . . . . 7.4.4. Equipment with high-resolution detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5. Counter flow of electrolyte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.2. Counter flow with level regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.3. Counter flow with light-dependent resistor regulation ...................... 7.5.4. Counter flow with direct control on the pumping mechanism via the power supply . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.5. Counter flow with no regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.6. Counter flow regulated by the cur -stabilized power supply; the membrane pump . . . . . . . . . . . . . ..................................

IX 135 140 143 143 153 153 155 159 161 164 165 171 171 171 174 180 191 191 191 193 193 196 199 201 203 203 203 203 203 204 205 208 211 211 211 213 215 217 217 219 221 224 230 230 231 233 237 238 24 1

X

CONTENTS

APPLICATIONS 8.Introduction ............................................................... Summary ................................................................ 8.Introduction ............................................................

249 249 249

9 .Practical aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary ................................................................ 9.1.Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. Disturbances caused by hydrogen and hydroxyl ions ............................ 9.2.1. Disturbances from the terminator zone in unbuffered systems . . . . . . . . . . . . . . . 9.2.1.1. HI-MI boundary ........................................... 9.2.1.2. MI-MI, boundary . . . .................................... 9.2.2. Disturbances from the leading zone in unbuffered systems . . . . . . . . . . . . . . . . . . 9.2.3. Disturbances due to the presence of hydrogen and hydroxyl ions in buffered systems ......................................................... 9.3. Disturbances due to the presence of carbon dioxide ............................ 9.4. Enforced isotachophoresis ............................................... 9.4.1. Disc electrophoresis ................................................ 9.5. Water as terminator ..................................................... 9.6. Purification of the terminator ............................................. 9.7. Conversion of data measured with different detectors ........................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

260 263 264 265 267 268 210 271

10. Quantitative aspects ........................................................ Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.Introduction ......................................................... 10.2.Theoretical .......................................................... 10.3. Thermometric measurements ............................................ 10.3.1. Reproducibility ................................................. 10.3.2. Calibration constant ............................................. 10.4. Conductimetric measurements ........................................... 10.4.1. Reproducibility ................................................. 10.4.2. Calibration constant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.Conclusion ........................................................... References ...............................................................

273 213 273 214 215 275 215 219 279 280 281 282

11. Separation of cationic species in aqueous solutions ................................ Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1. Separation of cationic species in aqueous solutions using a thermocouple as detector . . 11.1.1. The system WHCl ............................................... 11.1.2. The system WHIO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1.3. The system WKAC ............................................... 11.1.4. The system WKCAC ............................................. 11.1.5. The system WKDIT .............................................. 11.2. Separation of cationic species in water and deuterium oxide using a conductivity detector (a.c. method) and a UV absorption detector (256 nm) ...................

283 283 283 285 286 288 289 293

12. Separation of anionic species in aqueous solutions ................................. Summary ................................................................. 12.1. Separation of anionic species in aqueous solut'ions using a thermometric detector . . . . . 12.1.1. Operational system histidine/histidine hydrochloride (pH 6) . . . . . . . . . . . . . . . 12.1.2. Operational system imidazole/imidazole hydrochloride (pH 7) . . . . . . . . . . . . . 12.2. Separation of anionic species in aqueous solutions using a conductivity detector (a.c. method) and a UV absorption detector (256 nm) ............................. 12.2.1. Introduction ...................................................

253 253 253 253 253 254 254 257

293 295 295 295 295 296 300 300

XI

CONTENTS

.....................................

301

13. Amino acids, peptides and proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary ....................... ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.1.Amino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.1.1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.1.2. Separation at low pH values in aqueous systems ........................ 13.1.3. Separation at high pH values in aqueous systems ........................ 13.1.4. Separation by use of complex formation ........................... 13.1.5. Separation in aqueous propanal solutions ............................. 13.2. Separation of proteins in ampholyte gradients ............................... 13.2.1. Introduction ................................................... 13.2.2. Experimental ................................................... 13.3. Separation of small peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.3.1. Introduction ................................................... 13.3.2. Experimental .................................................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

311 311 311 311 312 312 318 319 322 322 325 335 335 336 336

............................ 14. Separation of nucleotides in aqueous systems . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.1.1ntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2. Separation using a thermometric detector ................................... 14.3. Separation using a conductivity detector (ax . method) and a UV absorption detector (256 nm) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

337 337 337 337

15. Enzymatic reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 15.1.Introduction ......................................................... 15.2. Enzymatic conversion of glucose (fructose) into glucose-6-phosphate (fructosedphosphate) with hexokinase from yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.3. Enzymatic conversion of pyruvate into lactate with lactate dehydrogenase from pigheart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

347 347 347

....................... 16.Separations in non-aqueous systems. . . . . . . . . . . . . . . . . Summary .................................... ................ 16.1.Introduction ......................................................... 16.2. Separation of anionic species in methanol using a thermometric detector . . . . . . . . . . . 16.3. Separation of cationic species in methanol using a thermometric detector . . . . . . . . . . . . 16.3.1. The operational system MHCl ...................................... 16.3.2. The operational system MKAC ..................................... 16.3.3. The operational system MTMAAC .................................. 16.4. Experiments in aqueous methanolic systems using a conductimetric detector (a.c. method) and W absorption detector (256 nm) .........................

361 361 361 362 364 365 367 373 373

17. Counter flow of electrolyte .................................................. Summary ........................................................... 17.1. lntroduction ......................................................... 17.2.Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.3.Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

375 375 375 378 384 384

12.2.2. Applications

...........

342

348 355 360

APPENDICES A. Simplified model of moving-boundary electrophoresis for the measurement of effective mobilities ................................................................

387

XI1

CONTENTS

A.1.inrroduction .......................................................... A.2. Model of moving-boundary electrophoresis .................................. A.2.1. Electroneutrality equations ......................................... A.2.2. Modified Ohm’s law . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.2.3. Mass balances for all cationic species . . . . . . . . . . . . . . . A.3. Procedure of computation ............................................... A.4.Experimental ................................................ A.5.Discussion ............................................................ References ........................................................... ~

B. Diameter of the narrow-bore tube, applied for separation C. Literature

...............

......................................................... Symbols and abbreviations ..................................................... Symbols ................................................................. Subscripts ............................................................... Superscripts .............................................................. Examples ................................................................ Abbreviations ............................................................. Subjectindex ...............................................................

387 387 388 388 388 389 390 392 394 395 397 409 409 410 411 411 411 413

It is very well known that charged particles move under the influence of an electric field. Because the final velocity of such particles depends on numerous parameters, many scientists through several decades have applied t h s phenomenon to the characterization and separation of a variety of charged particles, with a wide range of molecular weights, both for analytical and preparative purposes. Because the vital components of electrophoretic equipment need to be made of insulating materials, in the early days it was a handicap for the further development of electrophoretic instrumentation that modern insulating materials such as Perspex, PTFE and fluoroethylene polymer were not available. Moreover, sensitive detection systems had not been developed, so that the minimum detectable amount was rather high in comparison with some other separation techniques. After World War 11, the chemical industry began t o show considerable interest in the development of chromatographic separation techniques for the analysis of hydrocarbons and other (complex) organic compounds. It may have bezn due to this development that the materials for the construction of the vital parts of the electrophoretic equipment, including the detectors, rapidly became available. Moreover, in the same period the electronics industry also underwent a phenomenal expansion. Although this book is devoted mainly to isotachophoresis, with which all kind of charged molecules can be separated (as is shown in the Section Applications), the instrument described can be used for other types of electrophoretic separations. Three main aspects of isotachophoresis are covered, in three different sections. In the first section, the theory of the isotachophoretic separation technique is given, and other electrophoretic techniques are briefly described. For isotachophoresis, both a simplified and a more complicated model are given. The latter model results in a computer program suitable for the qualitative and quantitative interpretation of the analytical results. In the Section Instrumentation, several detectors and the “isotachophoretic” equipment are described. Also, a means is described of formulating a simplified model rapidly, because for many problems simplified equipment is adequate. Moreover, not much cheap equipment is commercially available yet. In the last section, possible fields of application are considered. Analytical conditions (so-called operational systems) are presented and results are given in the form of both automatically recorded isotachopherograms and tables. The data in these tables can be used for the qualitative interpretation of isotachophoretic analyses. Because all of the values given were derived directly under the operational conditions considered, they cannot be used for the calculation of, for example, mobilities at infinite concentration. All of the isotachophoretic zones have a well defined temperature, pH and composition of the electrolytes present, and these are constant in a chosen operational system but are different from each other. For further theoretical approaches, corrections need to be made. In the Appendices, a method is described for mobility determinations, the influence of the diameter of the narrow-bore tube is dealt with and a list of relevant papers concerning isotachophoresis is given. Each section can be used almost independently by scientists interested in fundamental aspects, by research groups who intend t o construct an instrument and by scientists whose main interest is in the analytical aspects.

XIV

PREFACE

In this book, most of the results given summarize about 12 years research, performed with a variety of instruments. Not only the authors but also the research students, who worked in the electrophoresis research group contributed to the development of this technique. In particular, we thank Ir. M. Geurts, who developed most of the electronic circuits described, and Ir. F. Mikkers, who helped greatly in the collection of many of the data presented and in the work with tKe instrument equipped with the UV absorption detector and the a.c. conductivity detector. Eindhoven

Schaesberg Eindhoven

FRANS M. EVERAERTS JO L. BECKERS THEO P.E.M. VEI~HEGGEN

Chapter 1

Historical review SUMMARY

Mens ugitat molem*

1. HISTORICAL REVIEW In the middle of the nineteenth century, Wiedeman [ 1 , 2 ] and Buff [3] reported on the phenomenon that charged particles migrate in a solution when an electric field is applied. Later experiments, carried out by Lodge [4] and Whetham [S, 61 , were the basis on which Kohlrausch [7] developed a theory of ionic migration. With the equation that he derived, all electrophoretic principles can be described**, including zone electrophoresis, moving-boundary electrophoresis and isotachophoresis. The discovery by Hardy [8,9] that many biocolloids, such as proteins, show characteristic mobilities that depend largely on the pH of the electrolyte solution in which the analysis is performed, greatly stimulated interest in electrophoretic work. The characterization of such substances on the basis of their electrophoretic properties, especially the p l points, increased interest in electrophoretic separation techniques. As an early example, the work of Michaelis [ 101 can be considered. He found that enzymes can be characterized on the basis of their isoelectric points, measured in migration experiments performed at various pH values; this work, of course, was carried out before pure enzymes were available. Although at first the terms cataphoresis and electrophoresis were introduced in order to indicate the migration of charged colloidal particles and the term ionophoresis was reserved for substances of lower molecular weight, nowadays most workers use the term electrophoresis to describe the migration of charged particles in aqueous and non-aqueous stabilized and free solutions. Perhaps owing to the major interest in compounds such as proteins and enzymes, or because high-resolution detectors had not been developed, most attention was paid to only one of the basic principles, as already described by Kohlrausch [7] , namely zone electrophoresis and few reports dealing with the other principles were published. It is a fact that substances such as proteins need appropriate stabilization by electrolytes, as discussed in Chapter 13. It was not until about 1923 that a principle of electrophoresis other than zone electrophoresis was described. Kendall and Crittenden [ 1I ] succeeded in separating rare *Motto, University of Technology, Eindhoven.

**In Chapter 2, isoelectric focusing is also briefly described because it is a separation technique that has many similarities with electrophoretic techniques, although once separated the charged particles do not migrate if the amphiprotic compounds have reached their isoelectric points (the overall charge is zero).

1

2

HISTORICAL REVIEW

earth metals and some simple acids by, as they called it, the ‘ion migration method’, which was, in fact, isotachophoresis. He stated that the ions not only separate, but also adapt their concentrations to the concentration of the first zone according to the Kohlrausch [7] regulating function, the ‘beharrliche Funktion’. It was also Kendall [ 12) who considered that it is necessary to be able to follow the separation in some convenient way and suggested that a coloured ion, with an effective mobility intermediate between those of the ions of interest, could be used. The end of the experiment could, by the addition of this coloured ion, easily be determined without the need for a detector. Kendall also suggested that other detection methods are possible, e.g., utilizing thermometric and conductivity detectors, and pointed out that, especially when analyzing metals, spectroscopic detection can easily be used. Finally, when appropriate, the measurement of the radioactivity can be used to obtain qualitative and quantitative information. The experiments in which he attempted to separate 35 Cl from 37Cl, as proposed by Lindemann [ 131 , failed, even when very long analysis times were used. Other isotopes also could not be separated. The ‘movingboundary method‘ of MacInnes and Longsworth [ 141 , which was used for the determination of transport numbers, was also based on the Kohlrausch [7] theory. For about 10 years, little relevant work was carried out on electrophoretic techniques other than zone electrophoresis, then in 1942 Martin [ 151 separated chloride, acetate, aspartate and glutamate by isotachophoresis, which he called ‘displacement electrophoresis’, because it was so similar to the displacement technique in chromatography. There was a further gap until 1953, when a paper was published by Longsworth [16], who realized the importance of Kendall’s work. In a Tiselius moving-boundary apparatus, he introduced a mixture of cations (Ca2’, Ba” and Mg2+)between two other zones, called the leading solution and the trailing solution. Once separated, the effective mobilities decrease on going from the leading solution towards the trailing solution. Detection via Schlieren scanning patterns showed very clearly the sharpness of the boundaries between the consecutive zones. Longsworth introduced a counter flow of electrolyte, because the separation chamber in a Tiselius apparatus is very short, and adjusted it in such a way that the zones remained in the detection region until they were separated. He also found that a steady state was reached, once the components were separated. The importance of the pH of the trailing solution was recognized. The work of Poulik [17] is important, although he was not aware that he was working along the lines of the Kohlrausch [7] regulating function. Kaimakov and Fiks [ 181 reported on experiments carried out in an electrophoretic equipment, the separation chamber being filled with quartz sand so as to eliminate convection problems. The separation chamber was initially filled with an electrolyte, called an indicator electrolyte, that was different from the test solutions. Again, their results showed that a decreasing sequence of mobilities was obtained once the steady Ftate had been reached. They also used a counter flow of electrolyte. Transport numbers were measured by Kaimakov [I91 and Konstantinov and Kaimakov [20]. Konstantinov et al. 121,221 extended the work of Hartley [23] and Gordon and Kay [24]. Konstantinov and Oshurkova [25,26] in 1963 described an analytical application based on the ‘moving-boundary method’. Their separation chamber was a narrow-bore tube of I.D. 0.1 mm and a wall thickness of 0.05 mm. Measurements of the refractive index of the various zones by photographic methods gave a recording of the zone boundaries.

HISTORICAL REVIEW

3

Independently in 1963, Everaerts [27] started, together with Martin, work that finally resulted in the appearance of this book on isotachophoresis. As Martin [ 151 , he used the term ‘displacement electrophoresis’. He performed the analysis in a narrow-bore tube of Pyrex glass of I.D. 0.5 mm and an O.D. of 0.8 mm. In order to prevent hydrodynamic flow between the two electrode compartments through the narrow-bore tube, caused by differences in levels, this tube was filled with an electrolyte the viscosity of which was increased up to 100 CPby addition of a watersoluble linear polymer, e.g., hydroxyethylcellulose. This polymer was purified by shaking it with a mixed-bed ion exchanger. A thermocouple (30-pm copper-25-pm constantan) was used as the detector. Independently, and unaware of this work, Kaimakov and Sharkov [28] reported on the use of microthermistors to detect zone boundaries. In 1964, Ornstein [29] and Davis [30] introduced disc electrophoresis. They placed a protein mixture between an electrolyte with an anion of low effective mobility and an electrolyte with an anion with a high effective mobility. Owing to the concentration phenomenon of isotachophoresis, the proteins are stacked in narrow zones between the two electrolytes (‘steady-state stacking’). The zones, however, are so narrow that even a high-resolution detector cannot detect them. Therefore, in the second stage of the analysis, the principle of zone electrophoresis was used, which allowed every protein to move at a different velocity. Cross-linked polyacrylamide was used as a stabilizing medium and as a molecular sieve. The mobilities of the proteins could be controlled, moreover, by varying the pore size in the gel. Ornstein [29] derived several equations, with which it was possible to calculate the mobilities and the pH values of the electrolyte systems. In 1966, Vesterrnark [31] introduced a new term, ‘cons electrophoresis’, for the electrophoretic technique that makes use of Kohlrausch’s regulating function [7] . Vestermark also used the spacer technique. In 1966, Preetz [32] gave a theoretical treatment of the use of a counter flow of electrolyte in isotachophoretic systems. In 1967, Preetz and Pfeifer [33] described an instrument that was specially designed for measurements of potential gradients and ion concentrations. Preetz also performed analyses in narrow-bore tubes. A further development was the continuous counter flow equipment described by Preetz and Pfeifer [34]. Based on work of Everaerts [35] and Martin and Everaerts [36], Verheggen and Everaerts built an instrument and introduced the technique in Bergstr6m’s department at the Karolinska Institute, Stockholm, Sweden, in 1968. This was the basis of the commercial production of isotachophoretic equipment, produced by LKB Produkter AB in Bromma, Sweden. In 1969, Everaerts and Verheggen introduced the technique at the Charles University in Prague, Czechoslovakia, in Vacik’s research group. Up to 1970, several names had been used for similar electrophoretic techniques, including ion migration method, Kendall [ 121 (1928); moving-boundary method, MacInnes and Longsworth [ 141 (1932); displacement electrophoresis, Martin [ 151 (1942) and Everaerts [27] (1964); steady-state stacking, Ornstein [29] (1964); cons electrophoresis, Vestermark [31] (1966); and ionophoresis, Preetz [32] (1966). Together with Haglund [37], a group of research workers in the field introduced a new name, based upon an important phenomenon of the electrophoretic technique, namely the identical velocities of the sample zones in the steady state: isotacho-electro-phoresis*, or isotachophoresis for short. *r

LOO =

equal; ~ 0 1 x 0= ~velocity; @peeueorc = to be dragged.

4

HISTORICAL REVIEW

It is very difficult to summarize the individual contributions of the various scientists to the development of the technique after 1970. In Appendix B, we give an almost complete list of the relevant papers on the subject. Of all the papers, special note is made of two, in which new types of operational detector were described, representing landmarks in the development of isotachophoresis. In 1970, Arlinger and Routs [38] introduced an operational UV absorption detector, and in 1972, Verheggen et al. [39] introduced an operational conductivity detector.

REFERENCES 1 G. Wiedeman, Pogg. Ann., 99 (1856) 197. 2 G. Wiedeman, Pogg. Ann., 104 (1858) 166. 3 H. Buff, Ann. Chem. Pharm., 105 (1858) 168. 4 0. Lodge, Brit. Ass. Advan. Sci., Rep., 56 (1886) 389. 5 W.C.D. Whetham, Phil. Trans. Roy. SOC. London, Ser. A , 184 (1893) 337. 6 W.C.D. Whetham,PhiI. Trans. Roy. SOC.London, Ser. A , 186 (1895) 507. 7 F. Kohlrausch,Ann. Phys. (Leipzig), 62 (1897) 209. 8 W.B. Hardy, Proc. Roy. Soc. London. 66 (1900) 110. 9 W.B. Hardy, J. Physiol. (London), 33 (1905) 251. 10 L. Michaelis, Biochem. Z., 16 (1909) 81. 11 J. Kendall and E.D. Crittenden, Proc. Nut. Acad. Sci. US.,9 (1923) 75. 12 J. Kendall, Science, 67 (1928) 163. 1 3 A. Lindemann, Proc. Roy. Soc., Ser. A, 99 (1921) 102. 14 D.A. MacInnes and L.G. Longsworth, Chem. Rev., 11 (1932) 171. 15 A.J.P. Martin, unpublished results, 1942. 16 L.G. Longsworth, Nut. Bur. Stand. (US.). Circ., No. 524 (1953) 59. 17 M.D. Poulik, Nature (London), 180 (1957) 1477. 18 E.A. Kaimakov, and V.B. Fiks, Rum. J. Phys. Chem., 35 (1961) 873. 19 E.A. Kairnakov, Russ. J. Phys. Chem., 36 (1961) 436. 20 B.P. Konstantinov and E.A. Kaimakov, Rum. J. Phys. Chem., 36 (1962) 437. 21 B.P. Konstantinov, E.A. Kaimakov and N.L. Varshovskaya, Russ. J. Phys. Chem., 36 (1962) 535. 22 B.P. Konstantinov, E.A. Kaimakov and N.L. Varshovskaya, Russ. J. Phys. Chem., 36 (1962) 540. 23 G.S. Hartley, Trans. Faraday SOC.,30 (1934) 648. 24 A.R. Gordon and R.L. Kay, J. Chem. Phys., 21 (1953) 131. 25 B.P. Konstantinov and O.V. Oshurkova, Dokl. Akad. Nauk. SSSR, 148 (1963) 1110. 26 B.P. Konstantinov and O.V. Oshurkova, Sov. Phys.-Tech. Phys., 11 (1966) 693. 27 F.M. Everaerts, Graduation Rep., University of Technology, Eindhoven, 1964. 28 E.A. Kaimakov and V.I. Sharkov, Russ. J. Phys. Chem., 38 (1964) 893. 29 L. Ornstein, Ann. N. Y. Acad. Sci., 121 (1964) 321. 30 B.J. Davis, Ann. N. Y. Acad. Sci., 121 (1964) 404. 3 1 A. Vestermark, Cons Electrophoresis: An Experimental Study, unpublished results, 1966. 32 W. Preetz, Tdanta, 13 (1966) 1649. 33 W. Preetz and H.L. Pfeifer, Talanta, 14 (1967) 143. 34 W. Preetz and H.L. Pfeifer, Anal. a i m . Acta, 38 (1967) 255. 35 F.M. Everaerts, Thesis, University of Technology, Eindhoven, 1968. 36 A.J.P. Martin and F.M. Everaerts,Anal. Chim. Acta, 38 (1967) 233. 37 H. Haglund, Sci. Tools, 17 (1970) 2. 38 L. Arlinger and R.J. Routs, Sci. Tools, 17 (1970) 21. 39 Th. P.E.M. Verheggen, E.C. van Ballegooijen, C.H. Massen and F.M. Everaerts, J. Chromatogr., 64 (1972) 185.

THEORY

This Page Intentionally Left Blank

Chapter 2

Principles of electrophoretic techniques SUMMARY The principles of the four main types of electrophoresis, viz., zone electrophoresis, moving-boundary electrophoresis, isotachophoresis and isoelectric focusing, are described and a simplified mathematical model for isotachophoresis is given. The characteristics of these four main types are compared.

2.1. INTRODUCTION As already described in the Preface, ionic species will move, under the influence of an applied electric field, E, with a velocity, v , of

v=mE

(2-1)

where m is the effective mobility of the ionic species, which depends on several factors that will be discussed in Chapter 3. Differences in effective mobilities cause differences in velocities and, by utilizing, this effect, the ionic species can be separated. Separation techniques based on this principle are called electrophoretic techniques, which can be divided into three main types, viz., zone electrophoresis, moving-boundary electrophoresis and isotachophoresis. In isoelectric focusing, ionic species are not separated according to differences in mobilities, differences in PIvalues determining whether they can be separated. In the steady state, ionic species do not migrate. Because the ionic species migrate electrophoretically in order to attain that steady state, isoelectric focusing can also be considered to be an electrophoretic technique; hence four main types of electrophoresis can be distinguished. In principle, all of these electrophoretic techniques can be carried out in any electrophoretic equipment. Such an instrument generally consists of five units, viz.,the anode and cathode compartments, the separation chamber, the injection system and the detector. In this chapter, the principles of the four main types of electrophoresis are discussed briefly, the most attention being paid, of course, to isotachophoresis.

2.2. PRINCIPLE OF ZONE ELECTROPHORESIS For the description of the principle of zone electrophoresis, we shall consider a narrowbore tube as the separation chamber, which is connected with the anode and cathode compartments. The distinguishing feature of all zone electrophoretic systems is that the whole system (anode and cathode compartments and the separation chamber) is filled with one electrolyte, the so-called background or supporting electrolyte, which carries the

PRINCIPLES OF ELECTROPHORETIC TECHNIQUES

8

electric current and generally has a buffering capacity. The sample (a mixture of anionic and cationic species) is introduced into the system in this background electrolyte. In general, the concentration of this background electrolyte is high compared with that of the sample ionic species and therefore it provides a constant pH and voltage gradient in the whole system. The ionic species of the background electrolyte have a certain effective mobility and, when an electric current is passed through the system, these ionic species will migrate with specific velocities, cations migrating towards the cathode and anions towards the anode. The sample ionic species also migrate under the influence of the applied electric field, each ionic species having a characteristic velocity, depending on the conditions chosen. Because of the high concentration of the background electrolyte, the influence of the sample ionic species on the voltage gradient and pH is negligible and therefore all sample ionic species migrate with constant velocity in time, resulting in a flow of ions of the background electrolyte accompanied by a flow of sample ions. The ionic species of the sample will be separated after some time if the differences in effective mobilities are sufficiently great. Owing to the diffusion, the peaks are wide (tailing), and adsorption phenomena can cause further tailing. Often detection is effected by a specific method, e.g., measurement of the colour of the sample ionic species. Quantitative information can be obtained by measuring the intensity of the colour due to the reaction products, while qualitative information (identification) is obtained from the migration distance. Just as in paper chromatography, for example, here the R , values can be used for identification in standardised systems, where R, is defined as the migration distance (Z) of the ionic species in question related to the migration distance of a standard ionic species:

R,

= 'ionic species 'standard

A disadvantage of such a detection method is that the detection takes place after the separation procedure. In addition to the extra steps required and the long time involved, disturbances such as diffusion in the various zones often occur. In Fig.2.1, the separation of a mixture of anionic species A, B and C and a cationic species D is shown. The background electrolyte consists of an anionic species Q and a cationic species P. In Fig.2. la, the whole system is filled with the background electrolyte and the sample is injected. In Fig.2.lb, all ionic species of the sample are separated. The migration distances are l A , I, I , and I, respectively. The R , values relative to the anionic species C would be R,(A) =-'A and R,(B) = IB 'C

In Fig.2.2, the voltage gradients, temperatures and pH values for some zones are shown. The background electrolyte containing sample ionic species with low effective mobilities shows a higher voltage gradient over the zone than that for rapid ionic species. This influence is small for high concentrations of the background electrolyte and nearly constant pH and voltage gradients can be expected in practice. In this instance, a specific means of detection must be used (see Chapter 6). This method can be compared with elution chromatography.

9

MOVINGBOUNDARY ELECTROPHORESIS

a

S

/

b

P O

\

S

0

I I

I I I

L

L _

/ a

_I I

I I

I

/ A

J

Fig. 1. Electrophoretic separation of the anionic species A, B and C and -..e cationic species D along the lines of zone electrophoresis. All compartments are filled with the electrolyte PQ-.The distances l A , I g , lc and I,, can be used for the determination of the ionic species A , B, C and D. (a), Sample injection; (b), all ionic species of the sample are separated.

2.3. PRINClPLE OF MOVING-BOUNDARY ELECTROPHORESIS We shall first consider the separation of anionic species according to the method of Tiselius. In Fig.2.3a, the anionic species to be separated, mixed with a buffer solution, fill the lower part of a U-tube while the upper part is filled with the buffer solution. If an electric current is passed through such a system, the anionic species migrate in the direction of the anode and, after some time, a partial separation occurs. Two series of mixed zones are obtained; in front of the original zone are present the zones A and A + B and behind the original zone are present B+C and C (see Fig.2.3b), if the effective mobilities of the ionic species A, B and C decrease in the order mA > m B > mc. It is also possible to carry out moving-boundary electrophoretic experiments in the following way. Anionic species can be separated by using a narrow-bore tube as the separation chamber, connected with anode and cathode compartments. The anode compartment and narrow-bore tube are filled with an electrolyte, the anionic species of which is chosen to be more mobile than the anionic species to be separated. The sample is introduced into the cathode compartment (see Fig.2.4a). The anionic species migrate towards the anode, and the sample anions can never pass the anionic species of the leading electrolyte because its effective mobility is higher. The mobilities of the

PRINCIPLES OF ELECTROPHORETIC TECHNIQUES

10

E

T

PH

I l

I

I

Fig.2.2. Electric field strength (E), temperature ( T ) and pH in the different zones of the zone electrophoretic separation procedure. Theoretically, the zones show small differences in the electric field strength and temperature. The dotted lines are exaggerated. No general means of detection can be applied, e.g., conductimetric or thermometric. X refers to, the position in the separation chamber.

anionic species of the sample differ, however, so that some of them will migrate forward. Thus a situation as shown in Fig.2.4b will be obtained after some time. Substance A, which is more mobile than the other substances of the sample, is partially separated from

11

MOVINGBOUNDARY ELECTROPHORESIS

0

0

0

buffer

butter

buffer

n r

0

iuf ter

A

-

-C B+C

A+B A+B+C

A+B+C

a

b

Fig.2.3. Separation according to the Tiselius moving-boundary principle. In (a), the lower tube is filled with a mixture of the anions A , B and C. Specific buffers need to be applied for optimal separation. In (b), it is shown that zones exist on both the front and rear sides, vii., A, A + B and A+B+C, and A + B t C , B t C and C, respectively.

B and C. Substance B, mixed with A, forms the second sample zone after the pure A zone. The third zone contains the mixture A t B+C. This method can be compared with frontal analysis in chromatography. In movingboundary electrophoresis, the zones generally contain more ionic species of the sample. The composition of the sample plays an important role in the determination of the concentrations, pH values and conductivities of the different zones. This situation contrasts with that in isotachophoresis, where all of these quantities are independent of the quantitative composition of the sample. A quantitative description of this method is given in Section 4.2. As will be clear after the description of isotachophoresis, the first zone in moving-boundary electrophoresis has a self-correcting effect, so that the first boundary will be sharp. All other zones are not sharp, although this influence is generally smaller than in zone electrophoresis. In Appendix A, a method is given with which effective mobilities can be measured by using moving-boundary electrophoresis. Fig.2.4 shows the temperature, voltage gradients and electrical resistances for the different zones. All of these quantities show similar relationships.

PRINCIPLES OF ELECTROPHORETIC TECHNIQUES

12

1

U

I

1 I

I

I

I

I

I

t

I I I

I

E , 1,R.

I I I

I

I

I

I

I

L

A

A+B

A+B+ C

t-

X

Fig.2.4. (a) Separation according to the moving-boundary principle. The sample anions A + B + C are introduced into the cathode compartment. The separation chamber and the anode compartment are filled with a leading electrolyte, a suitable choice of counter ion needs to be made, because it determines the pH at which the analysis is performed. After some time, a partial separation is obtained, which is shown schematically in (b). The electric field strength Q, electric resistance ( R ) and temperature ( r ) are shown schematically for the different zones.

ISOTACHOPHORESIS

13

2.4. PRINCIPLE OF iSOTACHOPHORESIS 2.4.1. Introduction

We shall consider here the separation of anionic species in narrow-bore tubes. For the separation of anionic species, the narrow-bore tube and anode compartment are filled with the so-called leading electrolyte, the anions of which must have a mobility that is higher than that of any of the sample anionic species. The cations of the leading electrolyte must have a buffering capacity at the pH at which the analyses will be performed. The cathode compartment is filled with the terminating electrolyte, the anions of w h c h must have a mobility that is lower than that of any of the sample anionic species. The sample is introduced between the leading and terminating electrolyte, e.g., by means of a sample tap or a micro-syringe. #en an electric current is passed through such a system (see Fig.2.5a), a uniform electric field strength over the sample zone occurs and hence each sample anionic species will have a different migration velocity according to eqn. 2.1. The sample anionic species with the highest effective mobility will run forwards and those with lower mobilities will remain behind. Hence, both in front of and behind the original sample zone, the movingboundary procedure results in two series of mixed zones (comparable with the Tiselius method). In the series of mixed zones, the sample anionic species are arranged in order of their decreasing effective mobilities (see Fig.2.5b), The anionic species of the leading electrolyte can never be passed by sample anions, because its effective mobility is chosen so as t o be higher. Similarly, the terminating anions can never pass the anionic species of the sample. In this way, the sample zones are sandwiched between the leading and terminating electrolyte. In the mixed zones of the sample (see Fig.2.5b), the separation continues and, after some time, when the separation is complete, a series of zones is obtained in which each zone contains only one anionic species of the sample if no anionic species with identical effective mobilities are present in the sample. Of course, this series of zones is still sandwiched between leading and terminating electrolyte (see Fig.2.5~). The first sample zone contains the anionic species of the sample with the highest effective mobility, the last zone that with the lowest effective mobility. After this stage, no further changes t o the system occur and a steady state has been reached. In such a case, we can speak of an isotachophoretic separated system. (Of course, one or more unmixable ‘mixed zones’, i.e., zones that contain one or more anionic species with identical effective mobilities, may still be present.) In this state, all of the zones must run connected together, in contrast to zone electrophoresis, where all zones release. Here the zones cannot release as there is no background electrolyte that can support the electric current (a requirement for the solvent is that its self-conductance must be negligible; see section 5.2 .)* . *If it is assumed that the zones release, then the concentration of the ionic species at that position will decrease, the electric field strength will increase (working at a constant current density) and hence the migration velocity of the ionic species involved will be higher. Therefore, finally these ionic species will reach the preceding zone.

