MULTIDIMENSIONAL LIQUID CHROMATOGRAPHY · MULTIDIMENSIONAL LIQUID CHROMATOGRAPHY Theory and...

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MULTIDIMENSIONAL LIQUID CHROMATOGRAPHY Theory and Applications in Industrial Chemistry and the Life Sciences Edited by STEVEN A. COHEN Life Sciences R&D, Waters Corporation Milford, MA 01757, USA MARK R. SCHURE Theoretical Separation Science Laboratory Rohm and Haas Company Springhouse, PA 19477-0904, USA A JOHN WILEY & SONS, INC., PUBLICATION

Transcript of MULTIDIMENSIONAL LIQUID CHROMATOGRAPHY · MULTIDIMENSIONAL LIQUID CHROMATOGRAPHY Theory and...

  • MULTIDIMENSIONALLIQUIDCHROMATOGRAPHY

    Theory and Applications in IndustrialChemistry and the Life Sciences

    Edited by

    STEVEN A. COHENLife Sciences R&D, Waters CorporationMilford, MA 01757, USA

    MARK R. SCHURETheoretical Separation Science LaboratoryRohm and Haas CompanySpringhouse, PA 19477-0904, USA

    A JOHN WILEY & SONS, INC., PUBLICATION

    InnodataFile Attachment9780470276259.jpg

  • MULTIDIMENSIONALLIQUIDCHROMATOGRAPHY

  • MULTIDIMENSIONALLIQUIDCHROMATOGRAPHY

    Theory and Applications in IndustrialChemistry and the Life Sciences

    Edited by

    STEVEN A. COHENLife Sciences R&D, Waters CorporationMilford, MA 01757, USA

    MARK R. SCHURETheoretical Separation Science LaboratoryRohm and Haas CompanySpringhouse, PA 19477-0904, USA

    A JOHN WILEY & SONS, INC., PUBLICATION

  • Copyright � 2008 by John Wiley & Sons, Inc. All rights reserved.

    Published by John Wiley & Sons, Inc., Hoboken, New JerseyPublished simultaneously in Canada

    No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form orby any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except aspermitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the priorwritten permission of the Publisher, or authorization through payment of the appropriate per-copy fee tothe Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400,fax 978-750-4470, or on theweb at www.copyright.com.Requests to the Publisher for permission shouldbe addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ07030, 201-748-6011, fax 201-748-6008, or online at http://www.wiley.com/go/permission.

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    Library of Congress Cataloging-in-Publication Data:

    Multidimensional liquid chromatography: theory and applications in industrial chemistry and the lifesciences / edited By Steven A. Cohen, Mark R. Schure.

    p. cm.Includes index.ISBN 978-0-471-73847-3 (cloth)

    1. Liquid chromatography. 2. Chemical engineering. 3. Chemistry, Technical.4. Biochemistry. I. Cohen, Steven A., 1953- II. Schure, Mark R.; 1952-QD79.C454M85 2007543’.84–dc22 2007041576

    Printed in the United States of America10 9 8 7 6 5 4 3 2 1

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  • CONTENTS

    Foreword xiii

    Preface xv

    Contributors xvii

    1 Introduction 1

    1.1 Previous Literature Which Covers MDLC 41.2 How this Book is Organized 5References 6

    PART I THEORY 9

    2 Elements of the Theory of MultidimensionalLiquid Chromatography 11

    2.1 Introduction 112.2 Peak Capacity 132.3 Resolution 172.4 Orthogonality 192.5 Two-Dimensional Theory of Peak Overlap 212.6 Dimensionality, Peak Ordering, and Clustering 232.7 Theory of Zone Sampling 242.8 Dilution and Limit of Detection 262.9 Chemometric Analysis 27

    v

  • 2.10 Future Directions 28References 30

    3 Peak Capacity in Two-Dimensional Liquid Chromatography 35

    3.1 Introduction 353.2 Theory 373.3 Procedures 413.4 Results and Discussion 423.5 Conclusions 49Appendix 3A Generation of Random Correlated Coordinates 50Appendix 3B Derivation of Limiting Correlation Coefficient r 54References 56

    4 Decoding Complex 2D Separations 59

    4.1 Introduction 594.2 Fundamentals: The Statistical Description of Complex

    Multicomponent Separations 624.3 Decoding 1D and 2D Multicomponent Separations

    by Using the SMO Poisson Statistics 684.4 Decoding Multicomponent Separations by the

