LIQUID CHROMATOGRAPHY–MASS SPECTROMETRY:AN INTRODUCTION

LIQUID CHROMATOGRAPHY– MASS SPECTROMETRY: AN INTRODUCTION Robert E Ardrey University of Huddersfield, Huddersfield, UK LIQUID CHROMATOGRAPHY– MASS SPECTROMETRY: AN INTRODUCTION Analytical Techniques in the Sciences (AnTS) Series Editor: David J Ando, Consultant, Dartford, Kent, UK A series of open learning/distance learning books which covers all of the major analytical techniques and their application in the most important areas of physical, life and materials science Titles Available in the Series Analytical Instrumentation: Performance Characteristics and Quality Graham Currell, University of the West of England, Bristol, UK Fundamentals of Electroanalytical Chemistry Paul M.S Monk, Manchester Metropolitan University, Manchester, UK Introduction to Environmental Analysis Roger N Reeve, University of Sunderland, UK Polymer Analysis Barbara H Stuart, University of Technology, Sydney, Australia Chemical Sensors and Biosensors Brian R Eggins, University of Ulster at Jordanstown, Northern Ireland, UK Methods for Environmental Trace Analysis John R Dean, Northumbria University, Newcastle, UK Liquid Chromatography–Mass Spectrometry: An Introduction Robert E Ardrey, University of Huddersfield, Huddersfield, UK Forthcoming Titles Analysis of Controlled Substances Michael D Cole, Anglia Polytechnic University, Cambridge, UK LIQUID CHROMATOGRAPHY– MASS SPECTROMETRY: AN INTRODUCTION Robert E Ardrey University of Huddersfield, Huddersfield, UK Copyright  2003 John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England Telephone (+44) 1243 779777 Email (for orders and customer service enquiries): cs-books@wiley.co.uk Visit our Home Page on www.wileyeurope.com or www.wiley.com 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, scanning or otherwise, except under the terms of the Copyright, Designs and Patents Act 1988 or under the terms of a licence issued by the Copyright Licensing Agency Ltd, 90 Tottenham Court Road, London W1T 4LP, UK, without the permission in writing of the Publisher Requests to the Publisher should be addressed to the Permissions Department, John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England, or emailed to permreq@wiley.co.uk, or faxed to (+44) 1243 770620 This publication is designed to provide accurate and authoritative information in regard to the subject matter covered It is sold on the understanding that the Publisher is not engaged in rendering professional services If professional advice or other expert assistance is required, the services of a competent professional should be sought Other Wiley Editorial Offices John Wiley & Sons Inc., 111 River Street, Hoboken, NJ 07030, USA Jossey-Bass, 989 Market Street, San Francisco, CA 94103-1741, USA Wiley-VCH Verlag GmbH, Boschstr 12, D-69469 Weinheim, Germany John Wiley & Sons Australia Ltd, 33 Park Road, Milton, Queensland 4064, Australia John Wiley & Sons (Asia) Pte Ltd, Clementi Loop #02-01, Jin Xing Distripark, Singapore 129809 John Wiley & Sons Canada Ltd, 22 Worcester Road, Etobicoke, Ontario, Canada M9W 1L1 Wiley also publishes its books in a variety of electronic formats Some content that appears in print may not be available in electronic books Library of Congress Cataloging-in-Publication Data Ardrey, R E Liquid chromatography-mass spectrometry : an introduction / Robert E Ardrey p cm – (Analytical techniques in the sciences) Includes bibliographical references and index ISBN 0-471-49799-1 (cloth : alk paper) – ISBN 0-471-49801-7 (pbk : alk paper) Liquid chromatography Mass spectrometry I Title II Series QP519.9.L55 A73 2003 543 0894 – dc21 2002028084 British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN 0-471-49799-1 (Cloth) ISBN 0-471-49801-7 (Paper) Typeset in 10/12pt Times by Laserwords Private Limited, Chennai, India Printed and bound in Great Britain by Antony Rowe Ltd, Chippenham, Wiltshire This book is printed on acid-free paper responsibly manufactured from sustainable forestry in which at least two trees are planted for each one used for paper production Contents Series Preface ix Preface xi Acknowledgements xiii Abbreviations, Acronyms and Symbols xv About the Author xix Introduction 1.1 What are the Advantages of Linking High Performance Liquid Chromatography with Mass Spectrometry? 