PRINCIPLES OF ELECTROPHORETIC TECHNIQUES

14

b

0

L

C , conatant

,,,

Fig.2.5. Separation of a mixture of anions according to the isotachophoretic principle. The sample A+B+C is introduced between the leading anionic species L and the terminating anionic species T. A suitable cationic species is chosen as the buffering counter ion. The original conditions are shown in (a). After some time (b), some mixed zones are obtained according to the moving boundary principle. Finally (c), all anionic species of the sample are separated and all zones contain only one anionic species of the sample (‘ideal case’).

For this steady state, all zones must have identical migration velocities, determined by the migration velocity of the anionic species of the leading electrolyte. Considering the zones L, A, B, C and T (see Figure 2 . 5 ~ ) :

v, = V A = V B = vc = VT or

m,E, = mAEA = mBEB= m,Ec

= mTET

Eqn. 2.4 will be called the ‘isotachophoretic condition’ and it is characteristic of isotachophoretic separations.

(2.4)

ISOTACHOPHORESIS

15

As the anionic species are arranged in order of decreasing effective mobilities*, i.e., > mT, the electric field strengths increase on the rear side. Working at a constant current density, the product EI (a measure for the heat production) also increases on the rear side and therefore the temperatures increase in the preceding zones. In Figs.2.6~and 2.6d, the electric field strengths and temperatures are shown for the zones of Fig.2.6a. In Fig.2.6b, the variation of potential with position in the tube is shown. The increase in the voltage gradients in the consecutive zones induces two important characteristics of isotachophoretic systems. The first characteristic is the ‘self-correction’ of the zone boundaries. When a zone has attained the steady state, the boundary will not broaden further, which again is in contrast to zone electrophoresis, where the peaks are unsharp and broad owing to adsorption and diffusion phenomena. This effect can easily be understood. If an ion remains behind in a zone with a higher electric field strength, then it will acquire a hgher migration velocity according to eqn. 2.1, until it reaches its own zone. If it diffuses into a preceding zone, where the electric field strength is lower than the value that corresponds to its velocity, its velocity will decrease and it will be overtaken by its proper zone*. The second characteristic is the increase in temperature in the preceding zones, and by this feature the zones can be detected with a thermometric detector. In order to obtain a better understanding of isotachophoresis, we now give a much simplified model and subsequently some isotachopherograms, obtained with several detection systems, are shown and discussed in order to facilitate the understanding of later chapters.

mL > mA > mg > mc

2.4.2. Simplified model for isotachophoresis Let us first consider a boundary between two connected zones, containing anionic species A and B with mA > m B .The influence of diffusion, etc., will be neglected. Suppose the counter ionic species Q are similar in both zones and have a constant mobility mQ , all ions are monovalent and fully ionized and the influence of the presence of H and OH- ions can be neglected. Working at a constant current density, the following equations can be derived: (a) According to the principle of electroneutrality, the amounts of positive and negative ions in both zones must be identical, so

The subscripts indicate the ionic species and the zone, e.g., cA,l represents the concentration of anionic species A in the first zone. (b) According to the isotachophoretic condition, the zones must have identical *If not too large pH shifts occur in the consecutive zones considered (see Chapter 9).

PRINCIPLES OF ELECTROPHORETIC TECHNIQUES

16

V

-X

-X

dl

I

T

/

\

I

Fig.2.6. Graphical representation of potential V (b), electric field strength E (c) and temperature T (d) for the different zones, moving in the steady state of an isotachophoretic analysis (a). X = Position in the narrow-bore tube where the analysis is performed; s = position of introduction of sample.

17

ISOTACHOPHORESIS

velocities, so that v1

=v2

(c) According to Ohm’s law: I = constant = E l hl

= E2

(2.10)

h2

and the conductivities of the zones can be written as

+ mQ)

A1 = C A , ~ ~ A F + C Q , ~ ~ Q F = ~ A , ~ F ( ~ A

(2.1 1) (2.12)

(2.13)

(2.14) Replacing El lE2 with mBlm, according to eqn. 2.9: (2.15) From e q n 2.15. it can be seen that the concentration of all zones is determined by the concentration of the leading electrolyte, and depends on the mobilities of the ionic species concerned. In Chapter 4, a more accurate model will be derived, with corrections for the influence of pH, different temperatures in the zones, buffering counter ionic species, the pK values of the ionic species present, etc. Although the model here is greatly simplified, it can be stated that the concentrations in the zones are constant in a given system and that the ionic concentrations decrease to the rear side. The concentrations do not depend on the composition of the sample. If the sample is very dilute, then during analyses by other techniques (e.g., zone electrophoresis and gas chromatography), the concentrations will be further decreased. In isotachophoresis, however, the concentration always attains a value fixed by the composition of the leading electrolyte. Therefore, isotachophoresis is sometimes used with other techniques in order t o concentrate the sample in narrow zones. For example, in disc electrophoresis, the first stage in the analysis involves concentration of the sample

18

PRINCIPLES OF ELECTROPHORETICTECHNIQUES

by the so-called ‘stacking electrophoresis’*. The importance of this phenomenon is clear when it is realized that, because the concentrations in the zones are constant, the length of a zone (the distance between two differential signals) is a direct measure of the concentration of the sample ionic species. 2.4.3. Concentration adaptation

If zones migrate, they must have a concentration that is fixed by the preceding zones according to Ohm’s law, and we call them ‘adapted zones’. The effects on changes in concentration during an isotachophoretic analysis are shown in Figs.2.7a-2.7f for the separation of a mixture of anionic species A and B, introduced between the leading electrolyte L and the terminating electrolyte T. In Fig.2.7a, the original situation is shown. A mixture of A and B (Ao +Bo) is introduced between the leading ions L and the terminating ions T I . Of course, the zone A. +Bo is not adapted to L according Ohm’s law, and nor is zone T I . In Fig.2.7b, the situation is shown after a certain time, where the leading zone L has migrated over a certain distance, its concentration remaining constant, however. According to all movingboundary procedures, a zone containing the anionic species A is formed and the concentration in this zone A l is adapted to zone L. The mixed zone A+B that has passed the original boundary is also adapted. But behind the original boundary, the originzl mixture A. +Bo is present, still not adapted. Behind that zone A. +Bo, a zone B1 is formed that contains only the anionic species B and this zone is adapted to the zone A. + Bo. Also, the migrated zone Tz is adapted to zone A. +B,. In Fig.2.7c, the original mixture A. +B, has disappeared, but now there are two zones B, one adapted to the leading zone L(Bz) and one still adapted to the non-existing original zone A. + Bo. In Fig.2.7d, the terminator has passed the original boundary and from this time also a zone T3 exists, already adapted to the leading zone L. At this moment, three T zones exist, viz., a zone T3 adapted to the leading zone L, a zone T2 adapted to the non-existing zone A. +Bo and the original zone T I **. In Fig.2.7e, the same situationisshown, the mixed zone A + B being much smaller. In Fig.2.7f, the mixed-zone A+B has disappeared, ie.,anionic species A and B are separated. Three T zones still exist, marking the spot where the sample was introduced. It is important to understand this procedure, although we shall not take this effect into account, because it is of no importance at the position of detection, as the original boundaries do not move and never reach the position of detection. In fact, we can never detect these zones with electrophoretic equipment, as will be discussed later***. These three zones do not remain sharp, because the ‘self-correcting’ effect, characteristic of isotachophoresis, does not occur in these zones. *It should be noted that the proteins in the ‘stack‘ can be easily denatured, because the conditions are not ideal for proteins, as indicated in Chapter 1 3 where the separation of proteins is considered. **In the separation chamber, all zones are now adapted to the composition of the leading zone, although mixed zones are still present. If a counter flow of electrolyte (as described in section 7.5.5) is to be applied, it should be applied at this moment, because the zone of the terminating electrolyte, which has passed the boundary occupied originally by the sample, has already attained its isotachophoretic velocity. Even if 100%counter flow of electrolyte occurs, neither the sample zone nor the zone of the terminating electrolyte is flushed back. ***No scanning device has yet been constructed.

ISOTACHOPHORESIS

19

d.t I

X

’.t -X

Fig.2.7. Changes in concentrations for the different zones in an isotachophoretic analysis. The sample is a mixture of A + B (original concentration A,+B,). The sample is introduced between the leading electrolyte L and the terminating electrolyte T.Theoretically, three different zones, marking the terminator concentrations T,,T , and T,, are finally obtained, in addition to the zones of the sample species to be analyzed. For further explanation, see text. s = position of introduction of sample; R = increasing electric resistance; X = position in the separation chamber.

20

PRINCIPLES OF ELECTROPHORETIC TECHNIQUES

2.4.4. Some isotachopherograms In order to detect the zones in isotachophoretic separations, several detection methods can be used, some of which are described in the section Instrumentation (Chapters 6 and 7). In order to understand the isotachopherograms shown in later chapters, some of them are discussed here, although only a brief description will be given. The first isotachopherograrn (see Fig.2.8) was obtained by means of a thermocouple. As explained in section 2.4, the temperatures of the proceeding zones increase and these temperatures can be measured by means of a thermometric detector, e.g., a thermocouple (made of 15-pm constantan and 25-pm copper wire). A signal as shown in Fig.2.8 is obtained. The construction of the thermocouple is described in the section Instrumentation. In Fig.2.8, the differential of the linear trace is also given. This signal marks the zone boundaries more clearly. The distance between two differential signal peaks is a measure of the zone length and hence it is a measure of the amount of the ionic species in that zone, because the concentration of the ionic species in that zone is constant for a given operational system. The step heights to be measured on the linear trace of the thermocouple signal are a measure of the conductivity in that zone and are also a measure of the effective mobilities of the ionic species in the zones. Hence the step height can be used for the identification of the ionic species in the sample. For recording the isotachopherogram shown in Fig.2.8, a potential recorder with zero suppression was used, which is advantageous for the accurate determination of the various step heights, but may confuse the information if one is not familiar with it. Under the conditions chosen, the step heights h l and h2 are characteristic of the tetramethylammonium and the ammonium ion, respectively. It should be noted that h l and h2 refer to the temperature of the chloride zone, which is also constant under the conditions chosen. The data presented later (Chapters 11 and 12) are referred to the thermocouple signal at 0 PA. [Note that, e.g., in gas chromatography, the distances are a measure of the identification (retention times) and the peak areas are commonly a measure of the amounts present.] Fig.2.9 shows an isotachopherogram for the separation of some anions. The experiments were carried out in the operational system at pH 6 (Table 12.1). The analyses were performed in equipment that is described in section 7.4.4,using the two highresolution detectors: a conductimeter (a.c. method)* and a UV absorption detector (256 nm). The linear trace from the conductivity detector, as in the linear trace from a thermocouple detector, is a measure of the conductivities of the zones. Hence it is a measure of the effective mobilities of the ionic species in the zones and characterizes the ionic species. The ‘step heights’ that can be found in the linear trace can be used for the identification of the various ionic species in a well defined operational system. All of the step heights, as described in the section Applications (Chapters 8-17), refer t o the conductivity of the zone of the leading electrolyte, which is-adjusted to ‘zero’ with the electronic device described in Chapter 6 (Fig.6.18). The differential of the linear signal *For the difference between the a s . method, using a conductivity detector, and the d.c. method, using a potential gradient detector, see Chapter 6 .

ISOTACHOPHORESIS

21

t

Fig.2.8. Isotachopherogram of the separation of some cations in the operational system listed in Table 16.1 (methanol was used as the solvent), obtained by using a thermometric detector. For further explanation, see text. 1 = H’ (leading ion); 2 = (CHJ,N+; 3 = NH:; 4 = K+; 5 = Na+;6: Li,; 7 = Mn2+; 8 = Cuz+; 9=CdZ’ (terminating ion). h,=Step height (qualitative information) of the tetramethylammonium ion; h , = step height of the ammonium ion. These step heights are referred to the ‘temperature’ of the zone of the leading ion and x , and x , are a measure of these quantities. T = temperature; i = time.

of the conductivity detector is also given in order to mark the zones, as the zone length is a measure that can be used for quantitative determinations. .4s the signals from W absorption detector depend on the absorption properties of the ionic species in the zones (not depending on the conditions of the leading electrolyte), one cannot always obtain quantitative and qualitative information from w h c h the ionic species can be defined. It will be clear that the combination of two high-resolution detectors will give the maximum amount of information in isotachophoretic separations.

PRINCIPLES OF ELECTROPHORETIC TECHNIQUES

22 4-

’7i -

I nI

3

-. __

a

,

3

0

?

ISOELECTRIC FOCUSING

23

The four isotachophoretic separations shown in Fig.2.9. were obtained under identical conditions, Le., stabilised electric current (70 PA), operational system, thermostating (22°C) and speed of the recorder chart paper (6 cm/min). The isotachopherograms show that the step heights in the linear traces from the conductivity and UV absorption detectors are not influenced by the sample size, that a quantitative determination of the ionic species is possible and that the mutual influence of the various ionic species is zero. In Fig.2.9A, the difference in zone lengths is due to the fact that the pyrazole-3,5dicarboxylate has a greater electric charge than the acetate at the pH of the operational system chosen. In Fig.2.9B, it can be seen that both zone lengths increase as the amounts of the components increase.

2.5. PRINCIPLE OF ISOELECTRIC FOCUSING Amphiprotic substances (e.g., proteins), which contain acidic and basic groups in their molecules, have a so-called isoelectric point, pl, which is the pH value at which they have no net charge. At this pH, they are present mainly in the form of a zwitter-ion. In solution, with a pH equal to the p l value of the amphiprotic substances, they do not migrate when they are placed in an electric field. At higher pH, they lose protons and become negatively charged, so that they will then migrate towards the anode if an electric field is applied. At lower pH, their net charges will be positive and consequently they 4 1 migrate towards the cathode. The basic principle of isoelectric focusing is that a buffer gradient is used such that the pH in the separation chamber increases from one side to the other, the lowest pH being obtained at the anode and the highest pH at the cathode. When a mixture of amphiprotic substances is introduced into such a system, all substances d 1 acquire different net charges according to their p1 values and hence will have different mobilities. On applying an electric field across the system, each substance will migrate towards that position where the pH is equal to its pI value. For example, if a protein is introduced at a pH higher than its p1 value, it becomes negatively charged, migrates towards the anode, in which direction the pH decreases, and reaches finally the position where the pH is equal to its p1 value. At this position, its net charge is zero and its velocity decreases to zero. By isoelectric focusing, all substances in the sample will be concentrated into narrow zones. Because plvalues are characteristic of, for instance, proteins, they can be separated by this means; proteins with pldifferences of 0.02 pH unit have been successfully separated. Fig.2.9. Isotachopherogram of the separation of some anions in the operational system at pH 6 (Table 12.1). R =increasing electric resistance;A =increasing UV absorption; ?=time. 1 =Chloride; 2=pyrazole-3,5dicarboxylate;+acetate; 4=glutamate. A, 10 nmole of acetic acid and 10 nmob of pyrazole-3,5-dicarboxylateinjected (note the difference in step length, due to the difference in charge of the ionic species): B, 20 nmole of acetic acid and 20 nmole of pyrazole-3,5-dicarboxylate injected; C, 10 nmole of acetic acid and 20 nmole of pyrazole-3,5dicarboxylate injected; D, 20 nmole of acetic acid and 10 nmole of pyrazole-3,5-dicarboxylateinjected. A conductimetric (ax. method) and a UV absorption (256 nm) detector were used. The step heights are constant (qualitative information), while the distance between the peaks (= length of the corresponding step) varies (quantitative information).

24

PRINCIPLES OF ELECTROPHORETIC TECHNIQUES

s

a

0

L

pH

4

5

IffCREASlNO

7

6

8

c pH

INCRPASINO

Fig.2.10. Separation of the amphiprotic substances A, B and C with PIvalues of 4, 6 and 8, respectively, by isoelectric focusing. The amphiprotic substances are eventually concentrated into narrow zones where the pH of the buffer gradient is equal to the PIvalue of the amphiprotic substance in each instance. (a), Sample introduction; (b), separation of A, B and C. s = position of introduction of sample.

In Fig.2.10, t h s situation is shown for the separation of three substances with pf values of 4,6 and 8, respectively. The substances A, B and C are introduced at a pH of 6, Le. the net charge of A is negative (pH is higher than its pZ value), the net charge of B is zero (pH = pf) and the net charge of C is positive (pH is lower than its pf value). Substance A will migrate towards the anode until it reaches a pH of 4, substance B stays at the position where pH = 6 and substance C migrates towards the cathode until it reaches a pH of 8. After a certain time, A, B and C are separated and concentrated into narrow zones with pH values of 4 , 6 and 8, respectively. Further information on equipment, performances, carrier ampholytes, etc. is given in detad in the literature.

2.6. DISCUSSION

In the preceding sections, the four main types of electrophoresis have been described, and in Fig.2.11 their characteristics are summarized. In each instance a sample is introduced (at an injection point X) that consists of two anionic species B and C and one

DISCUSSION

25

Fig.2.11. Survey of the four main electrophoretic techniques: (a) zone electrophoresis; (b) movingboundary electrophoresis; (c) isotachophoresis; (d) isoelectric focusing. X indicates the position where the sample is usually introduced. For further information, see text.

cationic species D. h Fig.2.11a, the situation in a zone electrophoretic system after a certain time is shown. The background electrolyte AE is present in the whole system, and the anionic species B and C have migrated in the direction of the anode. Anionic species B, which has a higher mobility, has covered a greater migration length. Cationic

26

PRINCIPLES OF ELECTROPHORETIC TECHNIQUES

species D has migrated in the direction of the cathode. Fig.2.1 l b shows the movingboundary procedure. The sample mixture is introduced into the cathode compartment and the leading electrolyte AE fills the separation chamber and the anode compartment. The cationic species D thus remains in the cathode compartment and the anionic species B and C, partially separated, migrate behind the leading electrolyte AE. Note that the pure zone B and the mixed zone B+C have the counter ionic species of the leading electrolyte E. The first boundary (with a velocity V , ) is sharp (according to the isotachophoretic condition), whereas the separation boundary (with a velocity V*) is not sharp, as in zone electrophoresis. The zone velocities v* and V1 are different. Fig.2.1 l c shows the separation procedure for an isotachophoretic system. The sample is introduced between the leading electrolyte AE and the terminating solution TE. The anionic species B and C are separated and migrate between the terminator T and the leading ion A. All zones have equal velocities, and contain the same counter ionic species E. The cationic species D of the sample has migrated to the cathode. In Fig.2.1 I d , the sample is introduced in an isoelectric focusing system. The point of injection is not important. The ionic species are eventually separated according to their plvalues and are concentrated on spots where the pH values are identical with their p l values, their net charges and velocities then being zero. B and C have migrated towards the anode (lower pH) while the cationic species D has migrated towards the cathode (higher pH). Only amphiprotic substances can be separated by this method.

Chapter 3

Concept of mobility SUMMARY Electrophoretic migration is discussed and the ionic mobility is defined. The relationship between equivalent conductance and ionic mobility is shown, the concept of effective mobility is described and the influence of partial dissociation, relaxation and the retardation effect on the effective mobility is discussed. Some approaches are suggested for the determination of unknown mobilities.

3.1. INTRODUCTION

The concept of mobility plays an important role in electrophoretic techniques, as differences in effective mobilities determine whether or not ionic species can be separated. The concentrations and the voltage gradients of the different zones, related to the parameters of the leading zone, are also fixed by the effective mobilities. In this chapter, the concept of mobility is discussed. We do not intend to give here a complete survey of all of the mathematical theories proposed and experiments carried out on this subject, which have been described in various papers. We will consider mobility only so far as is necessary in order to understand and use the theory of electrophoresis. Further, some approaches will be given with which unknown ionic mobilities can be estimated by relationships with other parameters.

3.2. INTERPRETATION OF ELECTROPHORETICMIGRATION

If an electric field is applied to an electrolyte solution, charged particles will move and a stationary state will be reached in which the velocity of the particles, in the direction of the field, is constant with time. In this state, there are four different forces acting on a particle, called F I ,F 2 ,F3 and F4 (see Fig.3.1). F1 is a force exerted on the charge of the particle and can be denoted by

F, = q E

(3.1)

where E is the electric field strength. F2 is a friction force, which Stokes determined for a rigid spherical particle as

F2 = -f,v

= -6nqrv

(3-2)

where v is the electrophoretic velocity, r is the radius of the particle, q is the viscosity of the solvent andf, is the friction factor. For a non-spherical particle,f, is still proportional to Q, but a correction factor has to be introduced so as to allow for-the sizeTshape and orientation of the particle. 21

CONCEPT OF MOBILITY

28

Fig.3.1. Forces acting on a positively charged particle, which moves under an electric field, E , can be represented by F, ,Fz , F3 and F A .The originally symmetrical, in this instance negatively charged, ionic atmosphere(1) is shifted due to the electric field E(2). For further explanation, see text.

The forces F3 and F4 are due to the presence of oppositely charged particles, forming a so-called ionic atmosphere. For F 3 , the electric field exerts a force on the ions of the ionic atmosphere, which is transferred to the molecules of the solvent. The particles considered do not move through a stationary solvent, but through a solvent flowing in the opposite direction, so that the net velocity is decreased. This effect is called the ‘electrophoretic retardation’. F4 represents the ‘relaxation effect’. The distribution of ions in the vicinity of the particles is deformed when an electric field is applied, because the particles move away from the centre of the ionic atmosphere. The Coulomb forces between the ions tend to re-build the ‘atmosphere’ in its ‘proper’ place, which takes a finite time called the relaxation time. Hence, the centre of the ionic atmosphere of the particle constantly lags behind the centre of the particle in the stationary state, resulting in an electrical force on the charge of the particle. This force is called the relaxation effect. For the stationary state, the sum of these forces must be zero, so that

FI

+F2

+F3 +F4 = O

(3 -3)

01

or

From eqn. 3.5, the influence of electrophoretic retardation, the relaxation effect, the shape, charge and radius of the particle and the influence of the solvent on the electrophoretic migration velocity can be understood. In the next section, the relationship between ionic conductance and migration velocity is considered and the absolute ionic mobility is defined.

29

IONIC MOBILITY AND IONIC EQUIVALENT CONDUCTIVITY

3.3. IONIC MOBIUTY AND IONIC EQUIVALENT CONDUCTIVITY

We speak of an 'equivalent weight' of an electrolyte if, for complete dissociation, the total amounts of positive and negative charges are eN and -eN respectively, where N is Avogadro's number and e is the electronic charge. For example, one equivalent weight of potassium fluoride gives, for complete dissociation, one Avogadro's number of K’and of F ions. The conductance of such an amount of electrolyte is the conductance measured in a conductance cell with electrodes 1 cm apart and with such cross-sections that the volume of solution between the electrodes will contain exactly one equivalent of the electrolyte. This conductance is known as the 'equivalent conductance' and is denoted by A*. Kohlrausch showed that at a f n e d temperature the relationship between the equivalent conductance of an electrolyte and the square root of the concentration is nearly linear, especially for very low concentrations and strong electrolytes. At infinite dilution, the equivalent conductances can be interpreted in terms of ionic contributions, whereby the contribution of an ion is independent of the other ionic species of the electrolyte (the influence of retardation and relaxation effects can be neglected, as no ionic atmosphere is present at infinite dilution). At infinite dilution, we can therefore write

A: = Ax’

+ Ax-

(3.6)

where Ax’ and Ax- are the equivalent ionic conductivities of the anions and cations, respectively, and A: is the equivalent conductance, all at infinite dilution. If a voltage V is applied t o a cell as mentioned above (see Fig.3.2) a current I flows through the cell: I = V/R or I = VA*

(3.7)

Assuming that such a cell contains one equivalent of the electrolyte, N/z' positive and N/z- negative ions are present, where z’ and z- are the valences of the positive and negative ions, respectively. If the velocities of the ions are represented by’ v and v-, respectively, the positive ions present in volume B and the negative ions present in volume C (see Fig.3.2) will have passed the cross-section A in 1 sec. Because the cell is I cm in length, this means that the volumes B and C will contain v+/l and v-/l parts of the total amount of the positive and negative ions of the cell, w h c h is

v’(N/z’)

positive ions and v - ( N / z - ) negative ions

(3.9)

The currents corresponding to these flow rates are obtained by multiplying by the ionic charges ez' and ez- and by this:

r' = ez' ’ v (IV/z+)= e Nu+= Fv'

(3.10a)

r = ez-

(3. lob)

v- ( N / z - ) = eNv-

= Fv-

At infinite dilution, combination of eqns. 3.8 and 3.10 gives

CONCEPT OF MOBILITY

30

Fig.3.2. Conductance cell with electrodes 1 cm apart. For further explanation, see text.

(3.1 la) (3.1 Ib) The average velocity with which an ion moves under the influence of a potential of 1V is called the ionic mobility, and the ionic mobility a t infinite dilution is called the

absolute ionic mobility. Thus, (3.12) It can be concluded from eqn. 3.12 that absolute ionic mobilities can be calculated by dividing the equivalent ionic conductivities at infinite dilution by the Faraday constant. The equivalent ionic conductivities can be obtained measuring the transport numbers. As the transport numbers give the fractions of the total current carried by each ion, ie., the fraction of the total conductance that each ion contributes, we can write

hg+= t,'A,*

(3.13a)

and A,*- = ti A:

(3.13b)

where t = transport number. Data for conductances and transport numbers in order to

EFFECTIVE IONIC MOBILITY

31

obtain A*" and A*- for the calculations of the ionic mobilities at concentrations other than infinite dilution cannot be properly used, because the law of independent migration of the ions is invalid and the conductance is really a property of the electrolyte rather than of the individual ions of the electrolyte. This means that the ionic conductivities (and hence ionic mobilities) of a chloride ion in 1N calcium chloride solution and in 1 N sodium chloride solution are different. In such instances a correction must be made for the influence of relaxation and retardation effects and for incomplete dissociation (ion pair formation). Also, for "weak" electrolytes it is sometimes very difficult to obtain correct values for the equivalent ionic conductances at zero concentration (infinite dilution). For such solutions, we can calculate the correct values from the ionic contributions of strong electrolytes at infinite dilution. For example: A,*(HAc) = A,*(NaAc) + A: (Ha)- A,*(NaCl)

(3.14)

because the right-hand side can be interpreted as AX+ (N2) + A-: =

Ar(H+ +)A-:

(Ac- ) + Ag"(H)

+A-:

(Ac-) = A;f'(HAc)

(GI-) - A x ' (Na') -A,*-

(a-) (3.15)

This procedure is not valid at concentrations other than zero, but in practice it can be used in order to obtain conductivities and mobilities at concentrations other than zero. In fact, corrections for the differences in relaxation and retardation effects and ion pair formation in electrolytes are neglected and i t can be used only as a rough approximation.

3.4. EFFECTIVE IONIC MOBILITY The absolute ionic mobility, m:, is defined as the average velocity of an ion per unit of electric field strength at infinite dilution. This absolute ionic mobility is a characteristic constant for every ionic species in a certain solvent and is proportional to the equivalent conductance at zero concentration: A,*=AE'fX:-

=(m,'+m;)F

(3.16)

In practice, we are not working at infinite dilution and the influence of other ionic species present in an electrolyte solution cannot be neglected. The effective mobility of an ionic species is related to the absolute mobility. Corrections have to be made for influences such as the electrophoretic retardation and the relaxation effect, as described by Onsager (see ref. 1). By using the Onsager equation, a correction can be made for ion-ion interactions. Another influence is the effect of partial dissociation. Tiselius [2] pointed out that the effective mobility is the sum of all products of the degree of dissociation and the ionic mobilities: meff.=

7 aimi

(3.17)

where meff.is the effective mobility, ai is the degree of dissociation and mi is the ionic mobility.

32

CONCEPT OF MOBILITY

To summarize, we can state that the effective mobility of an ionic species depends on several factors such as the ionic radius, solvation, dielectric constant and viscosity of the solvent, shape and charge of the ion, pH, degree of dissociation and temperature. It is very difficult to give a precise mathematical expression for the effective mobility. When speaking about effective mobilities, we shall use the expression

meff.= Ci cui rimi

(3.18)

where cti is the degree of dissociation, yi is a correction factor for the influence of relaxation and retardation effects and mi is the absolute ionic mobility. The correction factors c+ and yi will be described in more detail. 3.4.1. Partial dissociation

If an ion does not exist in the free form, but is in an equilibrium with the undissociated form, its effective mobility is smaller than its ionic mobility. For example, acetate, in water, is always in equilibrium with acetic acid according to the equation HAc +Hz 0

* H3O++Ac-

(3.19)

and the equilibrium constant is (3.20)

As the degree of dissociation is defined as cu=

[Ac-] [HAc] + [Ac-]

(3.2 1)

then, during time t, the ionic species exists in the form of acetate during time cut. Therefore, the migration distance in time f is

s = v a t = cumEt

(3.22)

Normally, the migration distance of an ion is s = v t=m Et

(3.23)

From eqns. 3.22 and 3.23, it can be concluded that the effective mobility can be calculated as

meR.= a m (a ' ; 'FLlil'I:=l'STEP'I'UNTIL'A'DB' 'BEGIN' NI C I 1:=READ;MHC I 1:=READ;M0HC I 1: =IIEAD;MBI C I 1: =REAP;

ZINC 11: =ilEAD; ' FOR ' J: =1 'STEP ' 1 'UNT IL'NI C I 1'Dl3' 'REGIN'PKI C 1, J 1: =READ; MI C I, J 1:=READ; 'END'5 NF3 C I 1:=READ;M09 C I 1 :=ilEAD; ZRN C I 1:=READ; 'FBR * J: =1 'STEP' 1 'UYTIL'N5 C I 1 ' DO ' ' BEGIN'PKl3 L: I J 3 :=REAL); M3 C 1, J 1:=READ; 'END' J ' END ;

,

0400'CBMPENT' q a l c u l a t i o n f i r s t z o n e : 0410 HPL: = l oT ( -PHL)j UHL: =1 O t ( -1 4+PHL); KLI t 01:= 1 ;KTLI :=KMLX :=KILI :=K II%I :=O; OM0 0430 'FkJR'I:=l'STEP'l'UNTIL'NLI'DB' 'REGIN'KLI CI I: =KLI[I-1 1*10t( -PKLICI l)A-IPL;QR:=ZI-I; 0440 KTLI :=KTLI+KLIC I 3; KILI :=KILI+KLI C I l*QH; 0450 Fig.4.13.

I1

VALIDITY OF THE ISOTACHOPHORETICMODEL 0460 0470 0480 0490

0500 0510 0520 0530 0535 0540 0550

0560 0570

0580 0590 0600 0610 0620 0630 0640

0650 0660

0670 0680 0690

0700 07LO 0720 0730

KMLI:=KMLI+KLICI WMLICI 3*SIGN+KMLH)/R; BCBR:=(l+ARS(MLBl )/ABS(MLIl ))*CSTLB; T: k0LI* CARS( ZI ) *MBLI+K IMLI 3 TUW: =C0LB*(AW( ZR) *MQLR+KIMU31 ; KBL: =HPL*MHL+VIHL*M")HL+T+~~~~; PR INTTEXTC ’ ( ’ M0F3LE = ’ ) ’ 1;F IXT(614 r MLB 1 1i NLCR; ’ FBR ’ I :=O * STEP’ 1 ’ UNTIL* NLR ’ DO ’ ’BEGIN’FIXT~SrOrZR-I~~F’L0T~5r2rKlECII*CBLR~~NLCK~’END’~

PRINTTEXT(’/2;’G(ZIT0’SYSTEMCIl;’END’ ’ ELSE ’ ’REG IN’ PH CV 1 :=CQL+QH /2; ’GGTB’SYSTEKC 1 1; ’ END ’ ; 1300 1400 L6: PRINTTEXT( ’ C ’ rJex t o n e ’ 1 ’ ;FIXT(5rOrL) ;NLCR; 1410 PRINTTEXT’);FIXTC6r4rE"Il CV1);NLCR; 1430 ’FBH’I:=O’STEP’l’UNTIL’NI[VI’G0’ ’BEG IN’ F IXTC5r O r ZINCV 1-1 1 5 FLUTt5r2J KI C V r I ]%@I CV 1 i 1440 1450 NLCHJ’END’; 1460 PRINTTEXT(’(’ CSTI= '>');FLffiT3,256 nm; and ascorbic acid, 4.6> pH>3.6,280 nm. The principle of indirect W detection can also be applied in operational systems in which methanol is used as the solvent. Attention has so far been paid to the separation of cations in methanol because the mobilities of cations in water do not differ so much and separation according t o pK values is difficult because many of the cations have similar pK values in water. This subject is discussed more extensively in the Section Applications and the data can be found in Chapters 11-17. Sulphanilic acid was found to work well in methanolic systems at 256 nm. Sulphanilic *The ‘indirect UV method’.