    Autocovariance Function 744.5 Application to 2D Separations 78

    4.5.1 Results from SMO Method 814.5.2 Results from 2D Autocovariance

    Function Method 844.6 Concluding Remarks 88Acknowledgments 88References 88

    PART II COLUMNS, INSTRUMENTATION AND METHODSDEVELOPMENT 91

    5 Instrumentation for Comprehensive MultidimensionalLiquid Chromatography 93

    5.1 Introduction 935.2 Heart-Cutting Versus Comprehensive Mode 955.3 Chromatographic Hardware 97

    5.3.1 Valves 975.4 CE Interfaces 104

    5.4.1 Gated Interface for HPLC–CE 1045.4.2 Microfluidic Valves for On-Chip

    Multidimensional Analysis 105

    vi CONTENTS

  • 5.5 Columns and Combinations 1065.5.1 Column Systems, Dilution, and Splitting 108

    5.6 Detection 1095.7 Computer Hardware and Software 109

    5.7.1 Software Development 1105.7.2 Valve Sequencing 1115.7.3 Data Format and Storage 113

    5.8 Zone Visualization 1155.8.1 Contour Visualization 1155.8.2 2D Peak Presentation 1175.8.3 Zone Visualization in Specific Chemical (pI) Regions 1175.8.4 External Plotting Programs 1175.8.5 Difference Plots 1185.8.6 Multi-channel Data 118

    5.9 Data Analysis and Signal Processing 1195.10 Future Prospects 120

    References 121

    6 Method Development in Comprehensive MultidimensionalLiquid Chromatography 127

    6.1 Introduction 1276.2 Previous Work 1286.3 Column Variables 1306.4 Method Development 130

    6.4.1 The Cardinal Rules of 2DLC Method Development 1326.5 Planning the Experiment 1436.6 General Comments on Optimizing the 2DLC Experiment:

    Speed–Resolution Trade-off 143Acknowledgment 144References 144

    7 Monolithic Columns and Their 2D-HPLC Applications 147

    7.1 Introduction 1477.2 Monolithic Polymer Columns 148

    7.2.1 Structural Properties of Polymer Monoliths 1487.2.2 Chromatographic Properties of

    Polymer Monolithic Columns 1507.2.3 Two-Dimensional HPLC Using Polymer Monoliths 152

    7.3 Monolithic Silica Columns 1537.3.1 Preparation 1547.3.2 Structural Properties of Monolithic Silica Columns 1547.3.3 Chromatographic Properties of

    Monolithic Silica Columns 156

    CONTENTS vii

  • 7.4 Peak Capacity Increase by Using Monolithic Silica Columnsin Gradient Elution 158

    7.5 2D HPLC Using Monolithic Silica Columns 1597.5.1 RP-RP 2D HPLC Using Two Different Columns 1617.5.2 RP–RP 2D HPLC Using Two Similar Columns 1647.5.3 Ion Exchange–Reversed-Phase 2D HPLC

    Using a Monolithic Column for the 2nd-D 1667.5.4 IEX-RP 2D HPLC Using a Monolithic RP

    Capillary Column for the 2nd-D 1687.6 Summary and Future Improvement of 2D HPLC 171References 171

    8 Ultrahigh Pressure Multidimensional Liquid Chromatography 177

    8.1 Background: MDLC in the Jorgenson Lab 1778.1.1 Cation Exchange–Size Exclusion 1788.1.2 Anion Exchange–Reversed Phase 1808.1.3 Cation Exchange–Reversed Phase 1818.1.4 Size Exclusion–Reversed Phase 183

    8.2 Online Versus Off-Line MDLC 1888.3 MDLC Using Ultrahigh Pressure Liquid Chromatography:

    Benefits and Challenges 1898.3.1 An Introduction to UHPLC 1908.3.2 UHPLC for LC�LC: High Speed

    Versus High Peak Capacity 1918.3.3 LC�UHPLC for Separations of Intact Proteins 191

    8.4 Experimental Details 1938.4.1 Instrumentation 1938.4.2 Data Analysis 1948.4.3 Chromatographic Conditions 1958.4.4 Samples 196