1.2 What Capabilities are Required of the Combination? 1.3 What Problems, if Any, Have to be Addressed to Allow the LC–MS Combination to Function, and Function Effectively? References Liquid Chromatography 2.1 Introduction 2.2 High Performance Liquid Chromatography 2.2.1 Pump 2.2.2 Sample Introduction (Injector) 2.2.3 Mobile Phase 2.2.4 Stationary Phase 2.2.5 Detectors 2.3 Chromatographic Properties 2.4 Identification Using High Performance Liquid Chromatography 7 10 10 11 12 14 16 18 21 vi Liquid Chromatography–Mass Spectrometry 2.5 Quantitation Using High Performance Liquid Chromatography 2.6 The Need for High Performance Liquid Chromatography– Mass Spectrometry References Mass Spectrometry 3.1 Introduction 3.2 Ionization Methods 3.2.1 Electron Ionization 3.2.2 Chemical Ionization 3.2.3 Fast-Atom Bombardment 3.2.4 Matrix-Assisted Laser Desorption Ionization 3.2.5 Negative Ionization 3.3 Ion Separation 3.3.1 The Quadrupole Mass Analyser 3.3.2 The (Quadrupole) Ion-Trap Mass Analyser 3.3.3 The Double-Focusing and Tri-Sector Mass Analysers 3.3.4 The Time-of-Flight Mass Analyser 3.4 Tandem Mass Spectrometry (MS–MS) 3.4.1 Instrumentation 3.4.2 Techniques 3.5 Data Acquisition 3.5.1 Identification 3.5.2 Quantitation 3.6 Processing of Mass Spectral Data 3.6.1 The Total-Ion-Current Trace 3.6.2 Qualitative Analysis 3.6.3 Quantitative Analysis 3.6.4 The Use of Tandem Mass Spectrometry References Interface Technology 4.1 4.2 4.3 4.4 Introduction The Moving-Belt Interface The Direct-Liquid-Introduction Interface The Continuous-Flow/Frit (Dynamic) Fast-Atom-Bombardment Interface 4.5 The Particle-Beam Interface 4.6 The Thermospray Interface 4.7 The Electrospray Interface 4.7.1 The Mechanism of Electrospray Ionization 4.7.2 Sample Types 23 30 31 33 33 36 36 36 38 39 40 40 41 42 42 44 46 47 51 53 53 54 58 58 60 68 71 73 75 75 77 82 85 89 94 98 100 105 Contents 4.7.3 The Appearance of the Electrospray Spectrum 4.7.4 Structural Information from Electrospray Ionization 4.8 The Atmospheric-Pressure Chemical Ionization Interface 4.8.1 The Mechanism of Atmospheric-Pressure Chemical Ionization References Applications of High Performance Liquid Chromatography– Mass Spectrometry 5.1 Method Development 5.1.1 The Use of Experimental Design for Method Development 5.1.2 The Choice of Electrospray or APCI 5.2 The Molecular Weight Determination of Biopolymers 5.2.1 Electrospray Spectra of Co-Eluting Components 5.2.2 The Use of Selected-Ion Monitoring to Examine the Number of Terminal Galactose Moieties on a Glycoprotein 5.2.3 The Effect of Mobile-Phase Additives and Cone-Voltage 5.3 Structure Determination of Biopolymers 5.3.1 Amino Acid Sequencing of Proteins 5.3.2 The Use of Enzymes for Amino Acid Sequencing 5.3.3 The Mass Spectral Fragmentation of Peptides 5.3.4 Confirmation of Amino Acid Sequence Using the Analysis of LC–MS Data from an Enzyme Digest of a Protein 5.3.5 Determination of the Amino Acid Sequence of a Novel Protein Using LC–MS Data from an Enzyme Digest 5.3.6 Amino Acid Sequencing of Polypeptides Generated by Enzyme Digestion Using MS–MS 5.3.7 The Location of Post-Translational Modifications Using LC–MS Data from an Enzyme Digest 5.3.8 The Location of Post-Translational Modifications Using MS–MS 5.3.9 The Analysis of Polysaccharides Present in Glycosylated Proteins 5.3.10 Location of the Position of Attachment of a Glycan on the Polypeptide Backbone of a Glycoprotein 5.4 Molecular Weight Determination of Small (