167

Fig.6.31. Isotachopherograms for the separation of some anions that lack W absorbance. (a) Creatinine, for which the molaf absorptivity of creatinine is function of the pH. Leading electrolyte = HCl(O.01 M , pro analysi grade) t creatinine (purified); pH = 4.5. Terminating electrolyte = morpholinoethanesulphonic acid (MES) (re-crystallized three times). (b) Counter ion for which the change in pH of a zone does not influence the molar absorptivity (p-alanine). Leading electrolyte = HCl (0.01 M,pro analysi grade) t e-aminocaproic acid (purified); pH = 4.5. Terminating electrolyte = MES (re-crystallized three times). Non-W-absorbing ions can thus be detected by the ‘indirect UV method‘. A = Increasing UV absorption; R = increasing electric resistance; t = time. The current was stabilised in both instances at 30pA. The chart paper speed was 2 cm/min. An injection was made of 1 pl of the sample 0.01 Mchlorate t 0.01 Macetate + 0.01 M formate + 0.01 M glutamate. Peaks: 1 = chloride; 2 = chlorate; 3 = formate; 4 = acetate; 5 = glutamate; 6 = MES. In (a) the lower pH of the glutamate zone with respect to the zone preceding it and following it is clearly visible. From this isotachopherogram, the pH can easily be checked as it can be calculated with the computer program discussed in Chapter 4. The difference in the step height as found in the linear conductivity trace, due t o the difference in the counter ion, should be noted. The conductimeter used is discussed elsewhere (Fig.6.18); the measuring electrodes were mounted equiplanar. Various impurities commonly present in the chemicals can be observed in the traces from both the U V and conductivity detectors.

DETECTION SYSTEMS

168

acid has a low electrophoretic mobility in methanol, although it dissolved sufficiently in it. A solution of methanol saturated with sulphanilic acid can be used to indicate the pH differences in the succesive zones in cation separations if acetate is used simultaneously as the electrically conducting counter ion with buffering capacity. As already mentioned, the contribution of sulphanilic acid to the buffering capacity and conductivity is negligible. The W detector can also be used for the determination of trace amounts of Wabsorbing material. An arbitrarily chosen component, salicylic acid, is used because it shows moderate UV absorption. For the determination of trace amounts of, e.g., ATP or ADP, the following method works more satisfactorily because of the higher molar absorptivity of these ions. Suppose the concentration of the salicylic acid is so small that even with the concentration effect of isotachophoresis the zones are too small for complete qualitative and quantitative determinations t o be effected by the W detector o r any detector with equal resolution. One can decrease the concentration of the leading ion, because the subsequent zones will be more dilute than the leading ion and longer zone lengths can be expected. The contribution to the conductivity from the solvent itself will be greater the more dilute are the solutions, because the mobility of, e.g., H’and O H , is great if water is used as the solvent. While at a concentration of the leading ion of 0.01 N a pH of 3 is a critical value, at a concentration of the leading ion of 0.001 N a pH of 4 is critical. The use of a

Y

7

7 3

2

d

I

f

Fig.6.32. Isotachophoretic separation of phosphate, salicylic acid and a mixture of them in an operational system at pH 4.2. HCI (0.01 M pro analysi grade) was taken and p-alanine (re-crystallized) added until the pH reached 4.2; 0.05% of Mowiol was added to this electrolyte. The terminating ion was glutamate. Separations: (a) phosphate; (b) 1:l mixture of phosphate and salicylic acid; (c) salicylic acid. The shift in the step height of the UV traces should be noted. An enrichment of salicylic acid was revealed by the W detector in (b), but not by the conductivity detector. 1 = Chloride; 2 = salicylate; 3 = glutamate; 4 = phosphate. R = Increasing electric resistance;A = increasing UV absorption; t = time.

UV ABSORPTION METER

169

50

Fig.6.33. Plot of step heights (h mm) in the UV traces for the mixed zone (Fig.6.32b) against the concentration ratio of phosphate to salicylate (r). BY this ‘dilution’ technique, the W detector sensitivity is improved for the UV-absorbing ion. For salicylic acid, the sensitivity is improved at least 50-fold.

counter flow of electrolyte is feasible, but longer times of analysis are involved and very pure electrolytes and even more complicated equipment are necessary. Alternatively to these two procedures, a decrease in the concentration of the leading electrolyte and counter flow of electrolyte may be applied for both W-absorbiag and non-UV-absorbing ions. Because salicylic acid shows UV absorption, another approach is possible. An operational system is chosen such that a stable mixed zone can be made of salicylic acid and a non-UV-absorbing acid. At pH 4, phosphate has been found to be satisfactory. In Fig.6.32, the isotachophoretic separation of phosphate, salicylic acid and a mixture of phosphate and salicylic acid is shown. The leading electrolyte was 0.01 N hydrochloric acid (pro analysi grade) plus 0-alanine, re-crystallized from water+thanol (1 :l), adjusted

170

DETECTION SYSTEMS

to pH 4.2. The electric current was stabilised at 70 PA and W detection was carried out at a wavelength of 256 nm. The drop in the height of the UV trace is clearly visible. The trace from the linear conductivity detector does not resolve these two acids in the mixture. The step height in the UV trace for salicylic acid can be plotted against the concentration ratio of phosphate to salicylic acid, as shown in Fig.6.33’. A 50-fold dilution of salicylic acid could easily be determined, which means that the resolution improves by a factor of 50. The step height in the W trace may be influenced by the following factors: the pH of the ‘mixed zone’ may vary as a function of the composition, which may influence the step height if there is a large difference between the acidic and basic forms of the molar absorptivity; and the molar extinction coefficient of the W-absorbing component. It does not need to be explained that a larger improvement in the resolution may be expected if the molar absorptivity increases. Moreover, the use of a En-Log converter will further improve this method. Anticipating on analyses discussed later, Fig.6.34 demonstrates that salicylic acid and phosphate can be separated at another pH, the so-called separation according to pK values. T h e e isotachopherograms are shown, demonstrating the separation of phosphate and salicylic acid (1 :1) at pH values of 3.2,4.0 and 7.0. It can clearly be seen that these two acids can be separated at pH values both below and above 4.0. At a high pH, the influence of carbonate can be seen, because no precautions were taken.

-7

w4

3

2

7L i

l

1

a

I ~

-

2+3

L;@

R

, - 1

-L

I-

Fig.6.34. Isotachopherograms of the separation of phosphate and salicylic acid, demonstrating that the separation is possible at both a ‘high‘ pH (7) and a lower pH (3). Because no precautions were taken during the preparation of the operational system at pH 7, the influence of carbonate can be seen. This aspect is considered further in Chapter 12. (a) Separation at pH 3; (b) separation at pH 4.2: (c) separation at pH 7. Glutamate was used as terminator. 1= Chloride; 2 = phosphate; 3 = salicylate; 4 = glutamate. A = Increasing UV absorption; R = increasing electric resistance; t = time. *The signal-to-noise ratio of the W detector is such that an amplification of at least 1000-fold is possible.

ADDITIVES TO THE ELECTROLYTES

171

6.6. ADDITIVES TO THE ELECTROLYTES 6.6.1. Introduction

As soon as the high-resolution UV detector became avdlable and the results could be compared with those of the conductivity detector, with comparable resolution, the initially non-reproducible results of both the conductivity detector and the UV detector could be studied more intensively. The UV detector mainly does not disturb the isotachophoretic pattern by its presence, except for compounds that may be destroyed by the UV light applied or if the material of whch the narrow-bore tube is constructed is eventually affected by the UV light. The conductivity detector, however, may disturb the electrophoretic pattern as a result of the polarization initiated by the driving current or due to a leak current, or due to excessive heat produced by the measuring current. Because in our systems the last mentioned current is small compared with the driving current, the heat produced by the driving current can be neglected. The aim of making additions to the electrolytes may vary. The addition of surfactants, for instance, not only sharpens the zone boundaries by depressing electroendosmosis, especially visible if the combination of a UV and a conductivity detector can be applied, but also influences the overpotential against electrode reactions on the micro-sensing electrodes of the conductivity detector. Additives can be classified into three categories: additives that affect the electroendosmotic flow; additives that influence various electrode reactions; and additives that show both of these effects. A study was undertaken in order to elucidate these phenomena. Another purpose of the study was to show the difficulties that might arise if the electrophoretic equipment is not well constructed. Many of the problems that were initially present during the development of the conductivity detector have been overcdme, but more useful information can be gained from considering these problems than from presenting the final solution only. 6.6.2. Effect of additives on the electroendosmotic flow

Electroendosmosis is the movement of a liquid with respect to a solid wall as the result of an applied potential gradient. Although it is generally assumed that the electroendosmotic flow can be neglected in a single narrow-bore tube, with high-resolution detectors this is not so. In the beginning of isotachophoresis (displacement electrophoresis), the viscosity of the electrolytes was increased in order to suppress the electroendosmotic flow, to prevent hydrodynamic flow (semipermeable membranes were not used) and to suppress convection. The viscosity was increased by the addition of hydroxyethylcellulose, linear polyacrylamide, arrowroot, agar agar or methylcellulose. These viscous liquids were purified by shaking them with a mixed-bed ion exchanger. One of the disadvantages was that between analyses considerable time was needed for rinsing the narrow-bore tube. In the early days, a precise classification could not be made. Most electrokinetic phenomena have to be explained in terms of the interaction between a flow of liquid in the double layer, but the exact structure of the double layer may generally be left out

172

DETECTION SYSTEMS

of consideration, especially if one is interested only in the suppression of the electroendosmotic flow. In isotachophoretic analyses, the electroendosmotic flow is not constant in all zones, but increases in the direction of the terminating zone. Ths effect increases the turbulence of the liquid in each zone, but it is beyond the scope of this book to go into great detail. Because hydrodynamic flow in the narrow-bore tube is blocked at one side by the semipermeable membrane, a profile as shown in Fig.6.35 may be postulated for both the electroendosmosis and the temperature differences in the various zones. There are still some differences of opinion concerning the boundary conditions for the movement of liquids, especially if this is compared with the movement of the zone boundaries. As in all types of calculation, the potential at the wall is taken determinative for the electroendosmosis. This potential is often called the { (zeta) potential. When an

A

B

Fig.6.35. Profile of a zone boundary in a narrow-bore tube that is blocked at one side by asemipermeable membrane. The arrows indicate the movement of the zone boundary; Xindicates the direction in which the sampk zones move. In both (A) and (B), the parabolic profile due to the difference in temperature between the centre and the wall of the narrow-bore tube is in the same direction as the movement of the zone boundary (-.-.-). A correction must be made for the difference in temperature on both sides of the parabolic profile. In (A), the electroendosmotic flow is chosen to be in the same direction as the movement of the zone boundary, while in (B) this flow is in the opposite direction. The dotted line indicates this electroendosmotic proffle. A correction must also be made for the difference in electroendosmotic flow on both sides of the boundary, because the potential gradients in the two zones are not equal. The final profiie (hypothetical) is indicated in both (A) and (B) by a full line.

173

ADDITIVES TO THE ELECTROLYTES

electric field E is applied, a stationary state is reached after a short period of time. We can divide the forces that are responsible for the electroendosmosis into two main classes: the force exerted by the external fieldE on ihe ions in the double layer, the force being transferred by these ions by liquid friction to the layer as a whole; and the force exerted by the friction on the layer considered by the neighbouring layers, moving with a different velocity. The force due to the difference in electroendosmosis in the consecutive zones is not taken into consideration. In order to gain an impression of the electroendosmotic velocity, vE, the following equation can be given: (6.16) where E is the dielectric constant, { is the potential at the wall, E is the electric field applied and q is the viscosity. The volume of liquid moving by electroendosmosis can be measured for each zone, but the net result is zero, because one side is blocked. If this side was not blocked, this volume transport would be

Q=vEO,

(6.17)

where 0 is the cross-section of the narrow-bore tube. We can eliminate 0 by means of Ohm’s law:

I

OE=-

x

(6.18)

where I is the current through the narrowbore tube and h is the specific conductivity of the liquid. For a rough estimation, the volume transport in a system in which no semipermeable membrane is applied is (6.19) We have not considered the contribution of the surface conductance, because in the systems applied by us they can be neglected. Under normal operating conditions, e.g., if analyses are performed at pH 6 (Table 12.1), an estimation can be made of the values of Q in the leading and terminating zones, the values being about 40 and 80 pl/h, respectively. The final profde of a zone boundary is, of course, influenced by these values. By adding a suitable surfactant, the viscosity in the vicinity of the wall can be increased at least 100-fold, which suppresses the electroendosmotic flow sufficiently. Finally, it should be noted that the time of analysis is not influenced by the electroendosmosis, again because one side is blocked by a semipcrrneable membrane.

114

DETECTION SYSTEMS

6.6.3. Effect of additives on the micro-sensirrg electrodes The physical chemist usually distinguishes between two extreme types of ideal electrodes [27, 281. The first type is the reversible electrode, on which ions from the solution are actually charged and discharged, so that a steady current is possible. The d.c. potential of the electrodes has a well defined value, which depends on the current and the composition of the solution. The second type is the polarized electrode, in which no transformation of ions take place, n o steady current can pass and any current that does pass represents the charging and discharging of a double layer made up of the electrode and the ions very close to its surface. As is well known, the double layer is a structure that acts as a capacitance, the value of which is dependent on the potential across it. This second type of electrode has no well defined d.c. potential. It may vary greatly under apparently identical circumstances and is greatly influenced by trace amounts of substances or impurities, as will be shown. Metallic electrodes are, in fact, always combinations of both types and their impedance as a function of frequency shows the extent to which one or other mechanism dominates their behaviour. For instance, a bright platinum electrode in a fluid that is rich in adsorbable compounds has an impedance that is very nearly proportional to W' , over the frequency range from 1 to 20 kHz, and it is therefore nearly an ideal polarized electrode. For the same electrode in a saline solution, a more complicated behaviour is observed. By the addition of, e.g., Triton X-100 to this saline solution the relationship mentioned above is again obtained. Without the addition of an inhibitor for electrode reactions (redox reactions), the electrode reaction is rapid and the current is limited by diffusion of the reacting ions and the products between the surface of the electrode and the bulk of the solution. The impedance will decrease proportional to w-f. If the electrochemical reaction itself is slow, the impedance will be lower for small w and higher for large w than in the case when diffusion predominates. In practical cases, inhomogeneities in the electrode material will spread out the band of frequencies for which the impedance decreases only slowly with frequency, as the rate of the reaction is strongly dependent on the d.c. potential of the electrode, which varies over the surface. In instances in which insoluble reaction products cover the electrode surface, the multiplicity of diffusion paths may have a similar effect. This is the case with a silversilver chloride electrode in either a saline solution or a saline plus gelatin solution. It has an impedance that decreases approximately proportional to w-t over a wide range of frequencies. The d.c. potential of the electrode can make a considerable difference in the impedance function by changing the rate or even the nature of the reaction carrying current. Therefore, the balance between this potential and diffusion is responsible for the impedance. The real and imaginary components of the impedance of a particular electrode decrease as approximately the same function of w. A fluid-filled micro-electrode can be compared with a low-pass filter that is d.c.-stable. They must be used when signals are large and for which d.c. and low potentials are of interest. The metallic electrode is a high-pass filter, which is d.c. unstable. Its optimum use is when rapidly varying signals are of interest and the amplitude of the signals may be close to the noise level. It should be remembered that the micro-sensing electrodes described in this book are

ADDITIVES TO THE ELECTROLYTES

175

also a combination of both types. Of course, those micro-sensing electrodes are meant which have a direct contact with the electrolyte inside the narrow-bore tube. Owing to the driving current, polarization of the micro-sensing electrodes occurs, Le., these electrodes may act as charge-transfer electrodes. This depends on the potential gradient across the electrodes caused by the driving current and the composition of the electrolyte. In those cases when the driving current itself was used for measuring the conductivity (the so-called d.c. method), difficulties similar to those found by several workers who used metallic electrodes were observed. Partial polarization of the metallic electrode made the recording of the boundaries obscure. In order to study the interaction of the electrolyte. the driving current and the micro-sensing electrodes, electrodes were constructed such that the electrolyte inside the narrow-bore tube remained surrounded by an uninterrupted cylindrical wall (Fig.6.10). In t h s measuring probe, the distribution of the measuring current is much more linear than in other constructions considered, although the measuring current flows mainly along the wall of the narrow-bore tube. This is especially so if the results of current distribution are compared with those for the probe in w h c h the micro-sensing electrodes are mounted equiplanar (Fig.6.16). The capacitance of the measuring cell used was about 3 pF. In the experiments described, the value of R,, varies from approximately 15 kf2 at the beginning to 50 kS2 at the end*. While the driving current is kept constant, the potential varies during the experiment from 4 to 12 kV between the anode and the cathode of the electrophoretic equipment. The polarization of the micro-sensing electrode, which is in direct contact with the electrolyte, due to the driving current is shown schematically in Fig.6.36. Although the platinum has the same potential over all of its surface, it acts as a bipolar electrode with the cathode directed towards the anode compartment of the electrophoretic equipment and the anode towards the opposite side. Depending on the potential gradient and the composition of the electrolyte, the micro-sensing electrodes can be ‘ideally’ polarized or act as a charge-transfer electrode, as discussed above. Normally the electrodes, under the conditions used, fall between the two extremes. The moment at which oxidation and/or reduction starts depends on various factors: the roughness of the electrode surface, the composition of the electrolyte and the configuration and material of the electrodes. Minor effects are the temperature, the pretreatment of the electrode surface, the pressure and the current density. All of these values are determined in our equipment. Data from the literature show that under the conditions used in our equipment, an overpotential of about 70 mV is adequate for thz evolution of hydrogen, while the evolution of oxygen requires at least 700 mV (bright platinum electrodes). In many instances the chloride ion (0.01 N ) is chosen as the mobile ion, but for the evolution of chlorine a higher overpotential is required. For hard electrode material, hgher values for the overpotential can be expected. Of course, difficulties can sometimes be expected if the micro-sensing electrodes are directly in contact with the electrolytes. If, for instance, ions are present that can be oxidized more easily than the proton, e.g., the equilibrium Fe3+=+Fe2+,particularly *If the distance between the measuring electrodes (axially mounted) is decreased, or the microsensing electrodes are very thin, these values increase.

DETECTION SYSTEMS

176

V

f

C

t

I

t

I

I

Fig.6.36. Polarization of the micro-sensing electrode. The potential gradient inside the narrow-bore tube at the position where the microsensing electrode is mounted decreases if these measuring electrodes change from an 'ideal' polarized electrode to a charge-transfer electrode, owing to the neghgible resistance of the platinum electrode. As a result of this effect, the concentration of the electrolyte, again inside the narrow-bore tube at the position where the measuring electrodes are mounted (l),will decrease if the electrode changes its character in order to fulfil the isotachophoretic conditions (2). L = position in the narrow-bore tube; V = increasing potential gradient; c = increasing ionic concentration; Z = centre of the electrode.

obscure results are obtained. Although under normal conditions some hydrogen may be produced at the beginning of the experiment, the evolution stops because the difference in potential between the anodic side of the bipolar sensing electrode and the electrolyte, which is surrounded by this electrode, is insufficiently great to start the evolution of oxygen (provided that no anion is present that can be oxidized more easily than the hydroxyl ion). If a sensing electrode changes, for any reason, into a charge-transfer electrode, the zone length, as actually measured, of the ionic species present in that zone is longer than can normally be expected according to the concentration in the sample. If the micro-sensing electrodes are made of Pt-Ir, Pt, Pd or Au considerable amounts of hydrogen and/or oxygen can be bound in the first two metallic layers of the electrodes. If hydrogen is bound, the impedance of the electrolyte increases, but with oxygen the contact of the electrolyte and the metallic electrode improves, which causes an apparently lower impedance of the same electrolyte. If the overpotential against the formation of oxygen is exceeded, the bipolar electrode always starts to produce both hydrogen and

ADDITIVES TO THE ELECTROLYTES

177

oxygen. The zone boundary, which causes the electrodes to start the production of gas, is mainly recorded with an overshoot (Fig.6.37). Fig.6.37 shows a series of boundary passages. The leading electrolyte consisted of hydrochloric acid (0.01 M), an unbuffered system. The cations themselves were used each time as the terminating ions. The electric current was stabilized at 50 PA. For any ion that is slower than Li' under these conditions, including also the construction of the probe: the evolution of gas is such that it is produced continuously and cuts off the electric current. This happens if, for example, Fe3+ is used as the terminating ion. At the moment the electrodes start to conduct electricity, i.e., when the electrodes change from polarized electrodes to charge-transfer electrodes, the potential gradient over the electrolyte, surrounded by the charge-transfer electrode, decreases immediately. Owing to the electroneutrality principle, the ions in this zone must follow the equally charged ion of the same species in front of it. The only possible way in which the condition can be fulfiled is for the concentration to decrease. The electrodes are slowly coated with a layer of gas, and less of the driving current then passes through the sensing electrodes. The potential gradient adjusts again and so does the concentration. If the potential over the zone considered is not too great, the sensing electrodes change again into polarized electrodes at the moment when the electrode surface is entirely covered with gas. The evolution of gas in this case stops automatically, and the micro-sensing electrodes become protected against the electrode reaction characteristic for this zone. This can be seen in the lithium zone in Fig.6.37. A second effect may result from the micro-sensing electrodes being first covered with more hydrogen. As soon as enough oxygen has been produced, this influence on the electrodes predominates. Electrodes on which oxygen is bound always records the conductivity as a lower value than do electrodes on which hydrogen is bound. Another possible explanation of the overshoot is the following. The more the zone is situated towards the terminating zone, i.e., the smaller the net mobility is, the higher is the temperature and consequently the Joule heat produced by the sum of the direct driving current and the measuring current. The conductivity increases with temperature and so an overshoot may be expected if a boundary passage is measured with a fairly high difference in conductivity with respect to the leading zone, because time is required for warming up the detector. Later experiments showed, however, that this effect is negligible. Later experiments, in the period the conductivity detector has reached its final construction, showed, however, that sometimes an overshoot can still be obtained. Such overshoots were also seen with the W absorption detector if a buffering counter ion was taken, for which the extinction coefficient was a function of pH. The overshoot appeared in those instances when the buffering capacity of the counter ion was not sufficient. Experiments in which no buffer was used showed the effect more clearly. Some overshoots may therefore be ascribed also to the fact that two consecutive moving zones may have a mutual effect on the pH of the zones involved. A lower pH of the first moving zone may decrease the conductivity of the zone following at the front side if the buffer capacity is not sufficient. If n o buffer is used, even the higher pH of the zone moving in the second position may cause a small region of higher conductivity in the zone of lower pH moving ahead of it. Of course all of these effects may (partially) play a role. The change of a polarized electrode to a charge-transfer electrode may also be due to a *The measuring electrode used was rather thick (0.1 mm).

HCI :0.01 M

Li

CS

Mg I

Ca Sr

Ba

K+NH4 Rb

0

Fig.6.37. Step responses of several zones after the leading electrolyte HCl(O.01 M). The electric current was stabilized at 50 p A because rather thick measuring electrodes (0.1 mm) were used. Clearly visible is the decrease in the concentration if a slow terminating ion (Li+)is applied, possibly owing to the change in character of the measuring electrode. The potential gradient over the Li zone was not so great that the analysis was disturbed by the production of too much gas. The overshoot may be also explained by a pH jump, because a non-buffered system is applied (see Chapter 9). If, instead of Li’, Few was chosen, the electric current was cut off.

ADDITIVES TO THE ELECTROLYTES

179

different cause. The potential on the electrodes may be so high that a leak of current to earth results. This leak to earth often causes the micro-sensing electrodes to be coated with a polymer derived from the components of the electrolytes, and this coating is sometimes not easy to remove. Cationic buffers, which mainly bear reactive nitrogen-containing groups, are especially liable to produce these coatings. These coatings are easily recognized by the decay in resolution of the conductivity detector. This effect will be discussed later in section 6.7. If the surface of the bright platinum electrodes is covered with platinum black, the contact with the electrolyte improves but the overpotential against oxidation and reduction is decreased considerably. Hence low current densities must be applied for separation. The adsorption of ions m d uncharged substances can be distinguished in the treatment of isotherms. Accurate measurements need to be carried out, and it appears that, at present, the dependence of capacity-potential curves on the bulk activity of the adsorbate provides the best criterion. The dependence of the standard free energy of adsorption on the electrode potential (or charge) is different for charged and uncharged species. While ionic adsorption (specific) leads to a linear relationship, uncharged particles give a quadratic dependence. The addition of, e.g., Triton X-100 affects the d.c. measurement of the conductivity, of course, more than the a.c. measurement, especially with respect to the electrode reactions that may occur. In order to demonstrate this effect, two isotachopherograms of the separation of oxalic, citric and acetic acids are shown in Fig.6.38. The traces represent the conductivities of successive zones as measured by the a.c. method (curve b) and by the d.c. method (curve a). A mixture of histidine (0.01 M) and lustidine hydrochloride (0.01 M) was used as the leading electrolyte. The terminating electrolyte was glutamic acid (0.01M). The current was stabilized at 40 PA. Because thicker electrodes are used than under normal conditions*, a greater direct driving current would cause the prcduction of gas at the beginning of the experiment. In general, the mobilities of the anions are low in comparison with those (absolute values) of the cations. It can be seen in Fig.6.38 that the potential gradient from the oxalate zone is sufficiently great to start an electrode reaction. Gas may be produced by this electrode reaction and a layer of a ‘histidine polymer’ coating may be deposited on the electrode surface by a combination of both a leak current to earth and the affect of the bipolar electrode. A close look at the two traces shows that the resistance, as measured by the d.c. method, increases in each zone and the conductivity of each zone no longer seems to be constant. The increment is greater in zones that are situated nearer the terminating zone, which can be explained by the higher potentials that exist in these zones. The impedance as measured by the a.c. method is initially not or only slightly influenced. In a long run (30-50 experiments), the resolution of the a s . method of conductivity determination is smaller and effects characteristic of coatings are obtained (see section 6.7). Later experiments showed that a large proportion of the ‘histidine’ coating is rinsed off by simply refilling the narrow-bore tube, contrary to the effect using other buffers such as aniline and pyridine. *In this experiment a thickness of 0.1 mm was used, while the thickness of the electrode material used in other experiments was only 0.01 mm.

DETECTION SYSTEMS

180

R

t

1

t

Fig.6.38. Detection of zone boundaries in isotachophoretic analyses performed by the a.c. method (b) and the d.c. method (a) simultaneously. At the point marked with an asterisk, the micro-sensing electrodes change their behaviour from polarized to charge-transfer electrodes. In this particular instance, gas was produoed. ?he coating of the electrode is more visible in the stepcurves of the d.c. method. The difference in inclination in the trace of the d.c. method in each zone should be noted. 1 = Chloride; 2 = oxalate; 3 = citrate; 4 = acetate; 5 = glutamate.

If stable coatings are obtained for any reason, the electrodes must be cleaned by rinsing with aqua regia for about 10 sec. The effect of the increment in the d.c. trace in Fig.6.38 must be ascribed largely to the evolution of gas. The small layer of gas influences the d.c. method much more than the a.c. method of conductivity determination. 6.6.4. Additives

Components that inhbit electrode reactions are characterized by strong adsorption on the electrode surface. The electrode reactions may be inhibited in two ways: (a) the transport of ions involved in the electrode reactions towards the electrode decreases as a result of an increase in viscosity in the vicinity of these electrodes; (b) the inhibitor, as it is adsorbed, inhibits the reaction by its presence. In general, adsorption on the electrode surface is caused by the interaction of free-electron pairs (e.g., in oxygen, nitrogen and sulphur compounds) or by n-electrons (e.g., in aromatic compounds). Again, two groups can be distinguished; (1) surface-active compounds, including detergents; (2) organic nitrogen or sulphur compounds, commonly used as corrosion inhibitors.

181

ADDITIVES TO THE ELECTROLYTES TABLE 6.5 SURFACE-ACTIVE COMPOUNDS USED IN ISOTACHOPHORETIC ANALYSES Compound Commercial source Structure Triton X-100

(C, H, 0)9- monoisooctylphenol

Ethomene T/20

(C, H, O), -talc amine

Priminox 32 Serdox ZCA-10

(C, H, 01, -tertiary amine (chain length unknown) (C, H, O),,, -C,,-,, amine

Serdox NJADZO Nonic 21 8 Mowi018-88

(C,H, 01, -C,,-,, amine (C, H,O),-,,-tert-dodecylmercaptan (-CH, -CH-), (polyvinyl alcohol)

I

Rohm & Haas, Philadelphia, Pa., U.S.A. Armour Industrial Chem. Co., Chicago, Ill., U.S.A. Rohm & Haas Servo, Delden, The Netherlands Servo Pennsalt Chem. Corp., Philadelphia, Pa., U.S.A. Hoechst, Frankfurt, G.F.R.

OH

u-

\ (polyvinylpyrrolidone)

PVP

-CHPEG 200

CH,

Fluka, Buchs, Switzerland

-

(-CH, -CH, -O-),, (polyethylene oxide, molecular weight 200)

E. Merck, Darmstadt, G.F.R.

Because the additives are to be used in electrophoretic analyses, compounds must be selected that do not take part in the electrophoretic transport (non-ionogenic compounds). Exceptions are those compounds which both inhibit the electrode reaction and can be applied as the buffering counter ions, e.g. pyridine and related compounds. The surfaceactive components studied were not only detergents, but also some soluble polymers. Detergents consist of a polar and an apolar part. The non-ionic part consisted mainly of 8-20 units of ethylene oxide, condensed with units of 8-20 carbon atoms, with or without functional groups. The polar part can be formed by alkylphenols, alkyl alcohols, alkylamines, alkylmercaptans or alkanes. Some possibilities are shown in Table 6.5. Of these additives, Triton X-100 and Mowiol 8-88 are especially useful. All of these compounds were purified on a mixed-bed ion exchanger. The nitrogen and sulphur compounds were expected to be particularly useful, because they tend to show surfaceactive activity* and are used commercially as corrosion inhibitors. However, these compounds adversely affect the analysis, possibly because they take part in the electrophoretic transport. These additives could not inhibit the interaction between the microsensing electrodes and active components present in the electrolytes such as chromate or malonate. In order to give an impression of the effect of the addition of the different additives on the recording and/or separation of the various ions, a test mixture was prepared as *It should be borne in mind that not only the electrode reactions need to be suppressed, but also the electroendosmosis.

182

DETECTION SYSTEMS

described in the legend to Fig.6.15 and all of the separations were carried out in the operational system at pH 6 (operational system prepared with histidine, see Table 12.1). The amount of additive differs enormously from compound t o compound, and the optimal amounts were therefore established in separate series of experiments. The electric direct driving current was stabilized at 80 PA. In Fig.6.39 and later in this chapter, unless otherwise stated, the isotachopherograms were unfortunately obtained from an a.c. measuring circuit that was not as good as those now available; the linearity is shown in Fig.6.20. The a.c. measuring circuit was poor not only with respect to linearity, but also with respect to the insulation towards earth and the RC time. The measuring probe was constructed of 0.05-mm platinum foil (nowadays Pt-Ir of 0.01 mm thickness is used). Later experiments showed that the typical reaction during the passage of chromate and malonate, components of the test mixture, could be suppressed by addition of 0.05% of Mowiol8-88 (polyvinyl alcohol). In order to check separately the influence of additives on electrode processes, currentvoltage characteristics were obtained. The equipment used is shown in Fig.6.40. An improvement in the detection was found when either an a.c. or d.c. method was used for the determination of the conductivity, although clearly a difference in behaviour between these two modes was observed. In order to accentuate this difference, gold was used as the electrode material, because it is known that it can easily be passivated and the influence of possible electrode reactions is greater. Unusual effects were obtained when gold was used. In order to give an impression of the recording of isotachopherograms with a conductivity detector with the sensing electrodes made of gold, the test mixture of anions (Fig.6.1 5), in the operational system with histidine hydrochloride as the leading electrolyte at pH 6 (Table 12.1), was again examined (Fig.6.41). The isotachopherogams in Figs.6.39 and 6.41 show that additives need to be used. In the experiments shown in Fig.6.41, the direct driving current was stabilized at 80 PA: It should be noted that in the trace shown in Fig.6.41 (l), where the impression is given that the conductivity of the zones decreases towards the terminator zone, this isotachopherogram was obtained by using the ax. method of conductivity determination. The simultaneous detection of the conductivity with aid of the d.c. method, as in Fig.6.41(2), shows the normal isotachophoretic tendency, viz., a stepwise increase in the resistance. This difference must be ascribed to the dominating influence on the capacity of a passivated oxide layer of the gold surface of the micro-sensing electrodes. These effects are discussed in more detail in section 6.7. Many other coatings were also tested with these gold electrodes because they easily adhered to gold. Sometimes the coatings were formed automatically by the driving current during the electrophoretic process. Some other unusual effects occurred when surface-active compounds were added. For instance, the amount of a surface-active agent added does not influence the analyses substantially; The concentration of many of them can be varied from 2 t o 0.5% without any recognizable difference in the recording of the zone boundaries. Of course, the material added to the electrolytes must not contain ionic impurities. Another phenomenon that occurs is the ‘memory’ effect. When an analysis was carried out with Nonic 2 18 and the narrow-bore tube was then rinsed carefully, subsequent experiments without the addition of Nonic 218 gave a low resolution. However, when experiments with Mowiol

ADDITIVES TO THE ELECTROLYTES

183

Fig.6.39. Influence of additives on the final recording of the isotachophoretic separation of a test mixture of anions (see Fig.6.15), carried out in the operational system at pH 6 (Table 12.1). 1 = 0.05% Ethomene T/20; 2 = 0.05% Serdox ZCA-10; 3 = 0.05% Serdox NJAD-20; 4 = 0.05% Priminox 32; 5 = 0.05% Triton X-100; 6 = 0.1% polyvinylpyrrolidone; 7 = 0.05% Nonic 218; 8 = 0.1% Mowiol (polyvinyi alcohol). The isotachopherogam shown in the centre represents the analysis of the test mixture of anions after the narrow-bore tube and the detector had been rinsed well with doubledistilled water after a series of experiments carried out with Mowiol, to show the ‘memory effect’ with this surfactant. The resolution disappears again when about ten experiments have been carried out.

were performed, many subsequent analyses could be made without the addition of Mowiol, as Mowiol is difficult to remove. It can therefore be concluded that the adsorption of surface-active components is really important. Other workers have also reported

184

DETECTION SYSTEMS

Fig.6.40. Equipment used to characterize the influence of additives and coatings on the micro-sensing electrode via current-voltage curves. It includes a platinum double electrode, a calomel electrode (S.C.E.) together with a Luggia capillary filled with agar agar (3%)and potassium chloride (30%) and a counter electrode in a separate compartment filled with 0.1 Mpotassium chloride solution, provided with a ceramic filter. AU experiments were carried out in 0.1 Mpotassium chloride solution.

that polyvinyl alcohol (Mowiol) shows little desorption if adsorbed. From our experiments so far, Mowiol proved to be superior to the other additives, even Triton X-100,especially if the effect is studied in long runs. Triton X-100 shows a type of saturation effect after some time, which results in a poor resolution. Some corrosion inhibitors were also tested in two groups of experiments: (1) small amounts were added to the leading electrolyte, e.g., thiourea and benzothiazole; (2) compounds were used as the buffering counter ions, e.g., pyridine and 0-picoline. These compounds also sharpened the pattern, but were not better than Mowiol. When gold electrodes were used, coatings were formed during the analysis that were recognizable in the electrophoretic recording because coated electrodes become sensitive to doubly charged ions (Fig.6.47). As usual, the d.c. method, applied as before, gave a slightly different behaviour. When gold was used as the electrode material, a layer was formed more easily, possibly containing the sulphur component, as it is known that thiourea can easily form such layers. In the experiments in which pyridine and /3-picoline were used as the buffering counter

ADDITIVES TO THE ELECTROLYTES

185

Fig.6.41. Isotachopherograms of the test mixture of anions (Fig.6.15) obtained in the operational system at pH 6 (Table 12.1). The isotachopherograms were derived from a conductimeter (a.c. method) with the micro-sensing electrodes made of gold. 1 = k c . recording with passivated electrodes; 2 = simultaneous detection by the d.c. method; 3 = a.c. recording when no addition of surfactants was made to the leading electrolyte; 4 = a.c. recording with the addition of 0.05% of Nonic 218; 5 = resolution of the a.c. recording increases if experiments lasting several hours were carried out with the addition of 0.05%of Nonic 218; 6 = a s . recording with the addition of 0.1% of Mowiol (polyvinyl alcohol).