    8.5 Results and Discussion 1968.6 Future Directions for UHP-MDLC 202References 203

    PART III LIFE SCIENCE APPLICATIONS 205

    9 Peptidomics 207

    9.1 State of the Art—Why Peptidomics? 2079.2 Strategies and Solutions 2089.3 Summary and Conclusions 218References 218

    viii CONTENTS

  • 10 ATwo-Dimensional Liquid Mass Mapping Techniquefor Biomarker Discovery 221

    10.1 Introduction 22110.2 Methods for Separating and Identifying Proteins 223

    10.2.1 pI-Based Methods of Separation 22310.2.2 Chromatofocusing-A Column Based pH Separation 22510.2.3 Nonporous Separation of Proteins 22610.2.4 Electrospray-Time of Flight-Mass Spectrometry 22810.2.5 MALDI Peptide Mass Fingerprinting 22910.2.6 Data Analysis and Recombination 230

    10.3 Applications 23010.3.1 Proteomic Mapping and Clustering

    of Multiple Samples—Application toOvarian Cancer Cell Lines 230

    10.3.2 2D Liquid Mass Mapping of Tumor Cell LineSecreted Samples, Application toMetastasis-Associated Protein Profiles 233

    10.3.3 Identification Annotation and Data Correlation inMCF10 Human Breast Cancer Cell Lines 235

    10.4 Summary and Conclusions 237Acknowledgments 238References 238

    11 Coupled Multidimensional Chromatography and Tandem MassSpectrometry Systems for Complex Peptide Mixture Analysis 243

    11.1 SCX-RP/MS/MS 24511.2 SCX/RP/MS/MS 24811.3 MudPIT 25111.4 Alternative First Dimension Approaches 25411.5 Conclusion 255References 255

    12 Development of Orthogonal 2DLC Methodsfor Separation of Peptides 261

    12.1 Introduction 26112.2 Previous Work 26312.3 Developing Orthogonal 2DLC Methods 264

    12.3.1 LC Selectivity for Peptides: Experimental Design 26412.3.2 Investigation of 2DLC Orthogonality

    for Separation of Peptides 26612.3.3 Geometric Approach to Orthogonality in 2DLC 27112.3.4 Practical 2DLC Considerations in Proteome Research 27512.3.5 Evaluation of Selected 2DLC MS/MS Systems 276

    CONTENTS ix

  • 12.3.6 Peak Capacity in 2DLC-MS/MS 28012.3.7 Considerations of Concentration Dynamic Range 282

    12.4 Conclusions 284Acknowledgment 284References 284

    13 Multidimensional Separation of Proteins with Online ElectrosprayTime-of-Flight Mass Spectrometric Detection 291

    13.1 Introduction 29113.2 Chromatographic Parameters 29313.3 Analyte Detection and Subsequent Analysis 29313.4 Building a Multidimensional Protein Separation 294

    13.4.1 Selection of an Ion-Exchange–Reversed-PhaseSeparation System for Protein-Level Separations 295

    13.4.2 Chromatographic Sorbent Considerations 29513.4.3 Chromatographic Behavior of Proteins 296

    13.5 Comprehensive Multidimensional Chromatographic Systems 29613.6 Coupling 2DLC with Online ESI–MS Detection 299

    13.6.1 Interactions between the Two Dimensions ofChromatography (Step Vs. Linear) 304

    13.6.2 Recognizing Increased Selectivity in 2DLC Separations 30613.7 Expanding Multidimensional Separations into a “Middle-Out”

    Approach to Proteomic Analysis 30813.8 Future Directions in Protein MDLC 311

    13.8.1 Protein Chromatography 31213.8.2 MS Analysis of Proteins 31313.8.3 Data Interpretation 314

    13.9 Conclusion 314References 315

    14 Analysis of Enantiomeric Compounds Using MultidimensionalLiquid Chromatography 319

    14.1 Online Achiral-Chiral LC-LC 32014.2 Applications 323

    14.2.1 Analysis of Enantiomers in Plasma and Urine 32314.3 Amino Acids 328

    14.3.1 Physiological Fluids or Tissues 32814.3.2 In Food, Beverages, and Other Products 333

    14.4 Other Applications 33414.4.1 Analysis of Enantiomers from Plant

    and Environmental Sources 33414.5 Miscellaneous Applications 33614.6 Conclusion 338References 339

    x CONTENTS

  • PART IV MULTIDIMENSIONAL SEPARATIONUSING CAPILLARY ELECTROPHORESIS 345

    15 Two-Dimensional Capillary Electrophoresis for theComprehensive Analysis of Complex Protein Mixtures 347

    15.1 Introduction 34715.2 Previous Work 348

    15.2.1 Miniaturized IEF/SDS-PAGE 34815.2.2 One-Dimensional Capillary Electrophoresis

    for Protein Analysis 34915.3 Two-Dimensional Capillary Separations for Analysis of

