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  • Practical High-Performance Liquid Chromatography

    FIFTH EDITION

    Veronika R. Meyer

    Swiss Federal Laboratories for Materials Testing and

    Research (EMPA), St. Gallen, Switzerland

  • Practical High-Performance Liquid Chromatography

    Fifth Edition

  • Practical High-Performance Liquid Chromatography

    FIFTH EDITION

    Veronika R. Meyer

    Swiss Federal Laboratories for Materials Testing and

    Research (EMPA), St. Gallen, Switzerland

  • This edition first published 2010

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    Library of Congress Cataloging-in-Publication Data

    Meyer, Veronika.

    [Praxis der Hochleistungs-Fl€ussigchromatographie. English]

    Practical high-performance liquid chromatography / Veronika R. Meyer. – 5th ed.

    p. cm.

    Includes bibliographical references and index.

    ISBN 978-0-470-68218-0 (cloth) – ISBN 978-0-470-68217-3 (pbk.)

    1. High performance liquid chromatography. I. Title.

    QD79.C454M4913 2010

    543’.84–dc22

    2009052143

    A catalogue record for this book is available from the British Library.

    ISBN H/bk 978-0470-682180 P/bk 978-0470-682173

    Set in 10/12pt, Times Roman by Thomson Digital, Noida

    Printed and bound in Great Britain by TJ International, Padstow, Cornwall

    Cover photo:

    Allmenalp waterfall at Kandersteg, Switzerland (Veronika R. Meyer)

    www.wiley.com

  • To the memory of Otto Meyer

    Alles ist einfacher, als man denken kann, zugleich verschränkter, als zu begreifen ist.

    Goethe, Maximen

    Everything is simpler than can be imagined, yet more intricate than can be comprehended.

  • Contents

    Preface to the Fifth Edition . . . . . . . . . . . . . . . . . . . . . xiii

    Important and Useful Equations for HPLC . . . . . . . . . . . . . . 1

    1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1.1 HPLC: A powerful separation method . . . . . . . . . . 5 1.2 A first HPLC experiment . . . . . . . . . . . . . . . . . 5 1.3 Liquid chromatographic separation modes . . . . . . . 8 1.4 The HPLC instrument. . . . . . . . . . . . . . . . . . . 9 1.5 Safety in the HPLC laboratory . . . . . . . . . . . . . . 10 1.6 Comparison between high-performance liquid

    chromatography and gas chromatography . . . . . . . 11 1.7 Comparison between high-performance liquid

    chromatography and capillary electrophoresis . . . . . 12 1.8 Units for pressure, length and viscosity . . . . . . . . . 13 1.9 Scientific journals . . . . . . . . . . . . . . . . . . . . 14 1.10 Recommended books . . . . . . . . . . . . . . . . . . 15

    2 Theoretical Principles . . . . . . . . . . . . . . . . . . . . . . 17 2.1 The chromatographic process . . . . . . . . . . . . . . 17 2.2 Band broadening . . . . . . . . . . . . . . . . . . . . . 19 2.3 The chromatogram and its purport . . . . . . . . . . . 23 2.4 Graphical representation of peak pairs with

    different degree of resolution . . . . . . . . . . . . . . 30 2.5 Factors affecting resolution . . . . . . . . . . . . . . . 35 2.6 Extra-column volumes (dead volumes) . . . . . . . . . 40 2.7 Tailing . . . . . . . . . . . . . . . . . . . . . . . . . . 41 2.8 Peak capacity and statistical resolution probability . . . 46 2.9 Effects of temperature in HPLC . . . . . . . . . . . . . 49 2.10 The limits of HPLC . . . . . . . . . . . . . . . . . . . . 51 2.11 How to obtain peak capacity . . . . . . . . . . . . . . . 55

    vii

  • 3 Pumps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 3.1 General requirements . . . . . . . . . . . . . . . . . . 59 3.2 The short-stroke piston pump . . . . . . . . . . . . . . 59 3.3 Maintenance and repair . . . . . . . . . . . . . . . . . 62 3.4 Other pump designs . . . . . . . . . . . . . . . . . . . 63

    4 Preparation of Equipment up to Sample Injection . . . . . . . 65 4.1 Selection of the mobile phase . . . . . . . . . . . . . . 65 4.2 Preparation of the mobile phase . . . . . . . . . . . . . 67 4.3 Gradient systems. . . . . . . . . . . . . . . . . . . . . 68 4.4 Capillary tubing . . . . . . . . . . . . . . . . . . . . . 70 4.5 Fittings . . . . . . . . . . . . . . . . . . . . . . . . . . 72 4.6 Sample injectors . . . . . . . . . . . . . . . . . . . . . 74 4.7 Sample solution and sample volume . . . . . . . . . . 78

    5 Solvent Properties . . . . . . . . . . . . . . . . . . . . . . . 81 5.1 Table of organic solvents . . . . . . . . . . . . . . . . 81 5.2 Solvent selectivity . . . . . . . . . . . . . . . . . . . . 83 5.3 Miscibility. . . . . . . . . . . . . . . . . . . . . . . . . 83 5.4 Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . 84 5.5 Shelf life of mobile phases. . . . . . . . . . . . . . . . 87 5.6 The mixing cross . . . . . . . . . . . . . . . . . . . . . 88

    6 Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 6.1 General . . . . . . . . . . . . . . . . . . . . . . . . . . 91 6.2 UV detectors . . . . . . . . . . . . . . . . . . . . . . . 96 6.3 Refractive index detectors . . . . . . . . . . . . . . . . 99 6.4 Fluorescence detectors . . . . . . . . . . . . . . . . . 101 6.5 Electrochemical (amperometric) detectors . . . . . . 103 6.6 Light-scattering detectors . . . . . . . . . . . . . . . 104 6.7 Other detectors . . . . . . . . . . . . . . . . . . . . . 106 6.8 Multiple detection . . . . . . . . . . . . . . . . . . . 107 6.9 Indirect detection . . . . . . . . . . . . . . . . . . . . 108 6.10 Coupling with spectroscopy . . . . . . . . . . . . . . 109

    7 Columns and Stationary Phases . . . . . . . . . . . . . . . 117 7.1 Columns for HPLC . . . . . . . . . . . . . . . . . . . 117 7.2 Precolumns. . . . . . . . . . . . . . . . . . . . . . . 119 7.3 General properties of stationary phases . . . . . . . . 120 7.4 Silica . . . . . . . . . . . . . . . . . . . . . . . . . . 125 7.5 Chemically modified silica . . . . . . . . . . . . . . . 126 7.6 Styrene-divinylbenzene . . . . . . . . . . . . . . . . 129

    viii Contents

  • 7.7 Some other stationary phases . . . . . . . . . . . . . 133 7.8 Column care and regeneration. . . . . . . . . . . . . 136

    8 HPLC Column Tests . . . . . . . . . . . . . . . . . . . . . . 141 8.1 Simple tests for HPLC columns . . . . . . . . . . . . 141 8.2 Dete