186

DETECTION SYSTEMS

ions, the pH of the leading electrolyte was about 6, containing 0.01Nhydrochloric acid (pro analysi grade). The pKa values for pyridine and P-picoline are 5.25 and 5.69, respectively. Analyses of the test mixture of anions (Fig.6.15) were carried out and sharp zones were observed. Unfortunately, W detection cannot be used, because the W absorption of these counter ions is strong between 250 and 300 nm. It must be remembered that the effective mobilities of pyridine and 0-picoline are greater than that of hstidine. This results in the need for longer narrow-bore tubes for the separation of similar mixtures, because mixed zones are formed much more easily as components with a higher effective mobility transport a higher proportion of the electricity. Nevertheless, here also the disturbance in the detection of the chromate zone (electrode reaction) was found to be a function of the driving current, which illustrates further that an electrode reaction indeed occurs, as shown in Fig.6.42 (1-3). The electrode reaction is shown to be a part of the profile finally recorded if chromate is used as the terminating ion. The reaction time for the electrode is therefore of the order of seconds. All analyses, carried out with the 0.05-mm platinum electrodes and the coil, for galvanic separation of the high potential of the micro-sensing electrodes and the low potential of the conductivity-measuring electronics, which are not well insulated, show this typical reaction, but at low pH (e.g., 4) it is less pronounced. If the chromate zone has passed the measuring electrodes, these electrodes are (partially) passivated. All other zones following the chromate zone are measured correctly if the chromate zone instead of the leading electrolyte is taken as a reference. Thus a shift is obtained: all zones before chromate (of the test mixture of anions) are of correct height and all zones after chromate are of correct height. An extra impedance, reversible and stable, arises during the analysis, but if the narrow-bore tube is rinsed after the experiment has been completed, this impedance ‘disappears’ again. If malonic acid is injected as a sample or is used as the terminating ion, while n o chromate zone is created before the malonate, a similar behaviour to that found with the chromate is found, as shown in Fig.6.42 (4 and 5). If both chromate and malonate are present, and the chromate zone may be very small, the typical behaviour of the electrodes can be recognized only during the passage of the chromate zone, as shown in Fig.6.42(6). If we look more closely at the isotachopherograms in Fig.6.42, in which chromate and malonate are used as terminating ions, a typical shape can be seen in both traces. Even sulphate, when used as a terminating ion, shows this behaviour if the recording of the sulphate step is scaled up. The shape has three different and clearly distinguishable parts: an overshoot, a slow decrease and an increase towards a constant value. The following explanation can be put forward. The anionic constituent present in a zone, at the concentration and pH determined by the operating conditions chosen, may be the cause of a change in behaviour from a polarized micro-sensing electrode partially to a charge-transfer electrode, (during the passivation of the electrode by the chromate ion). This always causes an overshoot, because the concentration must decrease if a current is applied to give an electrode reaction in addition to the electrophoretic transport, in order to fulfil the isotachophoretic condition and the mass balance of the buffer. Especially if oxygen is generated, for instance as a result of the electrode reaction (which means passivation of the electrode), the gas diffuses into the metallic structure. We found that passivated electrodes record the conductivity of a particular electrolyte with an apparently

187

ADDITIVES TO THE ELECTROLYTES

R

L 1

2

3

4

5

6

Fig.6.42. The a.c. recording of isotachophoretic zones of chromate and malonate. In spite of the addition of Mowiol (O.l%), electrode reactions of these types could not be prevented. In later experiments, in which the thickness of the micro-sensing electrodes was reduced, and in which Pt-Ir has been used, these electrode reactions disappeared. In traces 1-3, the electrode reaction caused by the chromate ion is shown. The final step height of the glutamate (terminator) is not constant, but depends on the amount of chromate injected into the system. In trace 3, chromate itself was taken as the terminating ion. Similar behaviour was found with malonate (4 and 5 ) . When both chromate and malonate were present ( 6 ) , the typical effect was only found during the passage of the chromate ion. R = Increasing electric resistance; t = time.

lower impedance than non-passivated or even activated electrodes. The full explanation of the trace can be given as follows. Owing to the change in behaviour of the electrode, the isotachophoretic zone is recorded with an overshoot; owing t o passivation, the conductivity of the zone is recorded with an apparently higher value (simultaneously the electrode reaction stops, which means that the real conductivity of the zone between the micro-sensing electrodes increases); the oxygen is adsorbed more strongly to the electrode surface and a new equilibrium is established, which is why the trace shows a different inclination. This inclination is not observed if hydrogen can be produced as a result of an electrode reaction (activation of the measuring electrodes). During an isotachophoretic separation and recording by means of the a.c. method, these effects are often difficult to observe, because the zone length is often too small. In

DETECTION SYSTEMS

188

order to prove that electrode reactions of all types influence the recording of the conductivity, a leak current (lo4 A) was created artificially. The leak current is small compared with the direct driving current (lo4 A). The result is shown in Fig.6.43. Again the test mixture of anions (Fig.6.15) was separated in the operational system at pH 6 (Table 12. I). This isotachopherogram shows the separation as recorded if a leak current towards earth is permitted. The isotachopherogram was obtained with the linearized a.c. conductimeter as described in this chapter. The W detector was mounted after the conductimeter in this instance in order to check if the concentrations of the zones had really changed or not. Fig.6.43 shows that good construction and insulation of the conductimeter probe are necessary. That the material of which the equipment is made plays an important role in the resolution of the detector proves the following. When experiments were carried out in

I"

Fig.6.43. Isotachopherograms of the test mixture of anions (Fig.6.15) in the operational system at pH 6 (Table 12.1). This figure shows the disturbance of conductimetric detection (a.c. method) if leak currents towards earth are not prevented. The UV trace is given for comparison of the resolution. In the experiment shown,a leak current towards earth of 10- A was created artificially. The isotachopherogram is difficult to interpret, although from later experiments we know that, owing to the leak current towards earth, a coating is deposited on the micro-sensing electrodes (the electrodes are sensitive to the presence of doubly charged ions). R = Increasing electric resistance; A = increasing UV absorbance; t = time.

ADDITIVES TO THE ELECTROLYTES

189

narrow-bore tubes made of PTFE in combination with a conductivity detector made of Perspex, sharp isotachopherograms were obtained only when a surfactant was added (Fig.6.44). When the UV detector was used it was also found that an additive is necessary, as can be seen in Fig.6.44. When the conductivity detector was made of TPX, the additives showed much less influence on the detection with the conductivity detector. Now, the wetting capacity of

Fig.6.44. Resolution in the absence (below) and presence (above) of surfactants. When no surfactants were added to the electrolytes, the conductimetric detection had poor resolution. This fgure also shows that additives need to be added when a UV detector is used. This proves that the electroendosmotic profite is reduced by the addition of a surfactant, which increases the viscosity in the vicinity of the wall. Sample: test mixture (Fig.6.15).

190

DETECTION SYSTEMS

TPX was found to be very poor, even if surfactants in high concentrations were applied. Experiments in which the TPX was 'coated' with a small layer of silicone oil showed improved resolution, which means that the TPX itself makes a large contribution to the electroendosmotic flow, which is difficult to suppress. TPX also show a poorer performance in methanol compared with Perspex, although TPX must be used because Perspex is affected in a long run. No additives have so far been found that can be added to methanolic electrolytes to give improved resolution. The inhibition by the surfactants of electrode reactions, if the thickness of the electrodes is too great or the insulation towards earth is not sufficiently suppressed, is poor. Therefore, additives still need to be added for this purpose, or the electrode reactions must be prevented in another way: low current densities and thin electrodes of hard material (Pt-Ir). A disadvantage of most of the additives that are suitable for this purpose is that most of them show U V absorption, which can be neglected if they need to be added only in trace amounts, but they cannot be used as counter ions. No observable difference in the current-voltage curve could be found if trace amounts of surfactants were applied (even high concentrations gave a smaller effect than expected, as shown in Fig.6.45. This aspect, however, will be discussed in more detail in section 6.7.

Fig.6.45. Current-voltage curve measured with the equipment shown in Fig.6.40. (a) Bright platinum electrodes with the addition of 2%of Mowiol (polyvinyl alcohol). (b) Bright platinum electrodes with no addition of surfactant. The curves were measured in a 0.1 Mpotassium chloride solution. They show that the surfactant must reduce the electroendosmotic profile (compare the results shown in Fig.6.44); the effect on the electrode reactions is small.

COATING OF THE MICRO-SENSING ELECTRODES

191

6.7. COATING OF THE MICRO-SENSING ELECTRODES 6.7.1. Introduction

The second means of preventing electrode processes is to apply a polymer coating to the micro-sensing electrodes. The main problem is to find a method of coating that gives a uniform layer. Two different methods were tested: (1) the electrophoretic coating process and (2) the electrolytic coating process. In the electrophoretic process for preparing coatings of polystyrene, acrylic and epoxy resins, both water and methanol were used as solvents. The platinum metal is probably not suitable for these coatings, and it is known that the metal plays a very important role in the coating process. In the electrolytic process, the electrode metal is less important, and therefore only electrolytic coatings are considered below.

6.7.2. Experimental The anodic polymerization of aromatic amines was particularly successful. The more aromatic rings present in the compound, provided that a sufficient amount could be dissolved, the more stable the coatings were found to be. 1-Aminonaphthalene dissolved in ethanol (saturated solution at room temperature) was employed in several experiments. A few drops of this solution were added to 10 ml of 1M potassium chloride solution and water was then added to give a total volume of 100 ml. The solution was filtered in order to remove undissolved 1-aminonaphthalene. The filtrate was approximately 0.01 M i n the aromatic amine. During anodic oxidation at 700 mV, a violet-coloured layer was formed. The electric current was maintained at 0.1 mA for 5 min and an increase in the electrode potential up to 2000 mV was obtained; the cell constant increased from 0.68 to 2.5 cm-' . The layer formed was cathodically very stable; even after drying and heating (lOO°C), the quality of the electrode improved. While the results with the coating of 1-aminonaphthalene were satisfactory, the results with 1-aminoanthracene were even better. The colour of the coating layer was yellow, and the cell constant increased from 0.68 to 3.5 cm-'. For these electrodes, current-voltage curves were obtained in order to characterize this quality. However, as it is difficult to estimate the thickness of the layers, experiments in the isotachophoretic equipment were still carried out. A conductivity measuring probe was used in which the micro-sensing electrodes were mounted axially as shown in Fig.6.12. In order to discriminate between electrodes, only a selection of thin and thick coatings was examined, depending mainly on the electric current applied during the coating procedure. The isotachopherograms obtained with the a.c. method of conductivity determination were unusual for the separation of both anions and cations. In order to demonstrate the difference between the a.c. and d.c. methods, the influence of different coatings and the influence of changing the frequency of the measuring current and the

192

DETECTION SYSTEMS

Fig.6.46. Effect of a coating deposited on the micro-sensing electrodes on the final recording of the isotachophoretic separation of the test mixture of anions (Fig.6.15) in the operational system at pH 6 (Table 12.1). 1 = A.c. method (4 kHz) with a phenol coating; 2 = a.c. method (4 kHz) with a coating formed by ‘Kolbe electrolysis’ 3 = a.c. method (4 kHz) with a thin coating of 1-aminoanthracene; 4 = a.c. method (1 kHz) with a thick coating of 1-aminoanthracene; 5 = a.c. method (4 kHz) with a thick coating of 1-aminoanthracene. In the analysis shown in 5 , the same coating as in 4 was used but the frequency of the measuring current was altered. Traces 6 and 7 are simultaneously recorded isotachopherograms obtained by the d.c. method, corresponding to traces 5 and 1, respectively. No surfactants were added to the electrolytes.

DETECTION LIMITS

193

electric driving current, a series of experiments was performed with the test mixture of anions as described in Fig.6.15. Also in this series of experiments, a non-linear a.c. conductimeter was used. The operational system at pH 6 (Table 12.1) was chosen and the direct driving current was stabilized at 80 PA, unless mentioned otherwise in the figure captions. In Fig.6.46, a series of isotachopherograms are shown, which indicate that the a x . method gives unusual results for the test mixture of anions before the coating. The sensitivity (selectivity) of the combination of the a.c. method with coated electrodes for the doubly charged sulphate ion was such that the following zone of chlorate was measured with a negative step, which suggests that this zone has a higher conductivity than the preceding zone. This is in contradiction to the isotachophoretic principle, if these ions are involved. When the thickness of the coating layer was increased, this effect also increased, as shown in Fig.6.46 (3 and 4). An increase in frequency of the measuring current also demonstrates this effect. Even the acetate-adipate transition shown in Fig.6.46 (5) is recorded with a negative step. In Fig.6.46 (3 and 4), the acetate-adipate transition was recorded with a smaller difference than under normal conditions (without a coating). The difference between the simultaneous detection by the a.c. and d.c. methods of determination of the conductivity, as already found in some instances when passivated gold electrodes were applied, must be ascribed to the change in capacity of the conductivity cell. In all experiments, the simultaneously performed d.c. method of conductivity detection showed a normal isotachophoretic pattern, as does UV detection. Similar behaviour was found if cations were separated, as can be seen in Fig.6.47. Hence the coating layer is only selective for the difference between singly and doubly charged ions. If, during an isotachophoretic run, a coating is deposited on the micro-sensing electrodes by an electrode reaction due t o a leak current or a change in the nature of the sensing electrode from polarized to charge transfer due to the driving potential, similar effects can be expected. The effect occurs especially when this coating layer is formed very slowly after a series of experiments, even if the most stringent precautions are taken. Cleaning must therefore be carried out from time to time. That a coating is formed more quickly if a high current density of the driving current is applied is shown in Fig.6.48. Owing to the higher potential gradient, the electrode reactions typical of the chromate zone are a function of the driving current. Also, the ratio of step heights is changed more quickly than under normal conditions, for which a change in the ratio of step heights could not be observed.

6.8. DETECTION LIMITS 6.8.1. Introduction

Although i t is somewhat premature at the present stage of isotachophoretic development, brief information will be given on detection limits in isotachophoretic experiments. More research aimed at optimizing detectors, equipment and Operational systems will

194

DETECTION SYSTEMS

Fig.6.47. Influence of a coating deposited on the micro-sensing electrodes on the final recording of the isotachophoretic separation of the cations. Ba2+,Ca'+, Na', Cd'+ and ( C , H, l4 N+ in the operational system at pH 5.39. K+ (0.01 N) was used as the leading ion, acetate was the counter ion and Tris' was the terminating ion. The electric current was stabilized at 80 PA. 1 = No coating; 2 = with a thin coating of 1-arninoanthracene (4 kHz); 3 = with a thick coating of 1-aminoanthracene (4 kHz).

certainly improve the detection limits in the future. Particularly when micro-scale preparative equipment becomes available it will be possible to combine various specific detection techniques with isotachophoretic equipment. Because isotachophoresis is still in the development phase, it is impossible to determine the ultimate detection limit of this technique.

DETECTION LIMITS

195

Fig.6.48. Influence of the current density of the driving current on the final recording of the test mixture of anions (Fig.6.15) isotachophoretically separated in the operational system at pH 6 (Table 12.1). An addition of 0.05%of Nonic 218 was made to the leading electrolyte. The recording was made by the a.c. method (4 kHz): 1 = 40 PA; 2 = 80 PA; 3 = 150 MA.

The following advances will give improvements in detection limits: if it will be possible to reduce the diameter of the narrow-bore tube and the electroendosmotic flow can be suppressed efficiently, the transitions of the zones will become smaller, which will improve the sensitivity of the method (see Appendix B); and if the sensitivity (or selectivity) of the detectors can be increased, smaller zones of ionic material at lower concentrations can be recorded. Of course, some of these aspects overlap.

196

DETECTION SYSTEMS

Apart from the above areas of development of the method, we should consider the following. (1) Are the chemicals available pure enough? (2) Is the operational system well chosen, Le., such that the proportion of the electric current carried by the buffering counter ion is small and the buffering capacity large enough, and is the solvent well chosen? (3) Is the detector sensitive enough? An example of t h s is the isotachopherogram in Fig.6.32, where an enrichment of salicylic acid is shown by the W detector, whereas the conductivity detector gives an ‘ideal’ mixed zone. (4) Is the time of analysis well chosen? If, for instance, the difference in the effective mobilities of a given pair of ions is small, a longer narrow-bore tube must be applied. If the difference in effective mobility is critical, the counter flow of electrolyte will not give relief (this aspect is discussed in Chapter 7). (5) Has the equipment for a special application been constructed well? Convincing evidence that demonstrates the variations in detection limits that can be obtained is provided by the difference in resolution attained by thermometric, conductivity and W detectors. A low-resolution detector gives no information about the real detection limits of the isotachophoretic separation process. The use of electrolytes at low concentration limits the choice of pH and hence the operational systems to be used, because of the increasing influence of O H and H ions on the electrophoretic separation procedure. An increasing eluting effect will be the result and zone electrophoretic effects can be expected. This means also that theoretically isotachophoresis is impossible. However, other solvents may be explored, which alter these limits. In the following discussion, some information is given on detection limits in isotachophoretic separations carried out on the equipment developed in our laboratory with commercially available chemicals, although purification was necessary, especially in analyses at low concentrations. This aspect is dealt with in more detail in Chapter 10. The detection limits in thermometric detection are discussed in section 6.2; they provide no information on the detection limits of the isotachophoretic process. Special attention is paid to UV and conductivity detectors with the electrodes mounted equiplanar (0.01 mm, Pt-Ir) in direct contact with the electrolyte inside the narrow-bore tube in combination with the linearized electronic measuring circuit as considered in this chapter. For the W detector, a round slit of 0.3 mm diameter was used. 6.8.2. Experimental

In order to find the lower detection limit, a series of experiments was carried out with ADP, because its ion can be detected by both W and conductivity detectors. Moreover, it was found to be very pure and dissolved easily compared with other strongly Wabsorbing compounds available in our laboratory. The effect of the direct driving current, temperature and the concentration of the leading electrolyte were studied. The experiments were carried out at pH 4 (see the operational system at pH 4.5, Table 12.5), and some results are given in Table 6.6. All of the data given are average values from three separate experiments. The analyses were carried out for each concentration range of two batches of electrolytes. The minimal amount needed for detection by the W detector was found to be about the same as that in the a s . method of conductivity determination, although if two

197

DETECTION LIMITS TABLE 6.6 SURVEY OF THE MINIMAL AMOUNTS THAT CAN BE DETECTED BY HIGH-RESOLUTION UV AND CONDUCTIVITY DETECTORS

CLE= Concentration of the leading anion (M); Quv = minimal amount of ADP that can be detected by the UV detector (pmoles); Qa.,. = minimal amount of ADP that can be detected by the a.c. detector (pmoles); QKv = minimal amount of ADP necessary for qualitative and quantitative detection by a UV detector (pmoles); and Qf,. = minimal amount of ADP necessary for qualitative and quantitative detection by an a.c. detector (pmoles). The injected volume was 1 pl. By using the dilution method (Fig.6.33), the UV detection limit can be further decreased. CLE

0.01 0.005 0.001 0.0005

25 20 15 5

25 20 15 10

150 130 100

so

150 130 120 100

W-absorbing species were present the resolution was lower (Fig.6.49). This effect may be due to the fact that a longer zone boundary is detected due to the parabolic profile of the zones and the fact that the fan-shaped field lines of the a.c. detector are less than 0.3 mm (the diameter of the slit). Table 6.6 shows that the minimal amount that can be detected by diluting the concentration of the leading electrolyte is far less than expected. Dilution by a factor of 10 decreases the limit by a factor of only about 2. The reason must be ascribed t o the poor development of the profile in the low concentration of electrolytes, because the electroendosmotic flow cannot be suppressed sufficiently, and to the increase in the electrophoretic transport by the more mobile ions (impurities, H?,O H ) . Changes in the temperature of the thermostated narrow-bore tube have only a slight effect, although a drastic change (from 20 to 4°C) occasionally decreases the separating capacity by about 50%. This is to be expected because ionic mobilities increase by 2-3% per 1°C change in temperature. T h s means that the differences between two ions that are difficult to separate change in a similar way, although the effective separation length increases. The separating capacity was measured by comparing the times of analysis of the standard mixture of anions (Fig.6.15), as follows. At 25"C,an amount of the standard mixture was injected just such that no mixed zones were obtained. The mixed zones are found especially in the beginning of the isotachopherogram, for zones at the rear always have a longer time of separation compared with the zones at the front because the detector is mounted at a fixed position. If the temperature is decreased, mixed zones soon appear. The extra time required for complete separation, if a counter flow of electrolyte is used, is taken as a measure of the separating capacity. Of course, one has to take account of the fact that the counter flow of electrolyte always disturbs the profiles and that the difference in effective mobility between the various ions is affected in a negative way by the counter flow. This was checked by observing the profiles of dyes moving in the narrow-bore tube with and without a counter flow of electrolyte. The influence of temperature on the pK values of the counter ion and the sample ions was not taken into account. The influence of the diffusion constant is negligible because the diffusion constant is directly proportional to the absolute temperature.

DETECTION SYSTEMS

198

t

I

t.

Fig.6.49. Isotachopherogram in the operational system at pH 4, with HCI (0.01 N) and e-aminocaproic acid as the counter ion. The terminating ion was glutamate. While the UV detector (below) indicates only one of the two UV-absorbing components injected, the conductimeter (above) shows the separation of the two components. In the trace derived from the conductimeter, a further component is determined, which is an impurity from the electrolytic system. The current was stabilized at 80 PA. R = Increasing electric resistance;A = increasing UV absorption.

The influence of variations in the direct driving current were small in the range studied. In order to prevent the already discussed electrode reactions, this driving current has to be limited to 150 PA. However, experiments at 300 pA showed that the standard mixture of anions was separated in a few minutes, although the resolution decreased, The main reason for this effect must be that high current densities increase the radialnon-uniformity of the temperature profile inside the narrow-bore tube. This causes an increased parabolic profile, especially for the zones that are situated towards the rear side. Also, the electroendosmotic profile increases. The use of the dilution method of concentration determination improves the resolution of the UV detector (Fig.6.33) about 50-fold. This factor depends on the molar extinction coefficients of the ionic species involved. The disadvantage of this method of detection, as can be seen in the determination of salicylic acid in Fig.6.32, is that a small change in the pH of the leading electrolyte may change the effective mobilities between the two ions forming the mixed zone in such a way that an enrichment of one of the components is soon detectable in the UV trace*. This sometimes makes an accurate determination of the step height in the UV trace (quantitative determination) less accurate. If a W-absorbing ion can be sandwiched between two non-W-absorbing ions, minimal amounts of the UV-absorbing ion can be measured quantitatively because one can make use of the parabolic profile of the consecutive zones. The W-absorbing ion can be determined, in spite of its small zone length (e.g., less than 0.01 mm), because the *A similar effect can be expected if the pK, values of the components involved differ much.

199

CONCLUSION

average length of a parabolic profile is commonly about 0.4 mm. This is shown later in Fig. 17.3, in which the profiles are visible because coloured ions are used. Special calibration graphs must be prepared for each ionic species. When using equipment in which high-resolution detectors are mounted, for compounds present in the range from micromoles t o nanomoles (average molecular weight loo), full qualitative and quantitative results can be obtained; at the picomole level or even lower, in special cases quantitative results can be obtained, as is discussed above.

6.9. CONCLUSION The choice of the method of detection, especially in analytical isotachophoresis, must be carefully considered. All of the types of detectors discussed in this chapter are not always needed, and for many purposes only one type of detector (specific or universal, with low or high resolution) is sufficient. Especially if one is interested only in the amount and/or quality of a single component, a detector of very simple performance can be chosen*. The choice of the method(s) of detection determines to a great extent the final construction of the equipment, but also makes demands on the purity of the chemicals used in the various operational systems. To compare the various method of detection, Figs.6.50 and 6.51 can be considered. In Fig.6.50, three isotachopherograms of the test mixture of anions (Fig.6.15), in the

T

R

-

a

b

'

c

Fig.6.50. Isotachophoretic separation of the test mixture of anions (Fig.6.15) in the operational system histidine/histidine hydrochloride at pH 6 (Table 12.1). Detector used: (a) d.c.; (b) non-linear a.c.; (c) thermometric. Speed of the recorder chart paper: (a) and (b) 2 cmlmin; (c) 5 mm/min. Average time of analysis: (a) and (b) 15 min; (c) 45 min. The linear traces should be noted. R = Increasing electric reisstance; T = increasing temperature; f = time. *Moreover, the use of thin-wall narrow-bore tubes with a small I.D. (e.g., 0.2 mm) improves the resolution (Appendix B).

200

DETECTION SYSTEMS

f.

11L

1 ’

1’

Fig.6.51. Isotachophoretic separation of the test mixture of anions (Fig.6.15) in the operational system histidinelhistidine hydrochloride at pH 6 (Table 12.1). Conductimetric detection was carried out with a linear conductimeter. The UV trace was derived from a UV absorption detector (not chopped) at 256 nm. The speed of the recorder chart paper was 6 cm/min and the time of analysis was 12 min. The electric current was stabilized at 70 PA. R = increasing electric resistance; A = increasing UV absorption; t = time. 1 = Chloride; 2 = sulphate; 3 = chlorate 4 = chromate; 5 = malonate; 6 = pyrazole-3,5-dicarboxylate; 7 = adipate; 8 = acetate; 9: p-chloropropionate; 10 = glutamate; X = impurity, possibly propionate (a degradation product of p-chloropropionic acid).

operational system at pH 6 (Table 12.1), are shown. The traces of the linear signals have been equalized photographically in order that a valid comparison of the results can be made. The recording of the final sharpness of the zone boundaries and the difference in the time of analysis with a high-resolution and a low-resolution detector can clearly be

REFERENCES

201

seen. Again i t is clear that in order to observe a sample zone with a thermometric detector the zone length must be greater (Table 6.2) than if a high-resolution detector is applied (UV, conductimeter). Hence more sample has been introduced into the system and consequently a longer time of analysis is needed. In Fig.6.51, an analysis under conditions identical with those in Fig.6.50 is shown, recording being effected with a linearized conductimeter (Fig.6.18) and a W-absorption detector (256 nm) (Fig.6.26). It should be pointed out that the various isotachopherograms shown in this chapter are given mainly for pattern recognition, in order to show different effects such as sharpness of the profiles, electrode reactions and impurities. It is difficult to compare one isotachopherogram with another, although a single test mixture of anions was used. In preparing the manuscript, the various isotachopherograms were treated photographically in order to make comparisons simpler. In the Section Applications (Chapters 8-17) the time axis is also given on the relevant isotachopherograms, so that the qualitative and quantitative information can be deduced more easily. Although electrode reactions may sometimes still occur during detection with a conductimeter with the electrodes in direct contact with the electrolyte, its resolution is lugh compared with that of other types of universal detectors. Moreover, possible coating of the electrode can easily be observed by using a combination of an a.c. and d.c. detector (see also Fig.8.1.). We recommend that the entire system should be cleaned from time to time with a non-ionic surfactant, which can be purified by running it through a mixedbed ion exchanger, because all types of material may be adsorbed on walls made of Perspex, TPX, Pt or even PTFE. When adsorbed, these impurities change the {-potential and hence the electroendosmosis, and thus the resolution of both conductivity and UV absorption detectors is decreased. For effective rinsing of the electrophoretic equipment, we recommend the surfactant Extran (Merck, Darmstadt, G.F.R).

REFERENCES 1 F.M. Everaerts, Thesis, University of Technology, Eindhoven, 1968. 2 F.M. Everaerts and Th.P.E.M. Verheggen,J. Chromatogr., 53 (1970) 315. 3 F.M. Everaerts and Th.P.E.M. Verheggen, in P.G. Righetti (Editor), Progress in Isoelectric Focusing and Zsotachophoresis, North-Holland, Amsterdam, and Elsevier, New York, 1975, p. 309. 4 F.M. Everaerts, J.L. Beckers and Th.P.E.M. Verheggen, Ann. N Y . Acad. Sci., 209 (1973) 419. 5 F.M. Everaerts, Graduation Rep., University of Technology, Eindhoven, 1964. 6 F.M. Everaerts and Th.P.E.M. Verheggen, J. Chromatog., 91 (1974) 837. 7 F.M. Everaerts and P.J. Rommers,J. Chromatogr., 91 (1974) 809. 8 F.M. Everaerts and Th.P.E.M. Verheggen,J. Chromatogr., 73 (1972) 193. 9 F.M. Everaerts, internal report, University of Technology, Eindhoven, 972. 10 A. Vestermark and B. Sjodin, J. Chromatogr., 71 (1972) 588. 11 L. Arlinger and H. Lundin, Protides Biol. Fluids, Proc. Colloq., 21 (1973) 667. 12 A.J.P. Martin and F.M. Everaerts, Anal. Chim.A c ~ Q38 , (1967) 233. 1 3 A.J.P. Martin and F.M. Everaerts, Proc. Roy. SOC.,Ser. A , 316 (1970) 493. 14 B.P. Konstantinov and O.V. Oshurkova, Sov. Phys.-Tech. Phys., 37 (1967) 1745. 15 I. Vacik, J. Zuska, F.M. Everaerts and Th.P.E.M. Verheggen, Chem. Listy, 66 (1972) 545. 16 F.M. Everaerts, J . Vacik, Th.P.E.M. Verheggen and J. Zuska, J. Chromatogr., 49 (1970) 262. 17 M. Coxon and M.J. Binder,J. Chromatogr., 101 (1974) 1.

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DETECTION SYSTEMS

Publication No. 556f424,AGA, Infrared Instruments Department, Lidingo, Sweden, 1974. J.L. Fergason, Sci. Amer., 231 (1974) 76. J.L. Beckers, Thesis, University of Technology, Eindhoven, 1973. M. Demjanenko, J. Vacik and .I. Zuska, Chem Listy, in press. Th.P.E.M. Verheggen, E.C. van Ballegooijen, C.H.Massen and F.M. Everaerts, J. Chromatogr., 64 (1972) 185. 23 D.I. Shernoff, Rev. Sci. Instrum, 40 (1969) 1418.