    Peptides and Proteins 35215.3.1 Capillary Liquid Chromatography Coupled

    with Capillary Electrophoresis for Analysis ofUnlabeled Peptides and Proteins 352

    15.3.2 Two-Dimensional Capillary Electrophoresis forAnalysis of Proteins 352

    15.3.3 High-Speed Two-Dimensional Capillary Electrophoresis 35615.3.4 The Analysis of a Single Fixed Cell 358

    15.4 Conclusions 36015.5 Abbreviations 360References 360

    16 Two-Dimensional HPLC–CE Methodsfor Protein/Peptide Separation 365

    16.1 Introduction 36516.2 Off-line Versus Online 36616.3 HPLC Fractionation 36616.4 2D HPLC–CE 36716.5 CE–MS Detection 36816.6 Applications 37016.7 Concluding Remarks 380Acknowledgment 381References 381

    PART V INDUSTRIAL APPLICATIONS 385

    17 Multidimensional Liquid Chromatography inIndustrial Applications 387

    17.1 Introduction 38717.2 Principles of Multidimensional Liquid Chromatography

    as Applied to Polymer Analysis 390

    CONTENTS xi

  • 17.3 Experimental 39317.4 Analysis of Alkylene Oxide-Based Polymers 395

    17.4.1 Amphiphilic Polyalkylene Oxides 39517.5 Excipients 39917.6 Polyether Polyols 40317.7 Analysis of Condensation Polymers 40617.8 Polyamides 40717.9 Aromatic Polyesters 41417.10 Aliphatic Polyesters 417References 420

    18 The Analysis of Surfactants by MultidimensionalLiquid Chromatography 425

    18.1 Introduction 42518.2 Analytical Characterization Methods 428

    18.2.1 CE and CGE 42918.2.2 SEC 43018.2.3 NPLC 43118.2.4 RPLC 433

    18.3 Detection Methods 43418.4 2DLC 434

    18.4.1 RPLC Coupled to SEC 43518.4.2 NPLC Coupled to RPLC 435

    18.5 Conclusions 442References 443

    Index 447

    xii CONTENTS

  • FOREWORD

    The principal rationale for multidimensional separations is that they offer a moreeffective as well as efficient way to generate high peak capacity and thus permit morecomplete resolution of complex mixtures. I suspect, however, that there is anothermotivation that attracts people to multidimensional separations: the resulting two-dimensional chromatograms make fascinating pictures. Two-dimensional separationpatterns are somehow more satisfying than a series of peaks in a one-dimensionalchromatogram. The humanmind is highly adept at dealing with complex informationpresented in the form of images and, despite the complexity, is able to quickly spotdifferences among such patterns.

    Myown inspiration for pursuingmultidimensional separations came fromJ.CalvinGiddings. I was invited to present a seminar at the University of Utah in May 1987,where I spoke on our current research project concerning liquid chromatography inopen-tubular columns. That night over dinner Cal told me that he liked the work Ipresented and also ourworkon capillary electrophoresis, and suggested that I considermultidimensional chromatography as a more practical approach for the resolution ofcomplexmixtures.On the tripback toChapelHill, I thought ofnothingother thanCal’srecommendation and how I might set about to implement it. I was interested inanalyzing samples in their entirety by two-dimensional separations, so I didnotwant tosettle for thewell-established “heart-cutting” approach, where only a single portion ofthe effluent from the first separation dimension is subjected to a second dimensionof separation. Instead, I wanted to subject the entire sample to the full two dimensionsof resolution. Also, I did not want to do two-dimensional separations in space, as intwo-dimensional thin-layer chromatography, but to use coupled columns instead.Thislatter consideration was driven at least in part by the ready coupling to mass

    xiii

  • spectrometry that columns (but not slabs) provide. Upon arriving home, I made planswith my graduate student, Michelle Bushey, to initiate a project on protein separationby two-dimensional liquid chromatography. Michelle also went on to developcomprehensive two-dimensional liquid chromatography-capillary electrophoresis inmy lab. We searched for a suitable term to differentiate our approach from the “heart-cutting’’ style of two-dimensional separations and settled for “comprehensive’’ in orderto emphasize that all of the sample components were subjected to the full twodimensions of separation.

    Multidimensional separations have proven to be quite successful, as evidenced bythewealth of examples of hardware and applications described in this book. It is hopedthat increased awareness and use of multidimensional separations will open uppossibilities for meaningful analyses of truly complex samples and permit the routineanalysis of thousands of components from a single sample in a single run.