18 19 20 21 22

Chapter 7

Instrumentation SUMMARY This chapter is devoted to the electrophoretic equipment developed for isotachophoretic analyses. Many classifications can be considered but, of the series of equipment resulting from the development of the instrumentation, only three clearly distinguishable types have been selected. Because the various instruments generally are combinations of injection systems, counter electrode compartments and detectors, these components are considered insofar as they were not discussed in Chapter 6. Special attention is paid to the counter flow of electrolyte during isotachophoretic analyses. In particular, the optimal regulation of this counter flow of electrolyte and simple and accurate means by which it can be achieved are considered.

7.1. INTRODUCTION The design of isotachophoretic instruments, for analytical purposes is determined mainly by the detection systems used. The stabilizing effect of narrow-bore tubes makes the use of stabilizing agents, e.g., polyacrylamide, agar agar, arrowroot and dextran, unnecessary. The shape of the narrow-bore tube, cylindrical or flat, has some influence (Appendix B). So far, only narrow-bore tubes made of Pyrex glass, quartz glass, Perspex (acrylic) and PTFE have been tested. In this chapter, most attention is paid to the injection system, the counter flow compartment next to the compartment with the semi-permeable membrane, and the means by which a counter flow of electrolyte can be achieved. Some instruments as constructed by Verheggen and Everaerts and used in our laboratory are discussed.

7.2. INJECTION SYSTEMS 7.2.1. Introduction The method of introducing a sample into equipment for isotachophoretic analyses may have a great influence on the time of analysis and even the separation of the ionic species involved. The sample can be introduced sandwiched between the leading and terminating electrolytes with aid of a sample tap, in which case the ionic species are separated from the mobile leading ion and the less mobile terminating ion. The influence of both the leading and terminating ions is minimal and also the pH of both electrolytes has virtually no influence in the initial phase. The amount of sample, however, can be changed only by

203

204

INSTRUMENTATION

varying the concentration of the sample or by inserting a small piece of insulating material in the bore of the tap. The latter procedure is very complicated. Introduction of the sample with aid of a syringe seems to be the most commonly used technique, because the sample size can be vaned quickly and usually a smaller amount of sample is required. However, if the sample is introduced in the leading electrolyte, mixed zones can be expected between the leading ion and the fastest moving ion of the sample. If the sample is injected in the terminating electrolyte, ionic species with a low pK value (cationic separation) or a high pK value (anionic separation) can be retarded so much that considerable amounts of these ionic species can be missed or even lost. Reproducible quantitative results can hardly be expected. Particularly if experiments are carried out at low concentrations (0.001 N ) , special care must be taken in selecting the concentration and the pH of the terminating electrolyte. If the sample is mixed with a terminating electrolyte that has too high a concentration or an incorrect pH, both the qualitative and quantitative results will be poor. In addition, the influence of impurities in the electrolyte may play an important role, but this is not influenced by the method of sample introduction. More attention is devoted to this aspect in the Section Applications. Of course, if a syringe is used for sample introduction, some of the sample will always be mixed with the leading and terminating electrolytes if the sample is introduced at the boundary between these electrolytes, as this boundary is never well defined. 7.2.2. Four-way tap The principle of the four-way tap is shown in Fig.7.1. The mechanism is shown in four alternative positions. In position 1 the narrowbore tube is rinsed and can be filed with the leading electrolyte, in position 2 the terminating electrolyte can be introduced into the reservoir for the terminating electrolyte, in position 3 the sample tap can be rinsed and filled with the sample and in position 4 the sample is sandwiched between the leading electrolyte and the terminating electrolyte. The analysis can be performed with the tap in position 4, in which case the connections must fit exactly, because no dead volumes can be allowed (gas bubbles may stick to these connections and if the dead volume is located between the narrow bore and the sample tap the time of analysis is adversely influenced). The other connections are not important in this respect, because they are used only for rinsing and filling the various compartments of the electrophoretic equipment . The tap initially applied by us was made of Pyrex glass, although any other insulating material can be used. A combination of Kel-F and Arnite can be particularly recommended. The average volume of the tap applied by us was 20-100 pl. The volume of the tap was sometimes changed by inserting a piece of insulating material, but this procedure proved to be very complicated if good qualitative and quantitative results were to be obtained.

205

INJECTION SYSTEMS

II!

n I Fig.7.1. Principle of the four-way tap for rinsing and re-filling the electrophoretic equipment and for sample introduction. Position 1: the narrow-bore tube can be rinsed and re-fiied. Position 2: the reservoir with terminating electrolyte can be rinsed and re-fiied. Position 3 : the sample can be introduced. Position 4: the analysis can be performed.

7.2.3. Six-way valve Fig.7.2 shows the way a six-way valve is used, while Fig.7.3 shows an exploded view of this valve in order to demonstrate its construction. The conical plunger is made of Amite, while the plunger housing is made of Kel-F. This plunger housing is surrounded by a brass hexagon for mechanical stability. Moreover, this hexagon prevents any shift of the plunger and the plunger housing due to the weakness of the Kel-F and the forces on the plunger so that a liquid-tight connection is obtained. Holes are drilled in the brass hexagon for connection of the various components via the holes drilled in the plunger housing and the plunger. In each of these holes in the hexagon, a threaded base is soldered, so that with screw-caps and collars liquid-tight connections can be made, as shown in Fig.7.4. It does not need further explanation that the liquid inside the various bores may not have any electrical contact with the brass hexagon. By means of the special construction shown in Fig.7.3 (parts 5 and 6), the six-way valve can be turned only through 60°, so that the three canals inside the plunger are always connected with the bores inside the plunger housing. The connections with the narrow-bore tube and the piece of insulating material that provides the connection with the injection block or directly with the reservoir filled with the terminating electrolyte must fit exactly, otherwise a dead volume will occur that will decrease the effective length for separation enormously.

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INSTRUMENTATION

4 A

6

Fig.7.2. Principle of the six-way valve used for rinsing and re-filling the isotachophoretic equipment and sample introduction (J. Vacik, Prague, private communication). In position A, the narrow-bore tube is rinsed via an open Hamilton valve (1MM1)at the side of the counter electrode compartment; (3) is the connection towards drain. The sample is introduced via the syringe ( S ) , while (2) again is connected with the drain. The reservoir of the terminating electrolyte can be rinsed via (6); (1) is the reservoir for the terminating electrolyte. In position B, the valve is shown in the ‘running’ position.

By varying the central bore inside the plunger, the volume to be injected can be varied. The tap constructed in our laboratory had a volume of 5 pl. The narrow-bore tube is connected with the six-way valve without the use of any adhesive, using a piece of insulating material that has an outside diameter such that it fits exactly in a chamber made for it in the plunger housing. The length of tlus piece of insulating material is about 2 cm. In order to make a liquid-tight connection with the piece of insulating material, it must be smooth and flat on top. Moreover, an extra small O-ring made of rubber is mounted on top of this cylinder of insulating material. In the cylinder, a hole is drilled with a diameter equal to the outside diameter of the narrowbore tube in which the analyses are performed. The narrow-bore tube that is to be mounted is first stretched over a length of about 4 cm t o enable it t o penetrate the cylinder of insulating material via the central bore. The narrowbore tube is then pulled through this central bore until it fits exactly. After allowing for shrinking (this piece of insulating material with the narrow-bore tube is inserted in hot water), the narrow-bore tube is cut with a lancet. The cylinder of insulating material with the narrow-bore tube can be connected to the plunger housing by fitting a screw-cap over the threaded base. A water pressure of at least 7 atm can be applied without any visible leakage at this clamping piece. However, a pressure n o higher than 6 atm could be applied, because at this pressure the narrow-bore tube shows its porosity and droplets appear all over it. The pressures applied for rinsing and re-filling are, of course, much lower. The connection of the six-way valve with the injection block (or directly with the reservoir filled with terminating electrolyte) is achieved with a cylinder of insulating material with

INJECTION SYSTEMS

207

Fig.7.3. Exploded view of the six-way valve. 1 = Brass hexagon with screw-caps for connection of the various parts liquid-tight to the plunger housing (2), made of Kel-F; 3 = pins for locking the plunger housing in the brass hexagon (1);4 = Arnite plunger with three pardel canals; 5 = stainless-steel screw-cap provided with a ridge for the exact determination of the position of the plunger in the plunger housing, in combination with component (6); the plunger can be switched through 60"; 7 = handle; 8 = narrow-bore tube. The clamping device of the nanowbore tube, provided with a small O-ring (see section 7.2.3.), should be noted.

a bore of 1 mm. This cylinder of insulating material has two collars, and on both ends it is flat and provided with two small rubber O-rings. Again, liquid-tight connections can be made with screw-caps. In practice, the tap is very reproducible in sampling, especially when various people use the instrument. For the use of syringes, more ability is needed. It need not be explained that a shorter length of narrow-bore tube is needed for separation, because the sample is not mixed with the leading and terminating electrolytes. This six-way valve was very useful particularly when experiments were carried out in which the position of the sample is important, e.g., zone electrophoresis in narrow-bore

INSTRUMENTATION

208

/

\

2

6

\

A

Fig.7.4. Cross-section of the six-way valve in the ‘running’ position. 1 = Connection towards the reservoir of the terminating electrolyte; 2 = connection towards drain; 3 = connection towards drain; 4 = narrow-bore tube in which the separation is performed; 5 = position where the syringe filed with sample can be mounted; 6 = position where the syringe fiied with terminating electrolyte can be mounted. Materials: a = brass; b = Amite; c = Kel-F; d = Kel-F.

tubes or movingboundary experiments. It can also be recommended for automation purposes. 7.2.4. Injection block

A method for sample introduction with a micro-syringe is demonstrated in Fig.7.5, and a photograph of the injection block is shown in Fig.7.6. . The leading electrolyte can be introduced via an open tap at the side of the counterelectrode compartment, not shown in the figure (see Fig.7.9). The tap between the injection block and the connection towards drain (4) is opened during this procedure, while tap (2) is closed. The tap at the side of the counter flow compartment is then closed and tap (2) is opened. The terminating electrolyte can now flow towards drain. In general, no suction need be applied. Next, tap (4) is closed and a ‘well’ defined boundary is obtained between the leading and terminating electrolytes. A sample can now be introduced via the septum (3) with a normal micro-syringe. The sample introduction can be effected in the leading electrolyte, in the terminating electrolyte or at the boundary of the two electrolytes, as desired.

INJECTION SYSTEMS

209

Fig.7.5. Injection block suitable for isotachophoretic analysis. 1 = Reservoir for the terminating electrolyte; 2 = PTFE-lined Hamilton valve (1MM1);3 = silicone rubber septum; 4 = tap provided with a conical tip, which gives the connection towards drain; 5 = narrow-bore tube, provided with a Perspex clamping piece (see section 7.2.3). This clamping piece is provided with a small O-ring for a liquid-tight connection with the injection block.

Of course, as sharp a plug profile can never be obtained as when the sampling is performed with a sample tap. The connection with the narrow bore tube is made by the construction discussed in

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INSTRUMENTATION

Fig.7.6. Photograph showing the injection block in Fig.7.5 and the six-way valve in Figs.7.2-7.4.

COUNTER ELECTRODE COMPARTMENTS

21 1

section 7.2.3. The injection block was made of Perspex and PTX. The conical tip of tap (4) was made of silicone rubber (shape 90"). This tap is not available commercially as a single piece. The other taps used were commercially available PTFE-lined Hamilton (1MM1) taps (Hamilton, Bonaduz, Switzerland). 7.2.5. Simplified injection block Because the construction of the injection block described in section 7.2.4 is rather complicated, an injection block of much simpler construction (T-way) is shown in Fig.7.7. Such injection blocks have been made of Perspex, TPX, Kel-F, PTFE and polypropylene. The connection with the narrow-bore tube is similar to that described in section 7.2.3. A Hamilton (1MM 1) PTFE-lined valve is mounted between the injection block and the reservoir containing the terminating electrolyte. Another tap, also a PTFE-lined Hamilton (IMM1) valve, is mounted between the injection block and the drain. The equipment can be rinsed and re-filled with leading electrolyte via a tap at the side of the counter electrode compartment (not shown in Fig.7.7, but shown in Fig.7.16). Tap B is opened during this procedure, while tap A is closed. The tap at the side of the counter electrode compartment is then closed and tap A is opened, while tap B remains open. The terminating electrolyte now flows towards drain. No suction or pump need be applied. After this procedure, tap B is closed and the sample can be introduced via the septum with a standard micro-syringe. In this method of sample introduction, the injection can be made only in the leading electrolyte. A large amount of mobile ions of the leading electrolyte are behind the sample introduced and these ions must overtake the sample ions during the isotachophoretic separation procedure. This may be a complication, especially if the concentration of the sample ions is hgh. Because some of the leading electrolyte is mixed with the terminating electrolyte before the analysis, as a result of the introduction of the needle of the micro-syringe, then after the injection of the sample has been made, tap B must be opened in order to remove the leading electrolyte that is mixed with the terminating electrolyte in the horizontal canal. Because of its simple construction, this type of injection block can be recommended in many instances, especially when some precautions can easily be taken with respect to the leading and terminating electrolytes. The concentration of all of the sample ions must not be too high.

7.3. COUNTER ELECTRODE COMPARTMENTS

7.3.1. Introduction Because in narrow-bore tubing a stabilizing effect is obtained, in most experiments no stabilizing agents (e.g., agar agar, polyacrylamide, arrowroot, dextran, agarose) are added to the electrolytes. The counter flow compartment must therefore consist of a semipermeable membrane in order to prevent any hydrodynamic flow of electrolyte between the two electrode compartments owing to the difference in levels in these compartments.

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INSTRUMENTATION

Fig.7.7. Simpler injection block for electrophoretic equipment suitable for isotachophoretic analyses. Two Hamilton (1MM1) PTFE-lined valves are applied: between the reservoir of the terminating electrolyte and the I-mm narrow-bore tube (A), and between the 1-mm narrow-bore tube and the drain (B). The sample can be introduced into the narrow-bore tube filled with leading electrolyte, so that it is always mixed with the leading electrolyte. A considerable amount of the leading ion must be behind the sample if no intern standard is applied.

COUNTER ELECTRODE COMPARTMENTS

213

Even if the narrow-bore tube is arranged in a horizontal position, this membrane is needed. Moreover, gas will generally be produced at the electrodes as a result of the electric current necessary for electrophoretic separations. These gas bubbles may also introduce a hydrodynamic flow of electrolyte if the electrodes are not separated by a semipermeable membrane from the narrow-bore tube in which the separation is performed. It is of minor importance that the electroendosmotic profile is somewhat suppressed by the semipermeable membrane, as discussed in Chapter 6 . Moreover, mainly those conditions such that electroendosmotic flow can be prevented must be sought. Although semipermeable membranes need to be applied, one must bear in mind that their use always causes a shift in pH on both sides of the membrane, due to the potential gradient across the membrane and the difference in the ionic mobilities of the various ions through the membrane. This shift in pH may disturb or minimally influence the analysis in a long run, especially if a counter flow of electrolyte is applied.

7.3.2. Cylindrical counter electrode compartment Fig.7.8 shows schematically a cylindrical counter electrode compartment. The main parts of this electrode compartment should be made of material resistant to various solvents; so far, Perspex, Kel-F, Arnite and Pyrex glass have been used. The membrane, made of cellulose polyacetate, fits around the two cylinders that are provided with a central bore. The membrane is fixed with Araldite, which in fact does not really stick the membrane to the two central cylinders, but still prohibits any leakage from the side on which the electrode is mounted towards the narrow-bore tube, or vice versa. The cylindrical membrane is made by wrapping a sheet of cellulose polyacetate (0.1 mm) around a rod with an external diameter equal to the external diameter of the cylinders on which the membrane will finally be mounted. During the wrapping of the cellulose polyacetate, acetone, in which the membrane is soluble, is applied. In order to remove this acetone, the rod, with the wrapped sheet on it, is immersed in a stream of water. A white, small-pore cylindrical membrane is the result, which is easy to remove from the rod with aid of a sheet of abrasive paper. The mechanical stability of the membrane is very high, the thickness being approximately 0.3 mm. The main advantage of a cylindrical membrane is that during rinsing and re-filing of the instrument with leading electrolyte, possible gas bubbles can easily be removed and do not stick to the wall. Also, the washing of the entire system is very easily effected. If experiments with a counter flow of electrolyte are performed, this procedure is normally carried out via the tap, a common PTFE-lined Hamilton (1MM1) valve. The disadvantage is that the fresh electrolyte has t o pass the membrane, by which the existing pH jump is transported by the counter flow quickly into the narrow-bore tube. Of course, the separation is influenced by this effect. This has been partially overcome by the construction of a special connection for the counter flow of electrolyte between the counter flow compartment and the narrow-bore tube in which the separation is carried out. (In section 7.3.3, another counter flow compartment is described that is much more suitable for experiments with a counter flow of electrolyte.) The connection with the narrow-bore tube in which the separation is carried out is similar to that described in section 7.2.3. At the side on which the electrode is mounted, the counter electrode compartment is

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Fig.7.8. Cylindrical counter electrode compartment, provided with a semipermeable membrane. 1 = Piece of Perspex on one side of which the semipermeable membrane is mounted, provided with an Wing; 2 = brass screw to clamp component (1)liquid-tight to the electrophoretic equipment; 3 = brass support for component (1); 4 = cap for the electrode compartment, provided with an O-ring; 5 = electrode (Pt); 6 = cylindrical semipermeable membrane made of cellulose polyacetate; 7 = wall of the electrode compartment; 8 = bottom of the electrode compartment with a PTFE-lined Hamilton (1MM1) valve, a connection for the currentstabilized power supply.

COUNTER ELECTRODE COMPARTMENTS

21 5

generally filled with double-distilled water in order to decrease any interference from impurities formed by electrode reactions. Moreover, a rapid change from one operational system to another is possible because the membrane is not ‘soaked’ with different types of electrolytes. If another operational system is chosen, less attention needs to be paid to the membrane compartment, as explained briefly below. Suppose one is interested in anion separations and for a complete separation three different operational systems are needed. In general, in all systems chloride will be chosen as the most mobile ion, because it is pure, stable and cheap. Even if another anion is chosen as the leading ion, this will not affect the analysis because it migrates through the membrane in the direction of the anode. Of course, the buffering counter ions move in the opposite direction and in a different operational system another counter ion must be taken. If double-distilled water is placed in the reservoir surrounding the anode, no buffering counter ion coming from this reservoir will be present. Therefore, the membrane is not saturated with the buffering counter ions. In most instances, simple rinsing of the system is sufficient for cleaning the membrane. The disadvantage when double-distilled water is used in the electrode compartment with the semipermeable membrane is that a large potential drop is caused. Especially if a conductivity detector is applied, this potential drop may cause an electric leak towards earth because the detector electrodes may finally reach too high a voltage for which the insulation is not adequate. The disadvantage of the cylindrical construction of the counter electrode compartment is that sometimes small leakages may arise because the Araldite employed to fix the membrane does not really fix it. Also, if experiments with methanol are performed, the Araldite becomes brittle and electrolyte may flow from the reservoir of the terminating electrolyte towards the counter electrode compartment. If these leakages are small, they are hardly noticeable, but ultimately there may be a decrease in resolution. Because a decrease in resolution may have many origins [e.g., electroendosmosis by adsorbed material on the wall and electrode reactions (if the a.c. method is used for conductivity determinations), due both to the driving current (polarization) and to electric leakages to earth], the hydrodynamic flow cannot be directly localized in the initial phase often.

7.3.3. Counter electrode compartment with flat membrane A more advanced counter electrode compartment is shown schematically in Fig.7.9 and a photograph is shown in Fig.7.10. The electrode vessel is separated from the narrow-bore tube in which the analysis is performed by a flat membrane made, for instance, of cellulose polyacetate (0.2 mm thickness). This membrane is clamped by two screws and an O-ring. The tap used is a common FTFElined Hamilton (IMMI) valve that provides the connection with the reservoir of the leading electrolyte. This reservoir is generally an ordinary polypropylene syringe with a volume of 20 ml. If the entire bystem has to be rinsed or re-filled with fresh leading electrolyte, the liquid applied flows as well along the membrane as along the septum constructed for the experiments with a counter flow of electrolyte. Because the bore is relatively large compared with the inside diameter of the narrow-bore tube ( 2 mm), the potential drop in the canals is small. Therefore, a normal metal syringe can be inserted for the experiments with a counter flow of electrolyte without the risk that gas will be produced owing to polarization (Fig.6.36). In addition to

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1

2

8

L

8. 12

u

l7

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the large bore, a more direct connection between the narrow-bore tube and the electrode compartments exist, so that the potential gradient in the canal where the syringe will be inserted is negligibly small. Of course if any gas were produced, it would destroy the analysis. So far, n o other electrode reactions have been observed. The connection with the narrow-bore tube is as described in section 7.2.3, The great advantage of this counter electrode compartment is that a counter flow of electrolyte is permitted that does not pass the membrane. A considerable time is needed for the pH jump at the membrane to enter the narrow-bore tube, where the analysis is carried out, by the direct electric current, because again the bore, which forms a direct connection between the PTFE narrow-bore tube and the membrane, is relatively large, so that a small potential gradient exists in this bore. In addition, the surface area and thickness of the membrane are small tie., the disturbance is relatively small) and, moreover, the buffer capacity of the electrolyte present in the 2-mm bore is high, so that any disturbance can be counterbalanced easily. A further advantage, of course, is that no adhesive is used with the membrane, so that a membrane can be changed and experiments in, e.g., methanol can be carried out more easily.

7.4. ECUIPMENT 7.4.1. Introduction

With the injection systems and the counter electrode compartments briefly discussed in this chapter, and the detectors discussed separately in Chapter 6, many types of instruments can be constructed. Moreover, the different components are connected in such a way that no adhesive need be applied and therefore the different parts are interchangeable. The means of thermostating can also be taken into consideration, e.g., the narrow-bore tube may be free-hanging in air that is thermostated with circulating water, a thermostated aluminium block can be applied with the narrow-bore tube mounted on it in a helix, or the narrow-bore tube may be thermostated directly with, e.g., circulating kerosene. The development and combination of electrode compartments, injection systems and auxiliary equipment has, of course, resulted in a continuous gradation of types of instruments and modifications, and it is sometimes difficult to distinguish one type from another. Therefore, in this section only three types of equipment will be discussed, Fig.7.9. Counter electrode compartment with a flat semipermeable membrane and a septum for experiments with a counter flow of electrolyte. 1, Perspex connection between the central bore of the counter electrode compartment and the narrow-bore tube of the electrophoretic equipment, provided with an O-ring; 2 = brass screw for clamping component (1); 3 = brass support for component (1); 4, 14 = Perspex units for mounting the counter electrode compartment on a rail (see Fig.7.16) and for clamping 8 and 11; 5, 1 5 = brass pen with screw-thread; 6, 16 = bolts; 7 = cap of the electrode compartment, provided with a hole; 8 = electrode compartment; 9 = flat cellulose polyacetate membrane; 10 = rubber O-ring; 11 = central housing with canals of 2 mm diameter that pass along the flat membrane and the septum; 12 = septum; 13 = screw-head for clamping the septum in the central housing; 17 = PTFE-lined Hamilton (1MMl) valve.

218

Fig.7.10. Counter electrode compartment with flat membrane.

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belonging to three clearly distinguishable types in the development of the instrument& tion of analytical isotachophoretic equipment with a narrow-bore tube. 7.4.2. Narrow-bore tube surrounded with a water-jacket Fig.7.11 shows a photograph of the isotachophoretic equipment with which experiments were carried out in the early days of isotachophoresis (1964). Instead of the PTFE narrow-bore tube, as in Fig.7.11, a narrow-bore tube made of Pyrex glass was used. The equipment consists of a narrow-bore tube, which is fixed with adhesive (shellac if a Pyrex or Araldite if a PTFE narrow-bore tube is used) in a type of Liebig condenser. On the left-hand side a four-way tap is mounted, as discussed in section 7.2.2. The electrode compartment, which contains the semipermeable membrane as discussed in section 7.3.2, is not mounted as a counter electrode compartment as is usually done, but is mounted behind the sample tap and contains the terminating electrolyte. The reason for this is the fact that we still use this equipment for measurements of mobilities by the moving-boundary method and it is easier t o rinse if the membrane compartment is mounted at the position shown in Fig.7.11. By means of the cylindrical membrane electrode compartment, hydrodynamic flow of electrolyte is prevented. The connection between the reservoir containing the terminating electrolyte and the cylindrical membrane electrode compartment is made with a PTFE tube and a PTFE-lined Hamilton (lMF1) valve in which the syringe fits. In the Liebig-type condenser, there are three holes into which the thermocouples (linear and differential) may be mounted. Because these thermocouples are so thin, the narrow-bore tube is fured by a small spring, which fits the tube and which is fixed to the wall of the Liebieg-type condenser with hot shellac. The thermocouples are finally soldered on copper wires, also fixed to the wall of the condenser with hot shellac. In Fig.7.11, three thermocouples, all of the linear type, are mounted. In order to reduce the influence of temperature differences in the laboratory (due to movement, draughts etc.), the holes in the wall of the condenser are sealed with adhesive foil that is covered with aluminium foil. For optimal results, the whole equipment is covered with a blanket of cotton-wool. Thermostated water is circulated through the outer space of the condenser to give a constant temperature inside. The reference junction of the thermocouple is therefore mounted with some adhesive on the inside of the condenser wall, protected with a heat-sink compound. For thermostating in our work, a Hacke thermostat with an accuracy of +O.l"C is used. However, if the variations in the temperature of the laboratory are not too large, no thermostat is needed and the outer space of the condenser can simply be filled with water. The capacity of the outer space and hence the volume of water are large enough to keep the temperature constant in the space where the thermocouples are mounted during the isotachophoretic run. The procedure for filing and cleaning this equipment was described in section 7.2.2. The total length of the narrow-bore tube of the equipment shown in Fig.7.11 is approximately 1 m, while the thermocbuples are mounted at distances of 25, 50 and 75 cm. In our work, the signals derived from the thermocouples are amplified with a h i c k amplifier (type A) and recorded with a potentiometric recorder. If only the linear

Fig.7.11. Electrophoretic equipment suitable for isotachophoretic analyses, constructed in 1964 by Everaerts. Detection is effected with thermocouples (copper-constantan), wound a o u n d the narrow-bore tube at three different positions and fixed with adhesive. I?le narrow-bore tube is mounted in a type of Liebig condenser, filled with thermostated water. The injection can be made via a four-way tap (Fig.7.1.). On the left-hand side is mounted the cylindrical electrode compartment with the cellulose polyacetate semipermeable membrane (Fig.7.8.), which was added at a later stage to the equipment.

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signal of the thermocouple is needed, no amplification is necessary if a 100-mV potentiometric recorder is available. 7.4.3. Narrow-bore tube thermostated with an aluminium block Fig.7.12 shows the main difference between this equipment and that described in section 7.4.2. A PTFE narrow-bore tube (O.D. 0.75 mm, I.D. 0.45 rnm) is embedded in a groove in an aluminium block, and is wound around the aluminium block in the form of a helix.

Fig.7.12. Exploded view of the basic components of an electrophoretic apparatus suitable for isotachophoretic analyses in which the narrow-bore tube is mounted on a thermostated aluminium block. 1 = Narrow-bore tube; 2,3 = thermocouples (copper-constantan); 4 = aluminium block; 5 = cap for the aluminium block; 6 = Pt resistor; 7 = load. Tap water can flow through the block through four canals.

222

d

Fig.7.13. Electronic circuit for thermostating the aluminium block shown in Fig.7.12.

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No effect on the resolution of the fact that the narrow-bore tube is no longer straight could be observed on the thermometric detector. Later experiments with high-resolution detectors showed that the narrow-bore tube must be mounted as straight as possible, especially the last 2 cm before the detector. If even a small kink was present just before the detector, the resolution was decreased. Gaps between the narrow-bore tube and the aluminium block are carefully filled with a heat-sink compound (zinc oxide powder in silicone oil). Because the heat produced in the narrow-bore tube is transferred so quickly to the aluminium block, a special compartment (see Fig.7.12) is created where the thermocouples are mounted in order to ensure that there will still be a signal to detect. If this compartment is too small, a noisy baseline results because a very high amplification has to be used. Of course, a compromise must be sought, because if this compartment is too big a situation similar to that described in section 7.4.2 results, and the narrow-bore tube is cooled only by thermostated air surrounding it. The temperature of the reference junctions of the thermocouples is always the same as that of the aluminium block, because a certain amount of heatsink compound is smeared on the junction that is insulated with a PTFE spray, which is glued to the aluminium block in order to guarantee good thermal contact with the aluminium block. The narrow-bore tube and the heat-sink compound are fixed by a thin layer of shellac spray (Krylon). For thermostating the aluminium block, thermostated water (accurate to +O.0loC) is used. A temperature sensor (100-L2 Pt resistor) is mounted in the neighbourhood of the detector compartment. Also here gaps are filled with the heat-sink compound. In the centre of the aluminium block a load of 60 W is mounted. The Pt resistor and the load are both connected to the temperature control unit, as shown in Fig.7.13. Rubber O-rings are employed to prevent contact of water, circulating inside the aluminium block, with the electrical circuits. The narrow-bore tube protruding from the thermostat is connected at one side with a type of injection block as shown in Fig.7.7. The narrow-bore tube is connected to the injection block, without adhesive, by means of the clamping device discussed in section 7.2.3. As the counter electrode compartment, the cylindrical construction shown in Fig.7.8 was used. The proportional temperature controller used in the thermostat is based on the relatively high temperature coefficient of a Pt resistor, a Pt resistor of 100 52 at 0°C being used. This Pt resistor forms an a.c. bridge (R5) together with the resistors R1, R 2 , R3 and %. The temperature coefficients of all resistors in the a.c. bridge, apart from the resistor R5,must be chosen to be as small as possible. The smaller these values are, the more accurate will be the thermostatic control. Of course, one of the resistors of the a.c. bridge is variable so as to permit the a.c. bridge to be balanced. If the bridge is unbalanced, a signal, the result of the unbalanced position, will be fed to a pre-amplifier, the phase of this signal being dependent on the polarity of the imbalance of the bridge. The preamplifier generates a sinusoidal voltage and this will be transformed by a voltage limiter into a symmetrical square wave. By means of the very high amplification of the preamplifier and the voltage limiter, the amplitude of the bridge voltage is transformed into a phase-shifted square wave. The leading edge of this square wave is amplified and triggers two antiparallel-connected thyristors that control the amount of heat dissipated in the load, which is mounted in the direct neighbourhood of the Pt resistor (Fig.7.12).

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If the bridge approaches its balanced condition, the output voltage of the bridge decreases and the phase shift of the thyristor trigger pulse will thus be reduced. The thryistor will trigger later and the heat produced in the load will also decrease, and a steady state will result. The temperature (T, "C) can be selected by the variable resistor & of the a.c. bridge, according to the equation

T = 2.59

(R4

- 0.5835)

(7.1) (temperature coefficient R s = 0.003916). Some possibilities are shown in the Table 7.1. An RC filter in front of the input of the pre-amplifier corrects the phase of the trigger pulse, and the proportional band (system gain) can be changed by varying the a.c. voltage over the bridge. Incorrect temperature regulation may result if the temperature coefficients of the other resistors of the a.c. bridge are poor, leading to instabilities if the thermal resistance between the load and the Pt resistor is large or if the heat capacity of the object to be thermostated is too high. A photograph of the equipment with indirect thermostating via the aluminium block is shown in Fig.7.14, which also shows the power supply. A pressure system has been developed for rinsing and re-filling the different compartments of the equipment with the chosen electrolytes. In order to prevent the dissolution of air in these electrolytes, the surfaces of the electrolytes were covered with a layer of kerosene. An additional advantage is that negligible amounts of carbon dioxide dissolve in the electrolytes if a 'high' pH is chosen in performing the analysis (even pH 7). If experiments are to be carried out in parallel-mounted narrow-bore tubes, this method of mounting the narrowbore tubes on a thermostated aluminium block is recommended. 7.4.4. Equipment with high-resolution detectors

Fig.7.15 shows a schematic diagram of isotachophoretic equipment for use with a highresolution W absorption detector and a conductimeter, and a photograph is shown in Fig.7.16. The equipment consists of a PTFE narrow-bore tube in which the analysis is performed, the length being about 25 cm, although a longer tube can easily be mounted. It is produced by Habia (Breda, The Netherlands), with O.D. c.a 0.7-0.75 mm and I.D. ca TABLE 7.1 SOME VALUES FOR THE RESISTANCES (a)TO BE MOUNTED IN THE BRIDGE (FIG.7.13) OF THE TEMPERATURE-REGULATING UNIT TO SELECT A TEMPERATURE RANGE Resistors R, , R, and R, : 1%, 50 ppm/"C. Resistance

R , =R, R3 R,

Temperature range ('0 0-50

0-125

0-300

120 100

150 100 50

180 100

50

100

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Fig.7.14. Electrophoretic equipment suitable for isotachophoretic analyses, with an injection system comparable with the injection block described in section 7.2.5. A counter electrode compartment of the cylindrical type (Fig.7.8) is used. The narrow-bore tube is wound around a thermostated aluminium block (Fig.7.12.). Detection is performed with thermocouples (copper-constantan). A pressure system is applied for rinsing and re-filling the various compartments. This equipment was constructed in 1968 by Verheggen and Everaerts.