    James W. Jorgenson

    Chapel Hill, North Carolina, USA

    September 9, 2007

    xiv FOREWORD

  • PREFACE

    At least two driving forces have contributed to the recent increased use and develop-ment of multidimensional liquid chromatography (MDLC). These include the highresolution and peak capacity needed for proteomics studies and the independent sizeand chemical structure selectivity for resolving industrial polymers. In this regard,separation science focuses on a system approach to separation as individual columnscan contribute only part of the separation task and must be incorporated into a largerseparation system for a more in-depth analytical scheme.

    Separation techniques are increasingly used to resolvemolecular structure at a finerand finer scale and in chemical environments that are fundamentally complex. Thisapplies not only to small and medium-sized (

  • found to be integral to the separation/detection scheme study when a full analysis ofthe information present is to be performed.

    In these themes we have assembled a number of recent contributions that helpdefine the present state of the art and science of MDLC. The coverage of these topicsincludes instrumentation, theory,methods development, applications ofMDLC in thelife sciences, and applications of MDLC in industrial polymer chemistry.

    We have purposely narrowed the scope of all multidimensional chromatography tothose techniques that incorporate separations in the liquid phase and to those in whichthe use of the comprehensive mode prevails but is not exclusive. This text neitherincorporates elements of multidimensional thin-layer chromatography, multidimen-sional separations ingelmedia suchas those commonly employed for the separationofcomplex mixtures of proteins, nor the techniques that utilize multidimensional gaschromatography. Someof the sameprinciples apply, particularly in the theory section,but our emphasis is strictly on separations carried out in the liquid phase and bycolumns, rather than in the gas phase or in planar configurations.

    We would like to thank the contributors to this book for their chapters and for theeasewithwhichwecouldworkwith these authors.We thankHeatherBergmanof JohnWiley Interscience for the opportunity to contribute this text in the active field ofMDLC. Additionally we thank the production team of Brendan Sullivan and RobertEsposito from JohnWiley and Sons, and Ekta Handa from Thomson Digital, and ourrespective wives, Nancy Schure and Donna Cohen, for their infinite patience.

    We hope that this book serves as a useful guide to the field and that it functions asboth a reference and as an enticement to others to enter this field.

    Mark Schure, Blue Bell, PA, USASteve Cohen, Hopkinton, MA, USA

    xvi PREFACE

  • CONTRIBUTORS

    Hiroshi AokiDepartment of Polymer Science and Engineering, Kyoto Institute of Technology,Matsugaski, Sakyo-Ku, Kyoto 606-8585, Japan

    Daniel W. ArmstrongDepartment of Chemistry, Iowa State University, Ames, IA, USA

    Timothy J. BarderEprogen, Inc., Darien, IL 60561, USA

    Scott J. BergerLife Sciences R&D, Waters Corporation, Milford, MA 01757, USA

    Nathan S. BuchananDepartment of Chemistry, The University of Michigan, Ann Arbor, MI 48109, USA

    Kathleen ChoDepartment of Pathology, The University of Michigan, Ann Arbor, MI 48109, USA

    Steven A. CohenLife Sciences R&D, Waters Corporation, Milford, MA 01757, USA

    Amy E. DalyWaters Corporation, Milford, MA 01757, USA

    Joe M. DavisDepartment of Chemistry and Biochemistry, Southern Illinois University at Carbon-dale, Carbondale, IL 62901-4409, USA

    xvii

  • Francesco DondiDepartment of Chemistry, University of Ferrara, 1-44100 Ferrara, Italy

    Norman J. DovichiDepartment of Chemistry, University of Washington, Seattle, WA 98195, USA

    Charles R. EvansDepartment of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill,NC 27599, USA

    John C. GeblerWaters Corporation, Milford, MA 01757, USA

    Martin GilarWaters Corporation, Milford, MA 01757, USA

    Steven GoodisonDepartment of Pathology, University of Florida, Jacksonville, FL 32209, USA

    Attila FelingerDepartment of Analytical Chemistry, University of P�ecs, P�ecs, Hungary

    Melissa M. HarwoodDepartment of Chemistry, University of Washington, Seattle, WA 98195, USA

    Ken HosoyaDepartment of Polymer Science and Engineering, Kyoto Institute of Technology,Matsugaski, Sakyo-Ku, Kyoto 606-8585, Japan