0.4-0.45 mm. The variations in diameter per unit length are negligibly small. This narrowbore tube is clamped by a special clamping device, as discussed in section 7.2.3, in the injection block, the counter electrode compartment and on both sides of the conductivity probe. The narrow-bore tube is uninterrupted in the W detector.

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/ I

1

d

I

/

/ ,

/

\

Fig.7.15. Exploded view of more advanced electrophoretic equipment suitable for isotachophoretic analyses in which the injection block shown in Fig.7.5 and the cylindrical counter electrode compartment shown in Fig.7.8 are used. For the detection of the various zones, a UV absorption meter, a potential gradient detector (d.c. method) and a conductivity detector (a.c. method) are applied (see Chapter 6).

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Fig.7.16. Photograph of the more advanced electrophoretic equipment based on that shown in Fig.7.15. This photograph shows the flexibility of the construction. Various injection systems, detectors and counter electrode compartments can easily be combined, In the equipment shown, the injection block shown in Fig.7.5 and the counter electrode compartment with a flat membrane shown in Fig.7.9 are used. The six-way valve shown in Figs.7.2-7.4 is fitted in order to make a simple introduction via either a micro-syringe or a tap possible. For the detection of the various zones, a UV absorption meter, a potential gradient detector (d.c. method) and a conductivity meter (a.c. method) are applied (see Chapter 6). The equipment was constructed by Verheggen and Everaerts.

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The W light is generated by a microwave-powered low-pressure mercury lamp. This W light is transported by an optical quartz rod and fed into a cylindrical slit of width 0.05-0.3 mm. As already mentioned, the PTFE narrow-bore tube is not interrupted and is clamped by the slit, which is made of brass, and the W light passes through the narrowbore tube and is again transported via an optical quartz rod to a set of filters (an interference filter in combination with an end filter). The UV quanta then illuminate a UV light-sensitive photodiode (S330; Hamamatsu, Hamamatsu City, Japan). The quality of the PTFE narrow-bore tube is not sufficiently constant for the UV detector, because the PTFE material itself has a high W absorption. On mounting a particular narrow-bore tube, the amount of light that passes through it and finally reaches the detector may vary by a factor of up to three compared with a previously used narrow-bore tube owing to the difference in the thickness of the two tubes, if they are filled with a non-UVabsorbing liquid. On one hand the high absorptivity of the PTFE material in the UV range is a disadvantage, while on the other hand the dark current, i.e., the current that is transported by the PTFE wall and reaches the detector without passing through the narrow-bore tube, is reduced to a minimum. The signals are handled electronically and result in a trace on a potentiometric recorder. The trace does not have a continuous stepwise character if the isotachophoretic zones pass the detector. At the position where the conductimeter is mounted, the narrow-bore tube is interrupted by a piece of insulating material (Perspex or TPX) in which the micro-sensing electrodes are mounted (Chapter 6). As a result, there is always a slight difference in cell volume between the conductimeter and the UV detector. The sequence of mounting these two detectors was tested and it proved to be of no importance; experiments were carried out to prove this only with components that were stable in the UV region, and possible deleterious effects due to W light were not studied. The conductivity detector can be applied for measurements of the conductivity (a.c. method) or for measurements of the potential gradient (d.c. method) via two microsensing electrodes (10-pm Pt-Ir foil) mounted axially and in direct contact with the electrolytes inside the narrow-bore tube. In Fig. 7.16 can be seen the position where the coil is mounted in order to give good galvanic separation of the high potential on the micro-sensing electrodes from the circuit for measuring the conductivity (potential gradient) at low potential. As discussed in Chapter 6, a leak current towards earth (even lo-” A) must be prevented. For this reason the conductivity probe is surrounded by PTFE insulation. Even the wires connecting the micro-sensing electrodes with the electronic measuring circuit are provided with extra insulation by means of a PTFE narrow-bore tube. The signals derived from the conductivity probe are fed to a field effect transistor (potential gradient measurement) or directly to a well insulated transformer for good galvanic insulation. If the measuring electrodes are mounted equiplanar, only the conductivity can be determined of course. For the measurement of the conductivity (a.c. method) with axially mounted electrodes, these electrodes must be separated from each other via a capacitor, otherwise an electric current will flow, due to the potential gradient, and electrode reactions will result, e.g. ,coating or gas production. Even when the micro-sensing electrodes were

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mounted equiplanar, we found it advantageous to have this capacitor between the measuring electrodes; possibly an exact equiplanar construction is not always possible. Owing to the high potentials applied, some leak current will decrease the resolution after a small series of experiments. The signals derived from the transformer are handled electronically and result in a trace on the potentiometric recorder. This trace has a continuous stepwise character if the isotachophoretic zones pass the detector. If the trace does not have a continuous stepwise character, e . g , if a drift is obtained or dips and/or overshoots are recorded, something is wrong. A drift of the base line is obtained if, for instance, impurities are present that are more mobile than the leading ion, the buffer capacity of the counter ion is not sufficient or some electrode reaction occurs at the micro-sensing electrodes. Dips (or negative steps) can be expected if the buffer capacity of the counter ion is not sufficient, if an enforced isotachophoretic system is obtained or if the electrodes are coated with a polymer as a result of an electrode reaction or the physical adsorption of any material. Overshoots can be expected if the buffer capacity of the counter ion is not sufficient, if the temperatures of two adjacent zones are too different or if an electrode reaction occurs. A modified Brandenburg (Thornton Heath, Great Britain) power supply of the alpha-series is used. It is modified in such a way that it not only can be applied as a constant-voltage source (+30 kV), but for the isotachophoretic experiments can also be applied as a current-stabilized power supply (lt30 kV). Various current-stabilized power supplies, however, are commercially available nowadays, even up to 60 kV. Between the injection block, shown in detail in Figs.7.5 and 7.6, a six-way tap (Figs.7.2-7.4), w h c h can easily be removed without changing the narrow-bore tube if necessary, is mounted. In the equipment shown, an injection can also be made with a normal commercially available micro-syringe, as the sample can be introduced via the tap. Thus this instrument combines all the advantages of taps and syringes. Also, instead of the cylindrical counter electrode compartment (Figs.7.8 and 7.1 5), a counter electrode compartment with a flat membrane (Figs.7.9 and 7.10) can be used. These electrode compartments can easily be changed if necessary without any problems or the need to fit a new narrow-bore tube. All components of the isotachophoretic equipment shown in Fig.7.15 are replaceable because no adhesive is applied, rubber O-rings and screw-threads being used for clamping. We found that it is sometimes necessary to replace the narrow-bore tube as their life was found to vary from several years to only a few months. A narrow-bore tube needs to be changed if a decreased resolution cannot be improved. Owing to differences in the behaviour of the various operational systems, or compounds of the sample, even the ‘inert’ PTFE can become coated with material that is not easy to remove, and the resolution may decrease. Impurities, possibly building up over a long period, that are adsorbed on the walls of the injection system or the counter electrode compartment have less influence on the detection or the separation itself, because the bores in these parts of the instruments are considerably greater. The conductivity probe, if washing with a non-ionic detergent gives no improvement, can easily be cleaned with some metal polish and a cotton thread.

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7.5. COUNTER FLOW OF ELECTROLYTE 7.5.1. Introduction

Many of the papers describing electrophoretic techniques have dealt with the electrolytic counter flow of electrolyte, and a large number of other papers could be cited, especially relating to equipment filled with various stabilizing media. In t h s field, experiments are carried out t o increase the length of the separation, especially for improving the separation of isotopes, and it has been found that mainly enrichment could be obtained. The electrophoretic techniques applied usually involved a movingboundary system, in w h c h a complete separation cannot be expected. However, if an isotachophoretic system is chosen for the separation of isotopes, the separation procedure is also a moving-boundary system that may result in a complete separation, ie., the steady state. In this section, some possible methods for regulated and non-regulated counter flows are given, and a newly developed pumping system is described in which the gas production is used for pumping hydrodynamically the liquid needed for the counterflow of electrolyte. The driving current can be regulated by signals from the isotachophoretic equipment, by means of which the zones can be stopped in the separation chamber (the narrow-bore tube) if counter flow is applied. Of course, it is beyond the scope of this book to discuss all possible systems for electrolytic counter flows. We can consider a regulated counter flow in terms of the main basic principles, as follows. (a) The electric current is constant during the analysis, and the hydrodynamic counter flow of electrolyte is regulated and controlled by signals derived from the electrophoretic apparatus. (b) The hydrodynamic counter flow of electrolyte is constant during the time the counter flow of electrolyte is required, and the electric current is adjusted to this counter flow by means of signals derived from the electrophoretic equipment. During the detection, the electric current is stabilized again. (c) The electric current is constant in the initial phase and the hydrodynamic counter flow of electrolyte is started as soon as a pre-set value of the voltage of the current-stabilized power supply has been reached. The counter flow of electrolyte is then adjusted until n o further increase in voltage is obtained. If for any reason a lower pre-set value is reached, the counter flow of electrolyte is stopped. It should be pointed out that although the length of separation is generally increased, the counter flow of electrolyte disturb the electrophoretic separation (Chapter 17). The method of producing the counter flow can vary widely, and syringe pumps, peristaltic pumps, level differences or ‘gas pumps’ can be applied. Two main reasons can be given for wanting a counter flow of electrolyte, both originating from the fact that the narrow-bore tube is not long enough for a particular separation: (1) the concentration differences between the ions to be separated are too laIge; and (2) the differences in (effective) mobility between the ions of interest are small. Of course, these two factors may be combined in a specific instance. In those instances when the difference in (effective) mobility is minimal, the use of a counter flow of electrolyte will generally fail. More research needs to be carried out in order to determine the effect of the ‘disturbance factor’. It is not unlikely that in specific

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231

instances this factor may be zero or even that the separation may be positively influenced. Also, when the difference in effective mobility is minimal, one cannot expect always a complete separation because in some cases the ions have a mutual adverse influence on the pH of the mixed zone and give a poorer separation. Once the ions are separated they will form discrete zones owing to the difference in the pH values in the two zones. We found the counter flow of electrolyte to be successful especially when samples need to be separated with large concentration differences between the various zones. The use of a counter flow of electrolyte has also proved of value in elucidating whether a separation is completed or not (i.e.,mixed zones are present or not). Although the use of a counter flow of electrolyte in isotachophoretic experiments can be seen to be a valuable tool, it also has disadvantages. If a counter flow of electrolyte is to be considered, the chemicals must be of the highest purity available, and even then they often are not pure enough. The impurities may sometimes be collected betwetn the leading and terminating zones and influence the analysis. Sometimes the zone still undergoes a small migration and cannot be stopped owing to impurities present. The impurities in the leading electrolyte and/or in the terminating electrolyte must be removed by recrystallization, zone refining or electrophoretic procedures, etc., if

Eqn. 7.2 relates to the leading electrolyte and eqn. 7.3 to the terminating electrolyte, are the effective mobilities of the terminating ion, where meff,T, meff.,I and impurity and leading ion, respectively. 7.5.2. Counter flow with level regulation Fig.7.17 shows schematically the equipment with which a counter flow of electrolyte can be applied, and the circuit for the regulation of the counter flow of electrolyte is shown in Fig.7.18. The moment at which the counter flow is to be started can be selected with the lO-kf2 potentiometer. The switch A is provided in order to have the possibility of selecting a high potential on the side of the injection block of chosen polarity. It has to be borne in mind that for optimal functioning of the conductimeter, the probe must be at a ‘low’ potential, Le., less than 10 kV. As soon as the voltage selected by the lO-kS2 potentiometer has been reached, the level is controlled by the plunger (Fig.7.17) by means of a coil. Before the experiment, this level is adjusted approximately t o the level in the compartment of the terminating electrolyte, such that the sample zones still migrate in the appropriate direction by means of the electric field strength (possibly a small flow in the direction of the movement of the zones is permitted). Owing t o the construction of the counter electrode compartment, the pH jump across the membrane is of minor importance. Experiments with a counter flow of electrolyte showed that it is important that the compartment in which the driving electrode is mounted should contain electrolyte also. The electrolyte, containing buffer ions, decreases the potential at the measuring electrodes of the conductivity probe and diminishes the pH jump across the membrane.

232

INSTRUMENTATION

Fig.7.17. Equipment for producing a counter flow of electrolyte via level regulation. 1 = Electronic regulation circuit (shown in Fig.7.18); 2 = coil with which a movement of the plunger can be effected; 3 = set of detectors.

The counter flow is stopped by switching the PTFE-lined Hamilton valve, mounted in the plunger reservoir, to the closed position, and the device for regulating the counter flow of electrolyte can then be switched off. It should be noted that the coil is fed by a rectified electric current that is not smoothed by a capacitor. It has been found experimentally that the vibration of the plunger by the unsmoothed current eliminates the mechanical friction that could disturb the analysis at the moment the regulation is started by an abrupt lowering of the plunger.

233

COUNTER FLOW OF ELECTROLYTE

p High V lOOMR

50

2

Fig.7.18. Electronic circuit for regulation of the counter flow of electrolyte via level regulation in isotachophoretic analyses. The resistances are given in kn unless stated otherwise.

This method of regulation can also be applied if a counter flow of electrolyte is required that is regulated by signals derived from the detectors mounted directly on the narrow-bore tube. In this instance the zones are stopped at the regulating detector, which usually gives a better result.

7.5.3. Counter flow with light-dependent resistor regulation This method of producing a regulated counter flow of electrolyte is shown schematically in Fig.7.19 and the circuit for regulation is given in Fig.7.20. A light-dependent resistor (LDR) is attached in series with the narrow-bore tube and a constant potential gradient is applied over the narrow-bore tube and the LDR. Because in isotachophoretic experiments the total potential gradient over the equipment is higher than 300 V, which is the LDR limit, a series of LDRs is used so that one is able to work at 1 0 kV. The series of LDRs can be considered as a voltage source. The amount of light given by a lamp (see Fig.7.20) is regulated by a thermocouple (copper-constantan) mounted around the narrow-bore tube. A change in the temperature of the narrow-bore tube will automatically involve a change in the electric current through it. As is normal in isotachophoretic analyses, the

234

INSTRUMENTATION

Fig.7.19. Equipment for producing a counter flow of electrolyte with regulation via a light-dependent resistor. 1 = electronic circuit shown in Fig.7.20; 2 = light-dependent resistor; 3 = set of detectors.

total resistance of the electrolytes inside the narrowbore tube will increase in time owing to the progress of less conductive zones. The increase in the resistance of the narrow-bore tube during the analysis will produce a decrease in the current if a constant voltage is applied over it, and this decrease in current will cause a decrease in the temperature of the narrow-bore tube (quadratic relationship.) This decrease in temperature is recorded by the regulating thermocouple. Hence the total resistance of the LDR and indirectly the voltage drop over the narrow-bore tube are controlled by this thermocouple and a stabilized current will be the result. A means of obtaining a more stable regulation of the current is to arrange a resistor in series with the narrow-bore tube. The potential drop over this resistor can be used for current stabilization, and slowly moving concentration fronts, such as pH disturbances, will not influence the regulation of the current.

COUNTER FLOW OF ELECTROLYTE

235

Fig.7.20. ElecQonic circuit for stabilizing the electric current by signals derived from a thermocouple mounted around the narrow-bore tube in which the electric current flows. This circuit can also be applied for regulation of the various zones moving isotachophoretically in the narrow-bore tube in experiments with a counter flow of electrolyte. 1 , 2 = connections for the thermocouple (copperconstantan); 3 = + 15 V; 4 = common terminal; 5 = - 15 V,

Therefore, during the counter flow of electrolyte, a thermocouple mounted around the narrow-bore tube is applied and during the detection of the zones the current is stabilized by an extra resistor mounted in series with both the LDR and the narrow-bore tube. The counter flow of electrolyte can be produced in various ways, although only the syringe pump is shown in Fig.7.19. Particularly if the counter flow is produced by a difference in levels, a complication can arise because the level is not controlled, and the counter flow will thus change with time. As will be discussed later, there are two limits for the counter flow and if at a certain moment the lower limit is exceeded the counter flow of electrolyte is no longer able to stop the zones. For a counter flow over a long period of time, the electrode compartment that contains the counter flow electrolyte must be very large and it is preferable to use a pump, especially that discussed in section 7.5.5. It wdl be noticed immediately that the adjustment of the electric current as described here will automatically result in an oscillation of the zones around the regulating thermocouple. For thermometric recording, the zone must have passed the thermometric detector by about 1-2 cm for complete qualitative and quantitative determination, but for the regulation a much lower signal is needed. We found that this method of regulation gives a negligible oscillation; the experimental conditions were checked with coloured ions for which the sharpness of the boundaries was studied. Fig.7.21 shows two isotachopherograms for the separation of formate and acetate with and without a counter flow of electrolyte in order t o demonstrate LDR regulation

INSTRUMENTATION

236

-

Chloride min.

Time

Fig.7.21. Isotachophoretic separation of formate and acetate without (a) and with (b) a counter flow of electrolyte. The experiments were carried out in the operational system at pH 6 (Table 12.1) with glutamic acid as terminating electrolyte. Detection was carried out with a thermocouple mounted at a distance of about 50 cm from the injection point. The electric current was stabilized by the circuit shown in Fig.7.20. The thermocouple for regulation was mounted at a distance of about 25 cm from the injection point. The traces show that the electric current decreases (lower temperature) if a hot zone passes the regulating thermocouple. As soon as the terminating ion is below the regulation thermocouple, the electric current is stabilized at imin.. Trace (b) shows that equilibrium is achieved between the counter flow of electrolyte and the electric current. Traces (a) and (b) show that the isotachopherograms finally obtained are similar. These isotachopherograms are not given to show the usefullness of a counter flow of electrolyte, but only to demonstrate current stabilization via LDR and the possibility of using a counter flow of electrolyte (see Chapter 17).

and the isotachopherogram that can be expected. The recording is performed with a thermometric detector (copper-constantan thermocouple). In this experiment, therefore, two thermocouples were mounted around the narrow-bore tube, one at the beginning of the narrow-bore tube, which was used for the LDR regulation during the counter flow of electrolyte period, and the other at the end of the narrow-bore tube, which was used for the detection. Because more advanced systems are considered later, further isotachopherograms with this type of regulation will not be shown. The experimental conditions for the isotachopherograms shown in Fig.7.21, however, will be given. The analysis was performed in the operational system at pH 6 (Table 12.1) with chloride as the leading ion and glutamate as the terminating ion. The aluminium block

COUNTER FLOW OF ELECTROLYTE

231

around which the narrow-bore tube was mounted (section 7.4.3) was thermostated at 18°C. A 2-pl injection was made, containing 0.02 mole of sodium acetate and 0.02 mole of sodium formate. The current was 92 pA in the initial phase and the temperature of the zone of the leading ion was used as reference for the current stabilization (about 25°C). In the initial phase, the current is not yet stabilized, possibly owing to the movement of ions ahead of the zones of formate and acetate, which increase the conductivity. Subsequently the real zones of formate and acetate reach the regulation thermocouple. Because these zones are considerably hotter than the leading zone (see Fig.6.7), even if the aluminium block is applied as a thermostat, the electrophoretic driving current will decrease. Fig.7.21 shows clearly a drop in electric current from ima. = 9 2 p A towards imh. = 52 pA, because the temperature of the glutamate is used for stabilization of the electric current. In Fig.7.21 b, from the initial phase a counter flow of electrolyte is produced in such an amount that the zones still have a movement in the appropriate direction. Fig.7.21 shows that the temperature of the formate zone does not give such a low electric current that the zones are stopped by the counter flow chosen. Between the formate and acetate zones, however, a temperature is attained such that the zones are stopped. The counter flow produced was 200 pllh. After the counter flow of electrolyte has stopped, the current decreases further to the in,h. value and the experiment is completed, as shown in Fig.7.21a. It need not be explained that the thermocouple used for the detection of the various zones must be mounted as far as possible from the regulating thermocouple, otherwise some material may pass the recording thermocouple too soon, especially if components are present in the sample that normally have a temperature in the zone lower than that at which equilibrium is obtained.

7.5.4.Counter flow with direct control on the pumping mechanism via the power supply A counter flow of electrolyte can be obtained in this way if the initial and end voltage over the narrow-bore tube are known. If both of these values are known, the position of the zones as a function of the potential gradient and the approximate counter flow required in order to stop the zones can be calculated (Fig.7.22). A circuit such as that shown in Fig.7.23 can be applied, with which it is possible to select a voltage of the current-stabilized power supply at which the pump is started. Because the counter flow to be produced is calculated only roughly, a counter flow must be selected such that the zones are stopped and slowly pushed back. If the counter flow is insufficient, the zones are not stopped by the pump and finally reach the detector, while if the counter flow matches the movement of the zones the pump will be in action continuously. If the zones are pushed back, the voltage across the narrow-bore tube will decrease. As soon as a chosen lower limit has been reached, the counter flow of'electrolyte is stopped and the zones will again move in the required direction. It needs no further explanation that the range of voltage in which the operation of the pump is planned must be very small. Experiments with coloured ions showed that the zone boundaries are less sharp during the period when they are being pushed back, but as soon as the pumping was stopped sharp boundaries were recorded very rapidly.

238

INSTRUMENTATION

Fig.7.22. Equipment for producing a counter flow of electrolyte by means of on-off regulation of the pumping mechanism by signals derived from the current-stabilized power supply. 1 = Electronic circuit shown in Fig.7.23; 2 = set of detectors.

7.5.5. Counter flow with no regulation

This method of producing a counter flow is comparable with the method discussed briefly in section 7.5.3.In this instance also the initial and end voltages must be known, and the current-stabilized power supply must have a voltage limiter. The procedure is demonstrated in Fig.7.24. A represents an experiment with no counter flow of electrolyte. The electric current is constant and the potential gradient increases continuously with time, because fewer conductive zones move and occupy more of the narrow-bore tube. This potential gradient does not, of course, increase regularly, because the bore of the injection block does not have a diameter identical with the inside diameter of the narrow-bore tube and the bore of the conductivity probe.

COUNTER FLOW OF ELECTROLYTE

239

p high V

i

1OOGR

r-

Fig.7.23. Electronic circuit for the on-off regulation of the pumping mechanism in isotachophoretic experiments with a counter flow of electrolyte. The common terminal should not be mounted such that the stabilization of the current in the narrow-bore tube is influenced (see Fig.7.26).

In B an experiment with a counter flow of electrolyte is shown. The voltage of the current-stabilized power supply is limited to V1, which is higher than the initial voltage and much lower than the final voltage. As soon as V , has been reached (after a time t l ) , the power supply is no longer able to keep the electric current stabilized and as a result the current will decrease. Depending on the magnitude of the counter flow produced, an equilibrium current (Ieq, 1) can be reached at which the zones are stopped after a time (tl*).After the counter flow of electrolyte has stopped (after a time tl**), the voltage is no longer limited and a stabilized current will be the result. In C, a similar experiment is shown with a limited voltage V , and a greater counter flow of electrolyte. If a well chosen counter flow of electrolyte is applied, i.e., a flow such that the zones will move in the appropriate direction if the current I , has been chosen and the zones can be stopped before the detector, no further regulation need be used. Nevertheless, we found this method t o be difficult to apply in practice, particularly because the position at w h c h the zones are stopped is influenced by the size and c o m p o sition of the sample. If the sample consists of many ions with a high effective mobility, the increment in voltage is not great initially. If the regulation is not performed at a ‘low’ voltage, t!ie zones may be stopped if they have already passed the detector.

240

V

INSTRUMENTATION

t “2

Fig.7.24. Isotachophoresis with a counter flow of electrolyte without direct regulation of the process. A, Experiment without a counter flow of electrolyte. The voltage increases because less mobile ions enter the narrow-bore tube. In practice, this increment is not as smooth as is shown here. The electric current is kept constant during the experiment at I, B, Experiment in which a counter flow of electrolyte is applied. The electric current is stabilized at I, up to V, (fJ, then the voltage is stabilized (limiter). This results in a decrease in the electric current to ieq, (t;). At time ff*, the counter flow of electrolyte is stopped, the current is stabilized at I , again and the voltage can increase steadily. C, Experiment with a greater counter flow of electrolyte t b n in B. The electric current is stabilized at I, up to V , ( t 2 ) , then decreases to ieqz) ( t z ) .The counter flow of electrolyte is stopped at fz* and the electric current is stabilized again at I,. This figure does not relate to actual experiments, all values being chosen arbitrarily. For further explanation, see text.

24 1

COUNTER FLOW OF ELECTROLYTE

7.5.6. Counter flow regulated by the current-stabilized power supply; the membrane Pump

This method is the most accurate and simple, and is therefore discussed in more detail. The principle is shown in Fig.7.25. Fig.7.26 can be used to explain the principle of the method and also the principle by which the electric current through the narrow-bore tube (Ic) is stabilized. If through the narrow-bore tube, filed with a suitable electrolyte (leading electrolyte), an electric current is stabilized at Ic, the total voltage needed (V,) is then increasing during

T

I

1

Fig.7.25. Equipment for producing a counter flow of electrolyte regulated by the currentstabilized power supply. This method of pumping and also the regulation were found to be optimal in combination with the narrow-bore tube, in spite of the fact that the membrane pump does not have linear characteristics. 1 = Electronic circuit shown in Fig.7.29; 2 = set of detectors. If the counter flow of electrolyte is also to be applied for micro-preparative purposes, another means of pumping can be sought (e.g., an electroendosmotic pump).

242

INSTRUMENTATION

Fig.7.26. Principle of regulation of a counter flow of electrolyte, via a membrane pump (as shown in Fig.7.25). Attention should be paid to the common terminal and the earth, which prevent disturbances to the current stabilization (electrophoretic driving current) by the counter flow regulation. This could destroy, or at least obscure, the final result.

the isotachophoretic run. If gas is now produced in the electrolysis cell of the membrane pump, the volume of this electrolysis cell tends to expand. The volume can expand easily because between the electrolysis cell and a cell filled with leading electrolyte, mounted. next to it, a thin membrane (e.g., a rubber contraceptive) is mounted. This membrane is mounted with pre-stressing. In Fig.7.27, the construction of the membrane pump is shown in more detail, and a photograph is shown in Fig.7.28. The flow of liquid caused by the production of gas in the electrolysis cell of the membrane pump counteracts the increment in V,. This gas is produced by an electric current I,, in an electrolyte (e.g., 0.01NKC1). Because V, is of the magnitude of kilovolts, V, is reduced to a value B V, with the aid of two resistors of 100 Ma and 56 ka.An electronic circuit (Fig.7.29) compares this value B V, with an adjustable V, If BlV,l< V,, (V,,> 0), then I, = 0. If BlV,l> V&., then I, # 0 and the increment in V, is counteracted. The regulation is such that I. will reach a value such that BIG1 becomes and remains approximately equal to VEf.. Thus the relationship betweenI,,, BI V,l and V,, is:

BI Vc I Q Vmf.

(7.4)

then I,, = 0, and

BI VCl> V,f.

(7.5)

then I , = A (BI V, I - VXt). The optimal value for the amplification factor, dl,/dl V,l = BA, is dependent, among other factors, on the electrolytic system chosen (operational system) and on the cross-

243

COUNTER FLOW OF ELECTROLYTE

I

I

31

I

Fig.7.27. Detailed diagram of the membrane pump. The pump can be used in isotachophoretic experiments with a counter flow of electrolyte. 1 = Cap for closing the electrolysis cell; 2 = electrolysis cell filled with a suitable electrolyte, e.g., 0.01 M KCl; 3 = nuts; 4 = the gas-producing electrodes; 5 = rubber O-ring; 6 = cap for closing the electrolysis cell; 7 = PTFE-lined Hamilton (1MM1) valve; 8 = cap for closing the compartment filled with leading electrolyte; 9 = rubber O-ring; 10 = electrode that can be used, if required, such that during the time the counter flow of electrolyte occurs, the electrode that is connected with the current-stabilized power supply is not separated from the narrowbore tube in which the analyses are performed by a semipermeable membrane; 11 = central body of the compartment filled with leading electrolyte; 12 = bolts for clamping components (11) and (2) together (in total four bolts and nuts are applied); 1 3 = needle; 14 = rubber membrane; 15 = rubber O-ring.

section of the narrow-bore tube, which may vary if a replacement narrow-bore tube is used. If BA has a too high a value, then the regulation will be unstable, while if BA is too small, the accuracy of the regulation will not be sufficient. Because the current, Z,., through the resistors of 100 MR and 56 ki2 may not influence the current through the narrow-bore tube (Ic), the resistors must be mounted as shown in Fig.7.26. Of course, the input current, Zi,of the electronic regulating circuit must be negligibly small compared with Z,. A galvanic separation of the electrodes of the electrolysis cell of the membrane pump with earth is arranged, because otherwise part of Z, would flow through the thm membrane. The membrane was found to be permeable for small

244

INSTRUMENTATION

Fig.7.28. Photograph of the membrane pump illustrated in Fig.7.27.

ions in a long run. In addition to the leak of the electric current, ions from the electrolyte of the electrolysis cell may also interfere if they can pass through the membrane due to poor galvanic separation of the electrodes of the electrolysis cell towards earth. The operational amplifiers (see Fig.7.29) ICl0, ICll and IClz form a differential amplifier with a high input impedance. The amplification factor of this differential amplifier is unity. With aid of a switch ‘polarity’, the output signal of the differential amplifier is always kept positive, depending on the polarity of V,. By means of a ten-turn potentiometer, the reference voltage Vref, can be adjusted. The trim potentiometer of 10 k n must have a value such that the output voltage of ICI3 is equal to zero if I V,I = 10 kV and V,, has its maximal value. If the absolute value of V, is greater than the selected value of VEf., a negative output voltage of IC13is obtained. The amplification factor of ICI3 is constant within 3 dB up to approximately 3 Hz. This frequency is sufficiently high to make stable regulation possible. By the low-pass characteristic of the amplifier, the eventual disturbance of the electric mains (50 Hz) is sufficiently suppressed. The transformer T, forms an oscillator with the two npn transistors. If the input voltage of ICI4 is negative, the sum of the average collector currents of both transistors is proportional to this voltage. The average value of the rectified current through L3, the electric current I, needed for the electrolysis cell of the membrane pump, is approximately proportional t o the input voltage of IC14. If this voltage is positive, I, is zero. By means of a resistor of 4.7 kSl between the connection points 4 and 5 of IC14, the offset voltage of ICI4 is changed in such a way that I , is certainly zero if the input voltage of IC14 is zero, in the case of manual regulation.

245

COUNTER FLOW OF ELECTROLYTE

max. 15 kV hishVl-+

*

a: 2 N 4 124

-- common Fig.7.29. Electronic circuit that can be used for the regulation of the electrolytic counter flow in isotachophoretic experiments, with aid of the membrane pump shown in Figs.7.27 and 7.28. Components IC,,, IC,,, IC,, ,IC,, and IC,, are all of the type rA741. All diodes are 1N4148 or 1N914. The resistances are given in kR unless stated otherwise. The specifications for the transformer are: L, = two times 10 turns; L, = two times 50 turns; L, = 65 turns. For the wires enamelled copper Wire, diameter 0.4 mm, is used. The potcore is of the type P 36/22,3B7, ~e (permeability) = 2030.

By means of a switch ‘auto-manual’, automatic or manual regulation of the membrane pump can be selected. The maximal value of I , is approximately 2 mA, and the voltage needed is low (+ 3 V). The amplification factor BA of the circuit (Fig.7.29) for experiments in the operational system at pH 6 (see Table 12.1) with the equipment as described in section 7.4.4. (a PTFE narrow-bore tube of I.D.0.4-0.45 mm and O.D.0.7-0.8 mm and a total length of approximately 30 cm) is approximately 0.1 5 mA/V. We can therefore calculate that I Vcl changes by approximately 14V if I , changes from 0 to 2 mA. Of course, other values can easily be taken, although we found the above values to be optimal.

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APPLICATIONS

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Chapter 8

Introduction SUMMARY In t h s chapter some practical information is given on the Section Applications, and a scheme is given for ‘trouble-shooting’.