    Tohru IkegamiDepartment of Polymer Science and Engineering, Kyoto Institute of Technology,Matsugaski, Sakyo-Ku, Kyoto 606-8585, Japan

    Haleem J. IssaqLaboratory of Proteomics and Analytical Technologies, SAIC-Frederick Inc.,National Cancer Institute at Frederick, Frederick, MD 21702, USA

    Megan JonesDepartment of Chemistry, University of Washington, Seattle, WA 98195, USA

    James W. JorgensonDepartment of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill,NC 27599, USA

    Hiroshi KimuraDepartment of Polymer Science and Engineering, Kyoto Institute of Technology,Matsugaski, Sakyo-Ku, Kyoto 606-8585, Japan

    James R. KralyDepartment of Chemistry, University of Washington, Seattle, WA 98195, USA

    Paweena KreuninDepartment of Chemistry, The University of Michigan, Ann Arbor, MI 48109, USA

    xviii CONTRIBUTORS

  • David M. LubmanDepartment of Surgery, Comprehensive Cancer Center, The University of Michigan,Ann Arbor, MI 48109, USA

    Egidijus MachtejevasInstitute of Inorganic Chemistry and Analytical Chemistry, Johannes GutenbergUniversity, Duesbergweg 10-14, 55099 Mainz, Germany

    Nicola MarchettiDepartment of Chemistry, University of Ferrara, 1-44100 Ferrara, Italy

    Fred R. MillerKarmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA

    Robert E. MurphyKroungold Analytical, Inc., Encinitas, CA 92024, USA

    Petra OlivovaWaters Corporation, Milford, MA 01757, USA

    Harald PaschDeutsches Kunststoff-Institut (German Institute for Polymers), 64289 Darmstadt,Germany

    Maria C. PietrograndeDepartment of Chemistry, University of Ferrara, 1-44100, Ferrara, Italy

    Frank RittigBASF Aktiengesellschaft, Polymer Research, 67056 Ludwigshafen, Germany

    Mark R. SchureTheoretical Separation Science Laboratory, Rohm and Haas Company, Springhouse,PA 19477-0904, USA

    Renee J. SoukupDepartment of Chemistry, Iowa State University, Ames, IA, USA

    Nobuo TanakaDepartment of Polymer Science and Engineering, Kyoto Institute of Technology,Matsugaski, Sakyo-Ku, Kyoto 606-8585, Japan

    Klaus K. UngerAm Alten Berg 40, 64342 Seeheim, Germany

    Timothy D. VeenstraLaboratory of Proteomics and Analytical Technologies, SAIC-Frederick Inc.,National Cancer Institute at Frederick, Frederick, MD 21702, USA

    Yanfei WangDepartment of Chemistry, The University of Michigan, Ann Arbor, MI 48109, USA

    CONTRIBUTORS xix

  • Michael P. WashburnStowers Institute for Medical Research, 1000 E. 50th St., Kansas City, MO 64110,USA

    Rong WuDepartment of Pathology, The University of Michigan, Ann Arbor, Ml 48109, USA

    xx CONTRIBUTORS

  • 1INTRODUCTION

    Mark R. SchureTheoretical Separation Science Laboratory, Rohm and Haas Company, Springhouse, PA19477-0904, USA

    Steven A. CohenLife Sciences R&D, Waters Corporation, Milford, MA 01757, USA

    Techniques commonly referred toas “MultidimensionalChromatography’’havehadalong and interesting history. One of the first examples of using two dimensions todevelop higher resolution separations utilized paper chromatography with mobilephases applied at right angles in twoseparate development cycles.Thiswas introducedbyMartin and coworkers (Consden et al., 1944). Thenovelty of this techniquewas thatthe separation space was increased because a component zonewas eluting through anarea, not just through a one-dimensional line-like separation axis.

    Theconcept that a separation couldbeconducteddimensionally, that is, separationsin a line (one dimension) and in an area (two dimensions), led to a number ofinnovations in developing separation techniques with an ever-increasing ability atresolving component zones. A great deal of thought regarding both the theoretical andexperimental work in separation science has taken place since this first experiment inplanar chromatography. If extractions are included in an analytical scheme, forexample, extractions of cations, anions, and neutrals, higher dimensional separationsare possible where the separation dimensions are greater than or equal to three. Thedimensional count can be further increased through detectors that detect along awavelength axis (e.g., a diode array detector for use in ultraviolet spectrometry) or amass-to-charge ratio axis (as inmass spectrometry). These detectors can be coupled to

    Multidimensional Liquid Chromatography: Theory and Applications in Industrial Chemistry and the LifeSciences, Edited by Steven A. Cohen and Mark R. Schure.Copyright � 2008 John Wiley & Sons, Inc.

    1

  • other detectors, for example, when mass spectrometers are coupled together (the so-called MS/MS detector), which increases the dimensionality even higher.

    Innovations in separation science continued on this theme and provided one of themost powerful separation techniques used in biochemistry, where proteins areseparated with isoelectric focusing (IEF) applied in one direction, and gel electro-phoresis (GE) applied at a right angle to the first separation direction (O’Farrell, 1975;Celis and Bravo, 1984). In this case, proteins are first separated according to theirisoelectric point,measured in pIunits, and then according to theirmolecularweight bygel electrophoresis. The size separation step is usually aided by addition of asurfactant, most typically sodium dodecyl sulfate (SDS), and the gel material is apolyacrylamide formulation.

    A typical two-dimensional (2D) gel electropherogram of proteins is shown inFigure 1.1.The resolutionof this technique is quite impressive; this resultwas stored inone of many databases containing thousands of protein electropherograms, whichhave been invaluable in molecular biology and medical research. One reason for thecontinued use of planar 2D gels is that it gives high resolution and permits a great dealof information to be extracted about proteins. Protein migration provides informationon the molecular weight and isoelectric point of the individual components. Inaddition, proteins can be isolated from the gel, identified, and cataloged. However,

    FIGURE 1.1 A 2D-gel electrophoresis map of colorectal epithelia cells proteins from theSWISS- 2DPage database (entry CATD_HUMAN, primary access number P07339) accessiblefrom http://www.expasy.org/swiss-2dpage.

    2 INTRODUCTION

  • this process is time consuming, often taking two or more days for separation andprocessing. The isolation of the component zones is not simple, and identification ofthe components can take significantly large amounts of timewhen the components aredigested and further analyzed by liquid chromatography/mass spectrometry to obtaintheir peptide signatures used for protein identification. This time burden does notpermit the planar techniques to be used as fast, sensitive biomarker discovery systemsfor routine investigations. Identification of trace-level zones is particularly problem-atic in planar systems.

    Much effort has beenmade over the past 15 years to replace planar techniques withmodern column-based techniques. The advantages of columns are many includingreproducibility, speed, selectivity, and ease of use amongothers.Another advantage ofcolumns is that they are much easier, in almost all cases, to interface to detectors suchas mass spectrometers.

    The column methods are much faster and are automated so that a much largernumber of samples can be processed per unit time. An example of this technology,described in more detail in Chapter 10 by Lubman and coworkers, is shown inFigure 1.2, where the first dimension is from a chromatofocusing column,which givesseparations in pImuch like isoelectric focusing, onlyhere thepI axis is in bands insteadof continuous pI increments. The second dimension is by reversed-phase liquidchromatography (RPLC).

    This technology is most important in demonstrating the huge capabilitiesthat column chromatography can bring to the separation arsenal. Althoughpredictions regarding the hugepeak capacity thatwouldoccurwhen couplingmultipledimensions were in place by 1984, as discussed in the theory chapter (Chapter 2),

    FIGURE 1.2 2D liquid protein expression map of the HCT-116 human colon adenocarcino-ma cell line. The x-axis is in pI units from 4.0 to 7.0 in 0.2 increments. The y-axis is percent B oftheRP-HPLCgradient. The gray scale of the bands represents the relative intensity of each bandby UV detection at 214 nm. FromYan et al. (2003) with permission of the American ChemicalSociety. (See color plate.)

    INTRODUCTION 3

  • many investigations in polymer separations had shown the power of off-linemultidimensional separations. It was the comprehensive coupling of columntechniques by Erni and Frei (1978) and Bushey and Jorgenson (1990) that lead theway for the continued development of automated column-based comprehensivemultidimensional methods. We use the term comprehensive to denote the repeatedapplication of sampling the kth dimensional column effluent by the (kþ 1)th columnin narrow volume elements. In this manner, the procedure resembles a planarseparation but run exclusively with column methods. The versatility of columns andthe understanding of how to interface columns in amultidimensional instrument wereevident in obtaining a three-dimensional separation implemented approximately 10years ago, as shown in Figure 1.3. Both life science and industrial applications benefitfrom the combination of multidimensional chromatography and advanced detectortechnology. Both of these areas of multidimensional liquid chromatography (MDLC)are covered in this book.