8. INTRODUCTION The Section Applications contains almost all of the practical information about isotachophoretic separations in narrow-bore tubes. In this section, applications and results are given for separations classified according to chemical compounds that belong to clearly distinguishable classes. The separations were carried out in so-called operational systems in which the electrolytes were shown to give optimal results. The operational systems are listed in tables, in order to make a comparison between them possible. The systems listed were chosen somewhat arbitrarily; many more possibilities could be given. Also, the separations considered were mainly chosen arbitrarily: many real problems from industry or hospitals proved to be much simpler. The separations are shown in order to indicate their possibilities and to make patterns recognizable. When a specific operational system is chosen, one always has to bear in mind that the pH in anionic separations by isotachophoresis tends to increase, while in cationic separations it tends to decrease, going from the leading zone towards the terminating zone. Therefore, the pH must always be chosen such that the optimal effect of the buffering counter ion is used. In some instances a buffering counter ion is not necessary, while in other instances two or more counter ions with overlapping buffer regions are needed. A difference of 0.5 pH unit can give an operational system that has completely different characteristics for a specific analytical problem, as shown in the following example. If a separation of anions is sought and the operational system at pH 6 (Table 12.1) is found to be suitable, one can adjust the pH of the leading electrolyte from its initial value of 6; the buffering capacity of histidine is sufficient until a pH of ca. 7. If, however, an anion is present with a very low effective mobility, which needs a terminator with an even lower effective mobility than the anion to be separated, it may be preferable t o adjust the pH of the leading electrolyte to 5.5. If the pH of the leading’electrolyte is decreased too much, sometimes difficulties can arise because too few counter ions are present and the buffering capacity may not be sufficient (see Fig.9.5). Experimentally, we found it best to adjust the pH of the leading electrolyte, with the chosen counter ion, to the selected value as accurately as possible and to check the pH of the solution again the following day. In most instances the pH is shifted (kO.1-0.2 pH unit). Step heights listed in the various tables are proportional to the effective mobilities of 249

The analysis can he carried out

The analysis of m e zest mixture of anions o r cations shows t h a t the step heights are n o t constant and/or the resolution is bad, and the base. line has a drift.

c

NO

I

I

The micro-sensing electrodes are coated. Depolarize the electrodes in 0 . 1 N HNOl. If this is not effective apply aqua regra. If this is not effective dismount the probe and apply metal polish. Wash the equipment after each procedure with a non-ionic detergent and thoroughly with douhledistilled water.

Perform an analysis c

NO

-

1

I

NO

NO

For the conductivity detector (n.c. method) only

detector (a.c. method).

The narrow-bore tube is

For the UV detector and t h e conductivity detector ( a x . method).

2

YES

YES

i 1

For the UV detector only

4

with a non-ionic detergent and rinse the entire equipment

If after several washings the analysis stlll does not improve, the entire equipment must be dismounted. Polish the various narrow bores with a suitable polish and use another narrow-bore tube. Before an analysis can he carried out, the entire equipment must be washed with a non-ionic detergent and rinsed with double-distilled water.

This easily can he checked. If the time hetween the start of the analysis and the appearance of the first sample zone varies (in our equipment dewrihed in section 7.4.4 the time is shorter). it indicates that a leak is present. Generally the time is constant within 10 sec in an average time of analysis of 15 min.

1

If only the resolution is had: (1)there IS not a liquid-tight connection between the Hamilton (1MM1) valve and the counter electrode compartment or between the counter electrode compartment and the narrow-bore tube; ( 2 ) the plunger of the Hamiltori (1MM1) valve is loose and leaks; (3)the semi-permeable membrane has a leak.

‘The instrument must be waslie; liiorougldy with a non-ionic aurfartant and then thoroughly rinsed with double.distilled water. 4

Fig.8.1. Flowsheet for ‘troubleshooting’. It is assumed that the electronics of the conductivity detector and UV absorption detector are perfect, and the electrolytes of the operational systems me pure and stable. For ‘trouble-shooting’,the electronics of the conductivity detector, the UV absorption detector and the UV source are as discussed in section 6.4.3 for the d.c. method, section 6.4.5 for the a.c. method, section 6.5.2 for the UV source and section 6.5.3 for the UV absorption detector. Later research shows that the sensitivity of the conductivity detector is improved if the complete equipment is rinsed with a solution of 5%silicon grease in pentadecane, followed by a normal washing procedure.

INTRODUCTION

25 1

the various ionic species in the operational systems and they indicate which ions can be separated in a given length of narrow-bore tube, assuming that the concentration differences are not too great. If the concentration differences are too great, a longer narrowbore tube or a counter flow of electrolyte must be used (see Chapter 17). It should be noted that no corrections for the temperatures of consecutive zones have been made to the results presented from either the thermometric or conductivity detector. If the influence of the counter ion chosen is great (high effective mobility in the operational system chosen), greater differences in effective mobility between the sample ions are needed for a complete separation. If the pH of the consecutive zones increases regularly (in anionic separations) or decreases regularly (in cationic separations), small differences in effective mobility are often sufficient because once the ions diffuse into the zone where the pH is higher (anionic separations) or lower (cationic separations), they may attain a greater effective mobility owing to dissociation. More attention is paid to this phenomenon in Chapter 9. It is sometimes easier and quicker to apply two or more operational systems, as the equipment can be rinsed in a few minutes and is then ready for another operational system, than to try to carry out a complete separation in one operational system. More attention is paid to this aspect in Chapter 11. Sometimes only the concentration of the operational system needs to be changed. The sulphate ion, for instance, in the operational system at pH 6 (Table 12.1) moves behind the chloride ion (concentration of the leading ion, C1- = 0.01 N), while it has a greater mobility than the chloride ion if the concentration of the ‘leading’ chloride ion is changed t o 0.001 N . Another example is given in Fig.12.7. In principle, we do not recommend the use of too long a narrow-bore tube, because the voltages needed are too high and electroendosmosis may dominate the separation. If two types of detectors are available, in many instances the analysis can be carried out, in spite of stable mixed zones (see Fig.6.33), in one operational system. From time to time the equipment used in isotachophoretic analyses must be washed well with a nonionic detergent followed by thorough rinsing with double-distilled water. Adsorption of many types of compounds may influence the detection (see Chapter 6), because amongst other effects the {-potential may be changed. Because the resolution of both the W absorption detector and the conductivity detector (ax. method) decreases in such instances, an electrode reaction only must be rejected. We prefer the a.c. method to the d.c. method because the detector indicates more quickly if something is going wrong and measures can be taken directly (see Chapter 6). We recommend that, before a series of analyses, a test mixture of anions or cations should be examined, because it can be seen very quickly if the resolution and reproducibility are adequate. If the test mixture indicates that non-reproducible data can be expected, appropriate measures must be taken, as shown in Fig.8.1. In our analyses, we pay special attention to the pH and the concentration of the terminating electrolyte, although from various papers one may obtain the impression that this is unnecessary. If too high a concentration of the terminating electrolyte is chosen, eluting effects due to the impurities can be expected, the sample zones may still migrate if a 100%counter flow of electrolyte is present (while the regulation is made via the

252

INTRODUCTION

current stabilizing power supply*) and sample ions can be flushed in the terminating electrolyte if the counter flow of electrolyte occurs too soon (Chapter 17). Moreover, quantitative results are non-reproducible if the injection is made at the boundary of the leading and terminating electrolytes because some of the sample is always mixed with the terminating electrolyte. Particularly if experiments are carried out at low concentrations (0.001 N C1-) problems can be expected (see Chapter 10). A wrongly chosen pH of the terminating electrolyte can cause eluting effects by H’ and OH- ions. Non-reproducible results can also be expected, especially if weak acids or weak bases are present in the sample and some of the sample is mixed with the terminating electrolyte. In the following chapters, much data are given on thermometric detectors, but also data obtained with conductivity and UV detectors are given. Data obtained with the thermometric detector can be applied directly, if a conductivity detector is available. However, the opposite is not true in some instances, because the resolution of the conductivity detector (and the W detector) is so much greater. So far, the information obtained with UV absorption detectors does not have qualitative uses. The electrolytes applied in the various compartments must not, of course, contain gas bubbles. Especially when the leading electrolyte is prepared, a surfactant (0.05% of Mowiol) is added, and as a result small gas bubbles can easily be formed. For de-gassing the electrolytes, in our laboratory we use an ultrasonic bath and still have a ‘trip unit’ on our current-stabilizing power supply, which cuts off the electric current immediately (faster than 0.1 sec) if the voltage increases too quickly. It was determined experimentally that the micro-syringes often need to be cleaned, because many impurities found in the isotachopherograms originate from dirty syringes. For washing the equipment and cleaning the syringes, we recommend the detergent Extran (E. Merck, Darmstadt, G.F.R.), which we purify by running it over a mixed-bed ion exchanger. The syringes are cleaned with this surfactant in an ultrasonic bath.

*For an extensive discussion, see the sections 7.5.5 and 17.1.

Chapter 9

Practical aspects SUMMARY

In experimental work on isotachophoresis, unusual effects are sometimes obtained. These effects can be caused when not all of the conditions that are required in order to obtain an isotachophoretic system are fulfilled. In this chapter, some of these phenomena are discussed and a method for comparing and converting results obtained with different types of apparatus is described.

9.1. INTRODUCTION In Chapter 8, some practical information was given concerning the use of the operational systems and the data presented in the Section Applications. It is difficult to give a complete survey of all phenomena that may obscure or disturb the analysis. It will be clear that, especially if narrow-bore systems are chosen, gas bubbles may affect the analysis if they occupy too much of the tube. Once present above a critical size, the temperature will increase, the gas bubbles will expand, and so on. Also, if small gas bubbles are present and if the narrow-bore tube is mounted horizontally instead of vertically, these gas bubbles may migrate, especially at the boundaries of two adjacent zones with a large temperature difference. Many obscure results in both qualitative and quantitative determinations can be expected if the operational systems are used in the wrong way and for the wrong application, e.g., if the buffer does not have a sufficient buffering capacity. Another possibility is that a leading electrolyte may be chosen of which the composition is not constant with time, e.g., owing to an increasing amount of carbonate in operational systems at high pH (if no precautions are taken), or an increasing amount of formic, acetic or propionic acid if the leading electrolyte consists of formaldehyde, acetealdehyde or propionaldehyde, respectively. This chapter summarizes some important general disturbances that can be found in almost all operational systems; specific disturbances are discussed in the chapters to which they belong.

9.2. DISTURBANCES CAUSED BY HYDROGEN AND HYDROXYL IONS 9.2.1. Disturbances from the terminator zone in unbuffered systems Sometimes disturbances can be caused by the presence of a large amount of H+ at low pH, especially in unbuffered systems. An unbuffered system for the separation of cationic species in isotachophoresis can consist of a strong acid as a leading electrolyte (e.g., hydrochloric acid) and a terminator such as Tris. After the introduction of a sample and 25 3

254

PRACTICAL ASPECTS

the separation of the sample ionic species, a series of zones is obtained containing one ionic species of the sample. Two kinds of separation boundaries can be distinguished, viz., a separation boundary between the leading ions (H') and the zone with ions of the sample (Ml), with the highest mobility (we shall call this boundary the 'HI-MI boundary'), and a separation boundary between two zones of sample cations (the 'MI-M,, boundary'). These two types of separation boundaries have different characteristics and are discussed below.

9.2.1.1. HI-MI boundary The zone of the cations M; will always contain H' ions, so that it is essentially a mixed zone of M; cations and H+ ions. The H+ ions are more mobile than the Mi ions and will therefore pass the HI-MI boundary. (In a buffered system they will be removed by the buffer, according to the equilibrium state.) Those H' ions which pass this boundary migrate into the leading electrolyte (hydrochloric acid) zone and create an H+ zone between the leading electrolyte zone and the first sample zone, Mi. Evidently the extra H+ zone has the same H' concentration as the leading electrolyte zone. In fact, this is a moving-boundary procedure. For the Mf zone, the isotachophoretic condition is no longer valid. The speed of this zone is lower than that of the leading electrolyte zone and the step heights will be smaller owing t o the effect of the H+ ions. If the H' concentration in the M; zone is low, the effect mentioned above is very small and almost no disturbances can be expected. If the pH is low in the M; zone, the original H' zone is elongated and the result is longer detection times and smaller step heights. Figs.9.la-9. Id show electropherograms for the situation with A13' as terminator after 0.01 N hydrochloric acid as the leading electrolyte in methanol, as obtained in practice. Fig.9.la shows the original situation, viz., the original leading ion zone H'(1) and the terminator solution A13+(3),which also contains H+. In Figs.9.lb-9.1 d, an increasing amount of H+(2) between the original solution of H'(1) and the mixed zone A13+-H+ is obtained after a longer time of analysis. The original concentration boundary, which will also be present, is neglected.

9.2.1.2. M,-M,, boundary Now two mixed zones are close together, both consisting of a cation of the sample and H' ions. The H' ions of the M;, zone will pass the boundary and will migrate into the M; zone. Calculation of the pH relationship for the two zones (for hypothetical values), including the mass balances for the H+ and OH- ions and the dissociation constant of water, gives imaginary data, assuming a stationary state. Hence no stationary state will exist. If the pH is about 7, the influence on a stationary situation will be small and almost no disturbances can be expected. If the concentrations of H' or OH- are high, elution phenomena will be dominant. If the pH of the second cation zone is low, the H+ concentration will pass the boundary and a mixed zone of Mf and the H+ coming from the Mi, zone is created.

DISTURBANCES CAUSED BY H+ AND OH

255

rt

Fig.9.1. (a)-(d): Simplified electropherograms of the leading electrolyte (HCl) and the terminator (A13') with methanol as solvent, obtained after different times. (e)-(h): Simplified isotachopherograms of the leading electrolyte (HCl) and the terminator (A13+),when a sample of K+ is introduced. Again the experiment is carried out in methanol and various phases are shown. (i)-(1): Simplified isotachopherograms of the leading electrolyte (KC1) and the terminator (A13+),when a sample of Na' is introduced. Again the experiment is carried out in methanol and various phases are shown. T = increasing temperature; t = time.

The step height in the electropherogram will decrease, which results in two zones of the cation M;, viz., the original M; zone and the mixed zone of H+ and M;. After some time, the H+ coming from the Mi, zone covers the whole Mi zone. A situation as described was obtained using a leading electrolyte of 0.01 N hydrochloric acid in methanol and a terminator of A13+.The sample K+ was introduced. Fig.9.le shows the original situation. The first zone is the leading zone consisting of H'( l), the second the original K' zone (2) and the last zone contains A13' plus H' ions (3). In Fig.9.lf the H+ ions have partially penetrated the K'(2a) zone, whereas in Fig.9.lg the H+ ions have nearly reached the leading zone. In Fig.9.lh an enlarged leading zone (la) can be seen. Zone 2 fits the isotachophoretic condition, while zone 2a does not. In Figs.9.li-9.11 a similar procedure is shown for a leading electrolyte of potassium chloride (l), a sample containing Na'(2) and a terminator of A13+(and H') (3). The H+ ions coming from the A13+zone enter the Na' zone (2a) and finally reach the K' zone (la). In order to check the influence of a low pH in the terminator quantitatively, experimental values are compared with theoretical values, as calculated with the model as described in Appendix A. As a terminator, mixtures of hydrochloric acid and potassium chloride at different pH values are used with a leading electrolyte of 0.01 N hydrochloric

256

PRACTICAL ASPECTS

acid. The current was 70 PA. The ratios f L / t Care i taken as a check*. In Fig.9.2, the relationship between the pH of the terminator and the r L / t U ratio is given for theoretical (solid line) and experimental (individual points) values. Good agreement is obtained, showing that a moving-boundary model provides a better description than isotachophoresis. If the influence of background electrolytes such as H' is too great, elution phenomena will appear after a certain time. The zone boundaries become less and less sharp and after a long time they release each other. The elution effects are often caused by electrode reactions when the electrode compartments are not renewed in time; using C1- as a counter ion in methanol (95%, wlw), the following reactions can be expected: 2C1-

* Clz + 2e

+HZO

HOCl HC1+

+CH30H

CHjOCl

In experiments with an unbuffered system, the H+ ions produced disturb the analyses. As an example, the separation of caesium, sodium and lithium with the leading electrolyte 0.01 N hydrochloric acid and terminator cadmium chloride is shown in Fig.9.3a. In Fig.9.3b, the separation of the same mixture in the same system after the terminator

Fig.9.2. Theoretical (line) and experimental (points) relationship between the pH of the terminator solution and the relative detection times for solutions of KCl applied as terminator, in a movingboundary system. * t ~ ' Time of appearance of a sample zone if no ionic species with the same charge as the species studied passes the separation boundary; tU = time of appearance of a sample zone if an ionic species with the same charge as the species studied passes the separation boundary. If tL/ru = 1, an isotachophoretic zone is obtained; if tL/tu< 1, a moving-boundary zone is obtained.

DISTURBANCES CAUSED BY H+ AND OH 5

4

3

2

1

25 I

5

4

3

2

1

1 T

Fig.9.3. Separation of a mixture of cations in an unbuffered electrolyte system. (a) With fresh terminating electrolyte; (b) with old solution the pH of which is changed by the electrode reaction. 1 = H'; 2 = Cst; 3 = Na+;4 = Li+; 5 = Cd". T = increasing temperature; r = time. A thermometric detector was used.

electrolyte has not been renewed for some time is shown. The terminator solution became increasingly acidic and a flow of r m i g r a t e s through all zones towards the cathode compartment. From the phenomena described above, it can be concluded that it is not advisable to work with unbuffered electrolyte systems, where regular renewal of the electrode compartments is necessary. The use of terminator solutions at low pH is undesirable in cationic separations. A similar disturbance can be expected in anionic separations in unbuffered systems as a terminator of high pH is used. A flow of OH- from the terminator zone penetrates all 7 n n ~ rr n i i r i n o rlirtiirhanrpq

RP

Clirriirwrl nhnvp

9.2.2. Disturbances from the leading zone in unbuffered systems In the previous section, disturbances caused by the presence of H' and OH- from the terminator zone and penetrating all preceding zones have been described. Sometimes disturbances can also be caused by H' and OH- from the leading zone penetrating all proceeding zones. Although it is sometimes difficult to recognize whether the disturbances originate from the terminator or from the leading zone, these disturbances have different characteristics. In order to recognize the difference, two detectors have to be used, and from the two signals obtained it can be concluded from which side the disturbance is coming. In this section we discuss the disturbances from the leading zone. In an anionic separation, this disturbance is due to a flow of H', whereas in a cationic separation it is caused by a flow of OH-. Some examples of the latter situation are considered below. If cations are separated in an unbuffered system, a leading electrolyte of, e.g., hydrochloric acid could be used. In such a case, hydrogen will be evolved at the cathode. OH-, possibly formed in the cathode compartment and migrating in the direction of the anode, will meet the H' ions of the leading electrolyte and will be neutralized because the

25 8

PRACTICAL ASPECTS

concentration of H' in the leading zone (normally about 0.01 N hydrochloric acid) is rather high. No disturbances can be expected. However, if a leading electrolyte that consists of a metal chloride, e.g., potassium chloride, is used, hardly any H+ is present in the cathode compartment and it can therefore be expected that OH- will be formed in the cathode compartment according to the equation 2 H z 0 + 2e + Hz + 2 OHThe OH- ions formed will migrate in the direction of the anode but will not meet H+ for neutralization to occur (the leading electrolyte is potassium chloride) and a flow of OHthrough the whole capillary tube, and all zones, will be the result. Of course, this disturbance will be visible if the concentration of the OH- formed is fairly high and if the time of analysis is sufficient for OH- to reach the detector. For a leading electrolyte of lithium chloride, an increase in pH from 6.7 to 10.7 in the cathode compartment could be measured after some experiments. In such a case, disturbances can be expected and renewal of the cathode compartment is necessary. An example of such a disturbance is shown in Fig.9.4. The isotachopherogram shows the disturbances of a flow of OH- from the cathode compartment, moving in an opposite direction to the migration of the sample zones. The

*

rim.

Fig.9.4. Disturbance due to the presence of OH- formed in the cathode compartment in cationic separations. The leading electrolyte is KCl and the terminator is LiCl (unbuffered system). Thermocouple (2) is mounted closer to the cathode compartment and therefore records the disturbance by OH- first (marked with an arrow). The amplification of the signal of thermocouple (1) is twice that of thermocouple (2). T = increasing temperature.

DISTURBANCES CAUSED BY H+ AND OH-

25 9

leading electrolyte was potassium chloride and the terminator was lithium chloride. The first thermocouple (close to the anode compartment) first detects the step height of lithium, but after some time this step height decreases because the flow of OH- has reached this thermocouple. A second thermocouple (close to the cathode compartment) first detects a decreasing step height of the leading electrolyte because OH- reaches this thermocouple first and after some time the step height of lithium appears. It is clear that the disturbance is coming from the cathode compartment, in contrast with the disturbances described in section 9.2.1. A similar disturbance can be expected in the separation of anionic species if H+ is formed in the anode compartment. Fig. 9.5 shows an example in which H' moves in the opposite direction to the anionic zones. Creatinine (pK = 4.88) was used as the 'buffering' counter ion. The leading electrolyte was 0.01 Nhydrochloric acid (pro analysi grade), adjusted to pH 4 by the addition of creatinine. Glutamic acid was used as the terminator. No sample was introduced. A UV absorption detector (256 nm) and a conductivity detector (a.c. method), both described in Chapter 6, were applied. The UV absorption detector was mounted closer to the reservoir of the terminating electrolyte, so that the isotachophoretic zones reached this detector first. As is well known, the W absorption of creatinine is influenced by the pH if it is approximately at its pK value, which is the reason why a disturbance by H' can be made visible. The length of the narrow-bore tube between the anode and cathode compartments

I.

2.

e -

pHe can be seen. In Fig. 12.7c, Fe(CN6)4- is moving in front of Cl-.

"f

2+3+ I--

-

-+t

c

!

t

r

I

: I

t Ib

1"

C

Fig.12.6. Separation of nitrate andsulphate in the operationalsystemat pH 6 (Table 12.1) withwater and deuterium oxide as the solvents. The difference in solvation of these two solvents is clearly visible. 1 = Chloride; 2 = nitrate; 3 = sulphate; 4 = acetate. The current was stabilized at 7 0 PA. A = Increasing W absorption;R = increasing resistance; t = time.

306

SEPARATION OF ANIONIC SPECIES IN AQUEOUS SOLUTIONS

3 @ 2+i

1

3 0

2

Fig.12.7. Isotachopherograms with Fe(CN,)4- as the leading ion at concentrations of (a), 0.01 N; (b), 0.005 N; (c), 0.001 N. Histidine was used as the buffering counter ion. (a) shows that C1- is more mobile than Fe(CN,)4-, but that the original concentration is not changed if this mobile chloride passes the first separation boundary; (b) shows that a mixed zone is obtained; (c) shows that Cl- is less mobile than Fe(CN,)4-. The current was stabilized at 70 MA.A = Increasing W absorption; R = increasing resistance; t = time. 1 = Fe(CN,)4-; 2 = C1-; 3 = MES-; 4 = mixture of anions.

Quantitative analyses could easily be performed, although the leading electrolyte must be freshly prepared each time as it is unstable. It should be repeated that the concentration change in the leading electrolyte of the operational system may give this system completely different properties. The concentration adjustment is not influenced if impurities with a higher mobility than that of the leading ion pass the first separation boundary. The test mixture of anions (Fig.12.5) was also analyzed in the mixture urea-water (1: 1). Differences similar to those in the isotachopherograms shown in Chapter 16 were obtained. Because of the high concentration of urea, these operational systems are difficult to work with, for practical reasons as the eqhpment soon becomes covered with urea. Nevertheless, this system can be used, because many organic substances are more soluble in it and it is not aggressive.

SEPARATION USING CONDUCTIVITY AND UV DETECTORS

307

TABLE 12.7 RELATIVE STEP HEIGHTS OBTAINED IN THE OPERATIONAL SYSTEMS LISTED IN TABLES 12.1, 12.4,12.5 and 12.6 FOR VARIOUS ANIONIC SPECIES The accuracy is better than 4%. The values given are to be used only for the identification of anionic species in isotachophoretic analyses in the operational systems considered. The chloride ion (leading ion) has a relative step height of 0, while the chlorate ion has relative step height of 100. At pH 3.0 the current was stabilized at 90 w A ;at pH 4.5 the current was stabilized at 80 MA;at pH 6.0 the current was stabilized at 70 PA; at pH 7.5 the current was stabilized at 80 MA.h = step height. - _ -- No UV absorbance; - = not measured. Ionic species

pH = 3.0

lOOh hchlorate

Aspartic acid, dl. benzyl Acetic acid Acetic acid, monochloro Acetic acid, dichloro Acetic acid, trichloro Benzoic acid Benzoic acid, p-amino Benzoic acid, 2,4-dihydroxy Benzoic acid, pnitro Butyric acid Cacodylic acid Capric acid Caproic acid Caprylic acid Chlorate Chromic acid Chromic acid, hi Citric acid Enanthic acid Formic acid E'umaric acid Glucaric acid Glucuronic acid Glu tamic acid Glycerinic acid Glycolic acid Gluconic acid Hippuric acid

pH = 4.5 UV absorption

lOOh

pH = 6.0

UV

lOOh

pH UV

7 absorp- 7 absorpchlorate tion chlorate tion

=

100h

hchlorate tion

-

3880

1090

1150 484

1139 466

745

516

487

465

578

537

542

524

656 2810

699 1200

620 726

596 7 23

6290

1780

836

768

1460

930

803

734

1600 5280 n.s.m.* n.s.m. 6520 7280 100 184

890 1240 6610 6260 1930 2320 100 173

771 751 1509 1486 905 1062 100 134

759 725 940 1410 859 1011 100 33

210 1370 6750 1010 980

1370

1180

132 259 988 208 217 355 1033 927 629 456 1023 962

35 178 94 8 209 230

2200 3750 1670 1620

168 51 9 2040 285 338 1310 1510 760 587

-

-

UV

-absorp-

-

-

7.5

_

1019 887 61 1 469 412

308

SEPARATION OF ANIONIC SPECIES IN AQUEOUS SOLUTIONS

TABLE 12.7 (continued) Ionic species

Iodic acid orKetoglutaric acid Lactic acid Laevulinic acid Maleic acid Malic acid Malonic acid Me thacrylic acid Naphthalene-2sulphonic acid Nicotinic acid Nitric acid Nitrous acid Orotic acid Oxalic acid Pelargonic acid Perchloric acid Phenylacetic acid Phosphoric acid Phthalic acid Picric acid F’imelic acid Pivalic acid Propionic acid Propionic acid, p-chloro Pyrazine, 2,3dicarbox ylic acid Pyrazole, 33dicarboxylic acid Salicylic acid Succinic acid Sulphamic acid Sulphanilic acid Sulphuric acid Sulphurous acid Tartaric acid Tartronic acid

pH = 3.0

pH = 4.5

lOOh

uv

‘chlorate

absorp tion

lOOh

pH = 6.0

uv

lOOh

pH = 7.5

w

100h

498

17

500

464

460

915 1910 4220 508 1460 750

__

__

505 1060 1390 491 5 24 469

259 609 744 305 259 209

250 593 719 207 253 169

3360

8

1100

619

6 24

830 5040 20 32 970 400 5970 69

89 100

910 1570 21 31 810 120 1880 63

798 731 35 25 738 93 1150 75

779 667 33 19 720 83 1023 72

823 614 382 766 450 859 635

814

-_ __ __

1210 750 5 00 890 1270 1910 1460

326 346 753 412 821 604

__

820

6 29

565

3680 840 1280 820 4330 7090 4480 1820

__-

50 __

__

__ 90 5

__ __

12

__

72 100

616

73

368

296

290

876 1060 2720 308 1260 33 364 1000 696

65 30

303 686 84 1 311 7 87 49 332 321 274

293 647 301 294 688 60 284 233 157

288 667 279 303 719 48 172 226 142

__ __

100 __

--

__ -_

*n.s.m. = not sufficiently mobile.

w

7 absorp- 7 absorp- - absorpchlorate tion chlorate tion %hlorate tion

SEPARATION USING CONDUCTIVITY AND UV DETECTORS

309

TABLE 12.8 RELATIVE STEP HEIGHTS OBTAINED IN THE OPERATIONAL SYSTEMS AT pH 6 (TABLE 12.1) FOR VARIOUS ANIONIC SPECIES WITH WATER AND DEUTERIUM-OXIDE AS SOLVENTS The accuracy is better than 4%. The values are given for comparison of the two solvents. The experiments with water were carried out at 70 PA and those with deuterium oxide at 80 PA*. In both solvents the chloride ion (leading ion) has a relative step height of 0, while the chlorate ion has a relative step height of 100. h = step height. -- = No UV absorbance. lonic species

40 100 h hchlorate

Aspartic acid, dl-benzyl Acetic acid Acetic acid, monochloro Acetic acid, dichloro Acetic acid, trichloro Benzoic acid Benzoic acid, p-amino Benzoic acid, 2,4-dihydroxy Benzoic acid, p-nitro Butyric acid Cacodylic acid Capric acid Caproic acid Caprylic acid Chlorate Chromic acid Chromic acid, bi Citric acid Enanthic acid Formic acid Furnaric acid Glucuronic acid Glutamic acid Glycolic acid Hippuric acid Iodic acid a-Ketoglutaric acid Lactic acid Laewlinic acid Maleic acid Malic acid Malonic acid Methacrylic acid Naphthalene-2-sulphonic acid Nicotinic acid Nitric acid Nitrous acid Orotic acid

1150 4 84 487 542 620 7 26 836 803 771 751 1509 1486 905 1062 100 134 132 259 988 208 217 1033 927 456 962 464 259 609 744 305 259 209 619 798 731 35 25 7 38

uv absorption (%)

100 h hchlorate

uv absorption

(%I

1244 519 526 583 669 7 86 923 873 840 818 1787 1814 998 1170 100 146 145 277 1099 217 225 1143 1015 503 1060 486 268 643 801 323 27 2 224 680 858 817 28 17 808

(Continued on p. 310)

310

SEPARATION OF ANIONIC SPECIES IN AQUEOUS SOLUTIONS

TABLE 12.8 (continued) Ionic species

HZ0 lOOh -

hcMorate

D*O

uv

lOOh

absorption (%)

&hlorate

W absorption (%)

88 1252 915 677 410 819 972 699 701 321 316 703 314 313 736 57 319 167

93 1150 823 614 382 766 859 635 6 29 296 293 647 301 294 688 60 284 157

Oxalic acid Pelargonic acid Phenylacetic acid Phosphoric acid Phthalic acid Picric acid Pivalic acid Propionic acid Propionic acid, p-chloro Pyrazine, 2,3-dicarboxylic acid Pyrazole, 3,5-dicarboxylic acid Salicylic acid Succinic acid Sulphamic acid Sulphanilic acid Sulphuric acid Sulphurous acid Tartronic acid

-

*The step height is not influenced by this difference in electric current, if conductivity detection is applied (a.c. method). TABLE 12.9 DIFFERENCES IN THE EFFECTIVE MOBILITIES OF SOME ANIONS IN THE OPERATIONAL SYSTEM AT pH 6 (TABLE 12.1) WITH WATER AND DEUTERIUM OXIDE AS THE SOLVENTS The values ( h )are the step heights (mm) that can be measured in the linear trace of the conductivity detector. Ionic species

'H,O

hD,O

Ionic species

hH,O

hD,O ~~

Sulphate Chlorate Chromate Malonate Pyrazole-3,5-dicarboxylate Adipate Acetate

5.6 9.3 12.4 18.8 27.5 37.2 44.9

5.6 9.8 14.2 21.8 30.8 42.8 51.4

p-chloropropionate Benzoate Naphthalene-2sulphonate Glutamate Enanthat e Benzyl-dl-aspartate

58.4 67.5 74.0 86.1 92.2 107.4

69.5 76.6 83.7 99.0 102.2 121.6

Chapter 13

Amino acids, peptides and proteins SUMMARY The separation of amino acids in aqueous solutions at low pH, at high pH and at ‘neutral’ pH when propionaldehyde is added to the electrolytes is discussed. Experimental data for the amino acids in several operational systems are given. The separation of proteins in an operational system at neutral pH is discussed. The addition of a mixture of amphiprotic substances, by which the proteins are diluted in their zone, stabilizes proteins of high moleculer weight, although this technique deviates from the originiil principle of isotachophoresis as discussed in Chapter 4. For small peptides, the addition of amphiprotic substances is not necessary. The time of analysis is approximately 15 min from the start of the experiment to the detection of the last zone.

13.1. AMINO ACIDS 13.1.1. Introduction

The analysis of amino acids is extremely important and nearly all separation techniques have been applied to them. Good results have been obtained by various research workers who analyzed these substances by liquid chromatography [ 1,2] , gas chromatography and electrophoresis. Many references can easily be found and they are not cited here because only an incomplete list could be given. So far, little attention has been paid to the separation of amino acids by isotachophoresis [3-51. In this section, we discuss various systems in which amino acids can be analyzed by isotachophoresis. The application of t h s technique to amino acids is particularly interesting because in theory they can be separated both as cations and as anions. The possibility of achieving a complete separation according to pK values (Chapter 5) is, of course, considered first. It is clear that n o systems at a neutral pH can be chosen, because most amino acids have their pZ values at neutral pH and hence will have a negligible migration in an electric field. It is also well known that amino acids form stable complexes, e.g, with metals and aldehydes. If such a complex is formed, not only the molecular size and solvation change, but also the pK values, and the effective mobility therefore changes in an operational system chosen. Several operational systems are considered below in order to show complex formation and the variations in the effective mobilities. Much more research, however, needs to be carried out. In particular, solvents or a combination of solvents in which the amino acids are more soluble than they are in aqueous systems must be sought. Unusual combinations of systems may be obtained e.g., a combination of urea and water to increase the solubility of the amino acids, to which an aldehyde must be added to decrease the p l 31 1

312

AMINO ACIDS, PEPTIDES AND PROTEINS

values of the amino acids (Schiff base formation) and methanol to stabilize the aldehyde (e.g , propanal). In this section, however, these solvent systems are not discussed as this topic is beyond the scope of this book. The analyses were carried out with the equipment described in Chapter 7, using the modified injection block, the counter electrode compartment with a flat membrane and hgh-resolution detectors. The conductivity detector (a.c. method) and the UV absorption detector (256 nm) were combined. The operational systems considered represent only a few of many possible combinations, and were chosen arbitrarily, although optimal characteristics were sought. Because the operational systems in the equipment can be changed quickly (it usually does not take longer than the normal rinsing and re-filling procedure) and the time of analysis is relatively short (10-1 5 min), sometimes a complete separation between the amino acids in a given mixture can be better achieved by applying two or more systems rather than by optimizing a single system. It will be recalled that a small difference in effective mobility will increase drastically the time of analysis and longer narrow-bore tubes or a counter flow of electrolyte must be applied. The last technique is effective only if the difference in the effective mobilities of the various amino acids is still sufficiently large (see Chapter 17). 13. I .2. Separation at low pH values in aqueous systems

At low pH, most amino acids will migrate as cations. However, most amino acids also have a low effective mobility, so that the pH of the amino acid zone will be lower than that of the leading electrolyte zone. Soon so many H ions are present that a significant proportion of the electricity is carried by the protons. As a result, the amino acids show only small differences in step height as measured in the linear trace of the conductimetric signal*. Only the amino acids L-lysine, L-arginine and L-histidine have a sufficiently high mobility that they can be separated without a visible disturbance of the protons. It does not need further explanation that the pH of the leading electrolyte cannot be decreased too far, because soon zone electrophoretic phenomena occur, e.g., ‘elution’ by the protons. For basic amino acids, an operational system is given in Table 13.1. 13.1.3. Separation at high pH values in aqueous systems

If the pH of the leading electrolyte is above 8, most amino acids will have an effective mobility suitable for a separation according to the isotachophoretic principle. A disadvantage is that at such pH values disturbances from carbon dioxide from the air can be expected. More information on this aspect is given in Chapter 9. If suitable precautions are not taken, the carbonate (hydrogen carbonate) may even obscure the analysis. For optimal results, we found that the electrolytes of the operational systems must be prepared under an atmosphere of nitrogen and stored in polyethylene bottles under *The step height is a measure of the qualitative information in isotachophoretic measurements. It indicates whether a complete separation can be expected, as it is a measure of the effective mobility.

31 3

AMINO ACIDS TABLE 13.1 OPERATIONAL SYSTEM AT pH 5.4 SUITABLE FOR CATIONIC SEPARATIONS

H, 0. Solvent: Electric current @A): Ca. 50-100. Electrolyte -~

Cation Concentration Counter ion PH Additive

Leading

Terminating

K+ 0.01 N CH, COO5.4 0.05% Polyvinyl alcohol (Mowiol)

DL-Ala’ Ca. 0.01 N OH- [added as Ba(OH),]

>I None

nitrogen. Moreover, barium hydroxide of pro analysi quality must be added to the terminating electrolyte in order to prevent any carbonate (hydrogen carbonate) from penetrating into the narrow-bore tube via the reservoir filed with the terminating electrolyte. The addition of barium hydroxide to the leading electrolyte did not improve the separation, however. Because of the high mobility of Ba2+,less sharp boundaries can be expected. If suitable precautions are taken, some carbonate (hydrogen carbonate) may still be detected if an anion with a lugher effective mobility is used as the leading ion. This carbonate (hydrogen carbonate), however, did not obscure both the qualitative and quantitative results. Only a leading ion with an effective mobility higher than or equal to that of the carbonate (hydrogen carbonate) ion can be used, otherwise the carbonate may no longer be visible but may still disturb or at least obscure the analytical result, if it is supported continuously. The resolution will decrease and zone electrophoretic phenomena can soon be expected e.g., ‘elution’ by the carbonate (hydrogen carbonate) ions. Several suitable buffers are commercially available that enable one to work at high pH. Some of them, including bis(3-aminopropyl)amine, trime thylenediamine, L-arginine, 1-methylpiperidine, octylamine, ethanolamine and L-lysine, were tested for purity and effective mobility*. Some of the compounds were found to be very impure and even could not be purified satisfactorily by the usual methods such as distillation, recrystallization or ion exchange. Some of the counter ions show unexpected phenomena, e.g., enforced isotachophoretic systems or undesirable complex formation. Of the compounds mentioned above, only 1 -methyl piperidine, L-arginine, L-lysine, octylamine and ethanolamine gave good results. In Tables 13.2 and 13.3, two operational systems are given in which analyses were performed and for which more data will be given later.

*If a counter ion (buffer) is too mobile, a considerable proportion of the electricity is carried by this counter ion, which results in less sharp zone boundaries and a decrease in resolution.

AMINO ACIDS, PEFTIDES AND PROTEINS

314 TABLE 13.2

OPERATIONAL SYSTEM AT pH ABOUT 9 SUITABLE FOR ANIONIC SEPARATIONS Solvent: H, 0. Electric current (/.LA):CQ. 50-100. Electrolyte Leading

Terminating __-

Anion

5-Br-2,4-diOH-C6H, COO-

Concentration Counter ion PH Additive

0.004 M HOC, H, NH ' 9.0, 9.2, 9.4, 9.6 0.05% Polyvinyl alcohol (Mowiol)

0-Ala- (recrystallized from waterethanol) CQ. 0.01 M Baa+[added as Ba(OH),] Ca. 10.5 None

TABLE 13.3 OPERATIONAL SYSTEM AT pH ABOUT 9 SUITABLE FOR ANIONIC SEPARATIONS Solvent: H, 0. Electric current (fiA): Ca. 50-100. Electrolyte Leading

Terminating

Anion

5-Br-2,4-diOH-C6H, COO-

P- Ala- (recrystallized

Concentration Counter ion PH Additive

0.004 M L-Lys+ 9.1, 9.2, 9.4 0.05% Polyvinyl alcohol (Mowiol)

from wa tere thanol) Ca. 0.01 M Ba2+[added as Ba(OH), J Ca. 10.5 None

The leading ion in these systems was chosen because its effective mobility is almost identical with the effective mobility of the carbonate (hydrogen carbonate) ion. In the linear trace of the conductivity detector, the carbonate (hydrogen carbonate) step is n o longer visible*. The leading ion, however, has a W absorption. If too much carbonate (hydrogen carbonate) is present, it is present in the UV trace, which is used only to mark the UV-absorbing zones and has so far not been applied for qualitative determinations. The carbonate (hydrogen carbonate), if present, is enriched just before the zones of the isotachophoretic 'train', as can be seen in Fig. 13.1. *This simplifies the qualitative information considerably.

315

AMINO ACIDS

20 5.c

8?-

I

654-

32-

f-

1"

Fig.13.1. Isotachopherogram of the separation of a mixture of amino acids obtakled with the operational system listed in Table 13.3. 1 = Chloride; 2 = Asp; 3 = Cys; 4 = I,-Tyr; 5 = Asn; 6 = Ser; 7 = Tyr; 8 = Gly; 9 = Trp; 10 = Ile; 11 = P-Ala. All are L-amino acids. R = Increasing resistance; A = increasing UV absorption; t = time.

AMINO ACIDS, PEPTIDES AND PROTEINS

316

TABLE 13.4 STEP HEIGHTS (mm) FOUND IN THE LINEAR TRACE OF THE CONDUCTIVITY DETECTOR SIGNAL IN THE ISOTACHOPHEROGRAMS OBTAINED WITH THE OPERATIONAL SYSTEM LISTED IN TABLE 13.2. Amino acid

L-Asp L-cys L-Glu I, -L-Tyr L-Ser L-Thr DL-Tyr DL-Met Gly L-His L-Phe L-Ma L-Val L-Trp 3-L-Hyp L-Ile L-Leu P-Ala

PH

9.00

9.20

9.36

9.55

40.5 51.5 49.5 76 112.5 113 135 133 138.5 145 147.5 180 184 191 185 203.5 205 239

33 39.5 40 63 88 98.5 114.5 116 119 124 127.5 154 159 161.5 162 172.5 172.5 210

30.5 35.5 34 59 84 90 107 112.5 106 118 121 147 151 157 151 162.5 164.5 20 1

38.5 34.5 35.5 58 85 94.5 105 117.5 110.5 123 126.5 149 155 161 154 167 170 190

The results obtained when using the operational systems specified in Tables 13.2 and 13.3 are given in Tables 13.4 and 13.5. Differences in step heights of about 15-20 mm are sufficient for a complete separation of the various amino acids. The differences found in the two operational systems considered must be ascribed mainly to the difference in effective mobility of the counter ion used. While in the operational system specified in Table 13.2 about eight amino acids can be separated in a single run, in that specified in Table 13.3 about ten amino acids can be separated simultaneously. L-Lysine has a very small effective mobility at the pH of the leading electrolyte chosen, while the effective mobility of ethanolamine is considerably greater. The idea that pure water, adjusted to a high pH by adding barium hydroxide, can be used as an optimal terminating electrolyte in operational systems at high pH is nearly always wrong. If double-distilled water adjusted to a high pH is applied as the terminating electrolyte*, one can expect the buffer capacity of the counter ion to be insufficient. If, instead, a suitable terminator, e.g., p-alanine, is added to the water, also adjusted to a high pH, the pH of the zone of the terminating electrolyte does not need to be increased so *OH- may carry the electricity because the pH in the zone is increased sufficiently as water is a weak acid in this electrolytic system. This, in combination with the high absolute mobility, will give OHa sufficient high effective mobility.

317

AMINO ACIDS TABLE 13.5

STEP HEIGHTS (mm) FOUND IN THE LINEAR TRACE OF THE CONDUCTIVITY DETECTOR SIGNAL IN THE ISOTACHOPHEROGRAMS OBTAINED WITH THE OPERATIONAL SYSTEM LISTED IN TABLE 13.3. Amino acid

L-Asp L-cys L-Glu I, -L-Tyr L-Ser L-Thr DL-Tyr DL-Met GlY L-His L-Phe L-Ala L-Val L-Trp 3-L-Hyp L-Ile L-Leu

p-Ala

PH

9.01

9.22

9.42

32 40.5 37.5 61 93 95.5 122 118.5 135 122 130 176 169 171 170 188 190 24 0

29 36 33.5 58 82 87.5 111.5 110.5 124.5 128.5 121 165 160 162 161.5 180 178 233

27.5 31 33.5 51 78 88 104 106 117 109 116 155

153 156 152.5 171 170 205

much because 0-alanine is a stronger acid than water under the conditions chosen. Conductimetric recordings of analyses in which double-distilled water, adjusted to a high pH, and analyses in which water plus a suitable terminator are applied, showed that the zone boundaries were less well defined and that the conductivity finally attained is smaller. An isotachopherogram obtained in the operational system specified in Table 13.2 is shown in Fig.13.2. Fig.13.1 and 13.2 also show the differences that can be found in Tables 13.3 and 13.4. Fig.13.3 illustrates the possible mixed zones that can be expected if a mixture of amino acids is analyzed. Two amino acids with close effective mobilities were injected in the system specified in Table 13.3, at a pH of the leading electrolyte of 9. A black square indicates that a mixed zone can be expected in a time of analysis of about 12 min (70pA). A decrease in the concentration of the leading electrolyte did not result in substantial differences in the effective mobilities of the various amino acids. An increase must be avoided because the amino acids are not sufficiently soluble. So far in our laboratory no research has been carried out to find electrolyte systems in which the amino acids are more soluble. Combinations of non-ionic substances with water seem to have good prospects, as the amino acids are not sufficiently soluble in methanol.

AMINO ACIDS, PEPTIDES AND PROTEINS

318 9-

8-

1

20 Sm2

7-

6-

54-

3-

2-

Fig.13.2. Isotachopherogram of the separation of a mixture of amino acids obtained with the operational system listed in Table 13.2. 1 = Chloride; 2 = Asp; 3 = I, -Tyr; 4 = Ser; 5 = Tyr; 6 = Phe; 7 = Ala; 8 = Leu; 9 = p-Ala. All are L-amino acids. A = Increasing UV absorption; R = increasing resistance: t = time.

13.1.4. Separation by use of complex formation

It is well known that amino acids form complexes with metal ions, e.g., Cu*+.In an aqueous solution of copper(I1) sulphate, the addition of various amino acids cause a colour change, which indicates that a complex is formed. Isotachophoretic experiments have shown that only a few of these complexes are sufficiently stable to be detected as real complexes. An isotachopherogram of a copper-histidine complex is shown in Fig.13.4C. In the isotachopherogram in Fig.13.4A, 0.2 pl of 0.01 M L-histidine solution was injected, in Fig.13.4B 0.2 pl of 0.005 M copper(l1) sulphate solution was injected and in Fig.13.4C 0.4 pl of the solution obtained by mixing equal volumes of these two solutions was injected, with the operational system specified in Table 13.1. When other amino acids were examined, however, their complexes were found to be too unstable. No further research was carried out on this aspect. It may be that the field strength applied in isotachophoretic analyses is too great or the complexation constants

AMINO ACIDS

319

Fig.13.3. Schematic diagram illustrating that mixed zones are found in isotachophoretic analyses if the differences in effective mobilities are too small. A black square indicates that a mixed zone is obtained between that pair of amino acids if they are present in one sample. The experiments were carried out in the operational system listed in Table 13.3.

are too small. If so, a method may be found for determining complexation constants in isotachophoretic analyses by varying the field strength in various experiments. 13.1.5. Separation in aqueous propanal solutions

It is well known that amino acids easily form complexes (Schiff bases) with aldehydes. If amino acids are dissolved in a solution that contains an aldehyde, differences in mobility can be expected because the solvent property changes, the amino acid molecule is larger after complex formation and its pZ value changes because the complex is formed with the amino group(s). The first two effects result in a small difference and the last effect in a large difference in mobility. The last effect occurs at very low concentrations of the aldehyde, assuming that the aldehyde character is great enough. Experiments with sugars did not show substantial differences in the effective mobilities of the various amino acids, especially if they were added in relatively low concentrations. Formaldehyde, which has a strong aldehyde character, and acetaldehyde were found to be very unstable. Even during analysis, formic acid and acetic acid, respectively, are formed. Research is continuing to find a suitable combination of a non-ionic stabilizer(s) in order to work reproducibly with formaldehyde and acetaldehyde.

AMINO ACIDS, PEPTIDES AND PROTEINS

320

/

c

/

B

A

Cu-His complex

His +

t

t

Fig.13.4. Isotachopherogramobtained with the operational system listed in Table 13.1. Species injected: A, histidine; B, CuSO,; C, a mixture of the two. R = Increasing resistance; f = time.

With propionaldehyde, experiments could be carried out satisfactorily, although it must be distilled several times under nitrogen. Even after distillation, propionic acid is present in small amounts, but it was found that the amount of propionic acid did not increase during the analysis. A similar disturbance to that discussed briefly in section 13.1.3, due to carbonate (hydrogen carbonate), can be expected; this disturbance does not obscure the analytical results either qualitatively or quantitatively. For optimal information, before the analyses were carried out, the pK values of the various amino acids were determined in aqueous propionaldehyde solutions of various concentrations, and the results are given in Table 13.6. It can be seen that the pK values of the acidic groups decrease, because the amino groups are blocked. The analysis of some amino acids was carried out in a solution containing 3% of propionaldehyde, with the operational system specified in Table 13.7. The leading electrolyte was adjusted to pH 7.2 because it was found that the pK value of ethanolamine was 7.2 in a 3%propionaldehyde solution. Only measurements at ‘neutral pH: are possible with an aqueous solution containing 3% of propionaldehyde. At a pH of the leading electrolyte, which contains propionaldehyde, of above 8, a white insoluble component is formed after some time. In Table 13.8, some step heights of amino acids obtained in the operational system

321

AMINO ACIDS TABLE 13.6 DETERMINATION OF pK VALUES IN AQUEOUS PROPIONALDEHYDE SYSTEMS Propionaldehyde concentration (mole%)

Amino acid

L-His L-G~U* L-AS~* L-Ile L-Leu L-Val L-Phe DL-Ala L-Met L-Ser L-Thr 3-L-Hyp L-Trp L-Tyr GlY L-Arg L-Lys

0.0

2.5

4.0

8.0

9.18 9.47 9.82 9.758 9.744 9.719 9.24 9.866 9.21 9.15

7.81 8.53 8.87 8.38 8.83 8.57 8.17 9.03 8.10 7.60 7.28 9.36 8.50

7.70 8.47 8.58 8.33 8.41 8.45 7.90 8.50 7.90 7.35 7.23 8.95 8.24 8.05 8.31 7.22 8.20

7.93 8.38 8.50 8.15 8.26 8.20 7.88 8.37 7.90 6.35 6.15

9.73 9.39 10.07 9.778 12.48 10.53

8.95 7.62 8.42

8.10 8.00 8.17 7.98

~

* P K ~value.

specified in Table 13.7 are given. Table 13.8 shows that the analysis can be performed at a relatively low pH. By this means, the disturbance due to the carbonate (hydrogen carbonate) is prevented, although in its place a disturbance due to propionic acid has to be dealt with. For the operational system specified in Table 13.7, the possibility of the formation of TABLE 13.7 OPERATIONAL SYSTEM AT pH ABOUT 7.5 SUITABLE FOR ANIONIC SEPARATIONS IN AQUEOUS PROPANAL' SOLUTIONS Solvent: H,0 + 3% C, H , CHO. Electric current k A ) : < 50. Electrolyte

Anion Concentration Counter ion PH Additive

Leading

Terminating

c10.01 N HOC, H, N+H

DL-Ma-

7.2, 7.8 0.05% Polyvinyl alcohol (Mowiol)

c4. 7 ( ms > m g , and the differences in mobility are sufficient for a complete separation, S acts as a ‘Spacer’ for ions A and B. If a component C is added to a sample consisting of ions A and B such that the effective mobility of C is equal to the effective mobility of B in the operational system chosen, component C acts as a ‘carrier’ for ion B. In specific instances it is possible for a component to be added such that a mixed zone is formed between the ions A, B and the component added, although generally an enrichment of A in front and an enrichment of B a t the rear can be expected.

question. One has to bear in mind that although the differences in effective mobility between the compounds of interest remain constant, the separation capacity decreases because compounds are added that have effective mobilities between those of the compounds of interest. The final result of the detection of the various zones, which really move with equal speed, will be less sharp than under ideal isotachophoretic conditions because the self-sharpening effect is much lower. Apart from the addition of, e.g., ampholytes to the leading electrolyte, the terminating electrolyte can also be doped with a suitable ion with an effective mobility higher than that of the most mobile protein. However, in some instances the elution effect due to the substance added may play a dominant role (i.e., isotachophoresis will gradually become zone electrophoresis). Experiments along these lines will not be discussed in this book, because they lie far outside its scope. 13.2.2. Experimental

All experiments described in this section were performed in the operational system specified in Table 13.9. Various operational systems can be used, depending mainly on the particular proteins to be separated. Only a general discussion is presented here but we hope it will be sufficient for scientists interested in the separation of proteins. Glutamic acid was chosen as the leading ion because it is commercially available in a very pure form (‘isotachophoretically pure’) and its mobility is sufficiently high in comparison with that of the most mobile protein at a pH of the leading electrolyte of 7.2. As already indicated in the analyses discussed in section 13.1, the influence of carbonate (hydrogen carbonate) on both the qualitative and quantitative results are negligible,

AMINO ACIDS, PEF'TIDES AND PROTEINS

326 TABLE 13.9

OPERATIONAL SYSTEM AT pH 7.2 SUITABLE FOR ANIONIC SEPARATIONS Solvent:

Electric current @A): Length of narrow-bore tube (cm): UV absorption detector wavelength (nm):

H, 0. Ca. 30-50. Ca. 15. 256.

Electrolyte

Anion

Leading

Terminating

Glu-

Gly- (adjusted to

a sufficiently Concentration Counter ion PH Additive

0.005 M Tris' 7.2 0.05%Polyvinyl alcohol (Mowiol)

high pH) Ca. 0.005M

Tris+ Ca. 9 None

assuming that the necessary precautions as mentioned in section 13.1.I are taken. These precautions must be taken because a less mobile ion is used as the termimator (low effective mobility) and hence the pH will increase considerably. In Fig. 13.8, isotachopherograms for several commercially available ampholytes (LKB) are given. The ampholytes were diluted with double-distilled water (dilution factor 1:20). In each instance 0.2 pl of ampholytes with the pZ ranges (b) 3 . 5 4 , (c) 4-6, (d) 5-8 and (e) 6-8 were injected, and the leading-terminating electrolyte boundary is shown in (a) when no sample was introduced. Fig.13.8a also shows the impurities in the electrolytes. Because the ampholytes were specially developed for use in experiments on isoelectric focusing, it would be fortuitous if they could be applied directly to experiments based on isotachophoretic principles. In order to obtain a better gradient between the leading and terminating electrolytes that would be more suitable for experiments with serum proteins, various commercially available ampholytes were mixed and several of the mixtures were found to be suitable. Obviously, if one is interested in the separation of mobile albumins the most suitable gradient will be completely different from one suitable for the separation of globulins. In the remainder of this section we show some isotachopherograms obtained in the analysis of normal serum and pathological human sera obtained from the St. Josef Hospital, Eindhoven, The Netherlands. The separations were carried out with the gradient shown in Fig. 13.9. The differential trace of the conductivity detector is given in order to show the amount of substances present, which is not so easy to see if only the linear trace is given. The gradient, as already mentioned, was determined experimentally by injecting 0.1 pl of a mixture of ampholytes with different pI ranges in the ratio indicated in Fig.13.9, using the operational system at pH 7.2 (Table 13.9). It proved to be important to include a larger proportion of the ampholytes with low p1 ranges.

SEPARATION OF PROTEINS IN AMPHOLYTE GRADIENTS

327

t

Fig.13.8. Isotachopherogram obtained with the operational system listed in Table 13.9. (a) Boundary between leading and terminating electrolytes; (b) separation of 0.2 rl (dilution 1:20) of ampholyte mixture of PI3.5-4; (c) separation of 0.2 pl (dilution 1:20) of ampholyte mixture of pI4-6; (d) separation of 0.2 pl (dilution 1:20) of ampholyte mixture of PI5-8; (e) separation of 0.2 p1 (dilution 1:20) of ampholyte mixture of PI6-8. A = Increasing UV absorption;R =increasing resistance; t = time.

In order to compare the results obtained from the analyses of sera, a zone electrophoretic separation was also carried out. The experiments were carried out on a porous cellulose polyacetate strip in veronal buffer, the separated proteins subsequently being rendered visible with amido black, washed with acetic acid solution and prepared for a densitometric scan. The results are shown in Fig.13.10 for both normal and pathological sera. The ratios of the proteins present in these sera are given in Table 13.10.

AMINO ACIDS, PEPTIDES AND PROTEINS

328

\

\

30sec

I

R

Fig.13.9. Isotachopherogramobtained with the operational system listed in Table 13.9. A 0.1-p1 volume of a mixture of ampholytes (LKB) with a ratio of pI3.5-4: PI4-6: PI6-8: water of 1:1.5:0.5:20 was injected. A = Increasing UV absorption;R = increasing resistance; 1 = time.

Figs.13.11 and 13.12 show the separations of the sera in Table 13.10 in a narrow-bore tube using a conductivity and a UV absorption detector (256 nm). The UV traces are not shown in Figs.13.1 l a and 13.12a as they do not give much useful information, but if they are of interest Fig.13.8a should be consulted. Fig.13.llb and 13.12b must be compared with Fig.13.10 in order to obtain a comparison of the isotachophoretic and zone electrophoretic separations of serum proteins. In order to obtain Figs.13.1 l c and 13.12c, the sera were diluted with the ampholytes by first injecting 0.2 p1 of the serum to be analyzed into the injection block (Fig.7.5) and then 0.2 p1 of the ampholyte mixture. This procedure was compared with a procedure in which the sera were diluted before the analysis, in a small bottle. The reproducibilities of both dilution techniques were identical, but in the first procedure only a small amount of ampholyte mixture is needed.

329

SEPARATION OF PROTEINS IN AMPHOLYTE GRADIENTS

i

A

l l

5

WL ~

....

--+

..--

Fig.13.10. Separation of a normal serum (A) and a pathological serum (B) by zone electrophoresis. The analysis was carried out on cellulose polyacetate, the proteins subsequently being coloured with amido black. The electropherogram was obtained with a Kipp (Delft, The Netherlands) densitometer. 1 = Albumin; 2 = 01, -globulin; 3 = a,-globulin; 4 = p-globulin; 5 = yglobulin. These sera were used in the analyses in the narrow-bore tubes. TABLE 13.10 COMPOSITIONS OF NORMAL AND PATHOLOGICAL SERA DETERMINED BY ZONE ELECTROPHORESIS ON CELLULOSE POLYACETATE STRIPS This mixture wasused in the analyses presented in Figs.13.11-13.15. Composition (%)

Protein

Albumin 0 1 ~-Globulin ~~,-Glob~~lin p-Globulin y-Globulin

___

Normal serum

Pa thological serum

45 4

47 6

12 17 22

5 4

38

At present it is difficult to draw a conclusion from the results shown in Figs.13.11 and 13.12 because the conditions can vary so easily, resulting in completely different isotachopherograms. The isotachopherograms in Figs. 13.1 1 and 13.12, especially the W traces, must be interpreted in a completely different manner to the traces in Fig.13.10. Although ampholytes are added to the serum proteins in the analysis shown in Figs.13.11 and 13.12, a relationship still exists between the amount of a species introduced into the system and the zone length finally occupied by it, with or without the presence of a supporting

330

AMINO ACIDS, PEPTIDES AND PROTEINS

Fig.13.11. Isotachopherogram of normal serum (Fig.13.10) in an ampholyte gradient (Fig.13.9) obtained with the operational system listed in Table 13.9. (a) Boundary between leading and terminating electrolytes; (b) 0.2 pl of normal serum injected; (c) 0.2 MI of normal serum and 0.2 pl of ampholyte mixture (Fig.13.9) injected. A = Increasing W absorption;R = increasing resistance; t = time.

electrolyte, assuming that it occupies a position between the boundary of the leading and terminating zones. Too easy the W traces in Figs.13.11 and 13.12 will be interpreted in a similar manner to the zone electrophoretic traces in Fig. 13.10. The differential trace from the conductivity detector is not given because it is rather complex and does not give any additional information. The application of a micro-preparative instrument will indicate if the isotachophero-

SEPARATION OF PROTEINS IN AMPHOLYTE GRADIENTS

331

Fig.13.12. Isotachopherogram of pathological serum (Fig.13.10) in an ampholyte gradient (Fig.13.9) obtained with the operational system listed in Table 13.9. (a) Boundary between leading and terminating electrolytes; (b) 0.2 pl of pathological serum injected; (c) 0.2 pl of pathological serum and 0.2 pl of ampholyte mixture (Fig.13.9) injected. A = Increasing W absorption;R = increasing resistance; r = time.

grams shown in Fig.13.11 and 13.12 have a practical value, because by applying specific techniques after the separation more information can be obtained. The isotachopherograms shown in Fig.13.11 and 13.12 may also be the result of the reproducible degradation of the various proteins. Fig.13.13 illustrates the reproducibility of the analysis. In order to show the difference if another ampholyte mixture is applied, the composition was changed, 0.2 pl of

332

AMINO ACIDS, PEPTIDES AND PROTEINS

Fig.13.13. Isotachopherograms of normal serum (Fig.13.10) in an ampholyte gradient obtained with the operational system listed in Table 13.9. The ampholyte mixture had the following composition: PI3.5-4: PI4-6: PI6-8: water = 1 : 1 :0.25 :20. In both A and B, 0.2 pl of normal serum and 0.2 p1 of ampholyte mixture were injected. A = Increasing UV absorption; R = increasing resistance; t = time.

serum* being diluted with 0.2 pl of the ampholyte mixture. The reproducibility in Fig.13.13 is acceptable. Fig. 13.14 demonstrates the effect of a variation in the amount of proteins, in an identical sample, on the shape of an ampholyte gradient. First 0.1 pl of the ampholyte mixture was injected and then (a) 0.1 pl, (b) 0.2 p1 and (c) 0.3 pl of normal serum*. Finally, we present some isotachopherograms that can be compared with those in Fig. 13.14. These isotachopherograms (Fig. 13.1 5) show that an optimum must always be sought in the dilution of the serum with the mixture of ampholytes. If too little ampholyte is added, the sample zones are small and denaturation will soon occur. Separate experiments in which a microscope was used to observe the narrow-bore tube showed that even with human albumin small solid particles are formed (denaturation) that move between the leading and terminating electrolyte zones, and the particles show convection. Under isotachophoretic conditions, thermal degradation of the albumin can soon be expected, as can be understood from Fig.6.7, where the temperatures of zones *The serum for which an analysis is shown in Fig.13.11 was used.

333

SEPARATION OF PROTEINS IN AMPHOLYTE GRADIENTS

t

i.

Fig.13.14. Isotachopherograms of normal serum (Fig.13.10) in an ampholytegradient obtained with the operational system listed in Table 13.9. The ampholyte mixture had the following composition: PI3.5-4: PI4-6: PI6-8: water = 1 : 1 : 0.5: 20. In each instance 0.1 pl of ampholyte mixture was injected, and (a) 0.1 pl, (b) 0.2 pl and (c) 0.3 p1 of normal serum. A = Increasing UV absorption; R = increasing resistance; t = time.

334

AMINO ACIDS, PEPTIDES AND PROTEINS

1 Fig.13.15. Isotachopherogram of normal serum (Fig.13.10) in an ampholyte gradient obtained with the operational system listed in Table 13.9. The ampholyte mixture had the following composition: pI 3.5-4: pI4-6: PI6-8: water = I : 1.5: 0.5 : 20. In each instance 0.2 pl of ampholyte mixture was injected and (a) 0.2 pl, (b) 0.05 pl and (c) 0 p1 of normal serum. A = Increasing UV absorption; R = increasing resistance; t = time.

in a narrow-bore tube, hanging free in air, are plotted. However, in another system than applied for the experiments with proteins. In the terminator zone of the operational system in which the proteins can be separated even higher temperatures can be expected. If too much ampholyte is added to the serum to dilute the protein zone, the resolution is decreased. The isotachopherograms presented in this section show that much more work must be carried out in this field. The compositicils of the ampholyte mixtures and their

SEPARATION OF SMALL PEPTIDES

335

reproducibility are very important, because with the ampholyte mixture a constant gradient, i.e., constant in slope and constant in length, between the leading and terminating electrolyte zones must be maintained. This is the most important rule in this typical version of isotachophoretic analysis. More information can be found in ref. 6.

13.3. SEPARATION OF SMALL PEP'IIDES 13.3.1. Introduction

Less attention will be paid to the separation of small peptides, because most of them can be analyzed both in the system in which amino acids can be separated (Table 13.3) and in the system in which the analyses with the proteins were performed (Table 13.9). A single isotachopherogram will be presented.

Fig. 13.16. Isotachopherogramof the analysis of some small peptides obtained with the operational system listed in Table 13.3. 1 = 5-Bromo-2,4-dihydroxybenzoicacid; 2 = chloride; 3 = glutathione; 4 = glycylglycine; 5 = glycylglycylglycylglycine; 6 = D-leucyl-L-tyrosine; 7 = L-alanine.

336

AMINO ACIDS, PEPTIDES AND PROTEINS

13.3.2. Experimental The operational system used was that specified in Table 13.3. L(+)-Alanine was used as the terminating electrolyte, adjusted to pH 9.8 by addition of barium hydroxide. The leading ion, 5-bromo-2,4-dihydroxybenzoic acid (0.004 M), was adjusted to pH 9.05 by addition of L-lysine. The current was stabilized at 100 MA,and the time of analysis was approximately 8 min. About 0.01 mole of glutathione (Merck, Darmstadt, G.F.R.), glycylglycine hydrochloride, glycylglycylglycylglycine and D-leucyl-L-tyrosine (Nutritional Biochemicals, Cleveland, Ohio, U.S.A.) was injected. The isotachopherogram of the analysis is shown in Fig.13.16. One should note the chloride*, which is more mobile than the 5-bromo-2,4-dihydroxybenzoic acid, which has passed the first separation boundary. Because the chloride is coming from the cathode compartment, it is not a pH disturbance, which may originate from the semi-permeable membrane, especially as the zone is reasonably well defined. It can clearly be seen in the linear traces of both the conductivity detector and the W absorption detector that the concentration of the leading electrolyte is not changed after the passage of the mobile chloride ion.

REFERENCES 1 A. Niederwasser and H. Curtius, Z. Klin. Chem. Klin. Biochem., 5 (1969) 4G4. 2 D.H. Spackman, W.H.Stein and S. Moore, Anal. C h e m , 30 (1958) 90. 3 F.M. Everaerts and A.J.M. van der Put, J. Chromatogr., 5 2 (1970) 415. 4 A. Kopwillem, J. Chromatogr., 82 (1973) 407. 5 A.J. de Kok, Graduation Rep., University of Technology, Eindhoven, 1975. 6 F.E.P. Mikkers, Graduation Rep., University of Technology, Eindhoven, 1974.

*The chloride is derived from the sample component glycylglycine hydrochloride.

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

SEPARATION OF NUCLEOTIDES IN AQUEOUS SYSTEMS

338

U-SLMP

G-