    1.1 PREVIOUS LITERATURE WHICH COVERS MDLC

    Two books of interest have covered some of the ground for multidimensional liquidchromatography in various forms. These include the book Multidimensional Chro-matography, edited by Cortes (1990) and Multidimensional Chromatography byMondello et al. (2002).

    Cortes’ book contains a collection of chapters that discuss many of the aspects ofthe modern MDLC system. Specifically, Giddings’ chapter covers many of thetheoretical underpinnings for themultidimensional techniques includingmulticolumn

    FIGURE 1.3 Three-dimensional representation of a tryptic digest of ovalbumin. The three-dimensional separation consists of size-exclusion chromatography (first dimension), reversed-phase LC (second dimension), and capillary electrophoresis (third dimension). From Mooreand Jorgenson, (1995) with permission of the American Chemical Society.

    4 INTRODUCTION

  • and planar chromatography modes of operation. The role of peak capacity isspecifically discussed and this has been one of the efficacy metrics to judge manyof the multidimensional techniques. These developments will be elaborated on inthe theory section of this book as new results have been obtained since Giddings’chapter in 1990. In addition, the chapter on multidimensional high performanceliquid chromatography by Cortes and Rothman has been a useful reference for earlyapplications where coupled-column systems were utilized. These were not exclu-sively comprehensive but employed heart-cutting techniques that solved chemicalproblems.

    In the second book, Multidimensional Chromatography by Modello, Lewis, andBartle, a number of contributions are made that cover MDLC. These include thechapter by Kazakevich and LoBrutto on industrial and polymer applications that useliquid chromatography (LC), gas chromatography (GC), and supercritical fluidchromatography (SFC) techniques. The introduction by Bartle is general to MDCand covers the topics of peak capacity, statistical overlap, resolution, and columncompatibility. Multidimensional and electrodriven separations are described byDegen and Remcho. However, the emphasis inMondello et al.’s book is undoubtedlyon multidimensional gas chromatography. In the present book, we exclusively dealwith liquid-phase techniques, mostly liquid chromatography with a brief focus oncapillary electrophoresis (CE).

    Other reviews ofmultidimensional separations havebeen published. These includea book on polymer characterization by hyphenated and multidimensional techniques(Provder et al., 1995), a review on polymer analysis by 2DLC (van der Horst andSchoenmakers, 2003), and two reviews on two-dimensional techniques in peptide andprotein separations (Issaq et al., 2005; Stroink et al., 2005). Reviews on multidimen-sional separations in biomedical and pharmaceutical analysis (Dixon et al. 2006) andmultidimensional column selectivity (Jandera, 2006) were recently published. Sug-gested nomenclature and conventions for comprehensive multidimensional chroma-tography were published in 2003 (Schoenmakers et al., 2003), and a book chapter inthe Advances in Chromatography series on MDLC was published in 2006 (Shallikerand Gray 2006).

    1.2 HOW THIS BOOK IS ORGANIZED

    This book is organized into five sections: (1) Theory, (2) Columns, Instrumentation,andMethods, (3)Life Science Applications, (4)Multidimensional SeparationsUsingCapillary Electrophoresis, and (5) Industrial Applications. The first section coverstheoretical topics including a theory overview chapter (Chapter 2), which deals withpeak capacity, resolution, sampling, peak overlap, and other issues that have evolvedthe present level of understandingofmultidimensional separation science. Two issues,however, are presented in more detail, and these are the effects of correlation on peakcapacity (Chapter 3) and the use of sophisticated Fourier analysis methods forcomponent estimation (Chapter 4). Chapter 11 also discusses a new approach toevaluating correlation and peak capacity.

    HOW THIS BOOK IS ORGANIZED 5

  • The columns, instrumentation, and methods chapters, Chapters 5–8, includepresenting the necessary background information for the reader to be brought to themodern literature on the instrumentation used in 2Dmethods. Specifically, Chapter 5gives a general overview,Chapter 6 discusses themethod development neededbesidesthe usual 1D optimizations for chromatographic operation, Chapter 7 discussesmonolithic columns used in 2DLC, and Chapter 8 discusses ultrahigh pressuremultidimensional LC.

    Perhaps the biggest increase in the application and development of the MDLCtechnique since Cortes’s book is in life sciences, which accounts for approximatelyhalf of this book.One reason for thismay be due to the high level of interest in studyingthe human proteome (proteomics). Proteomics is such a demanding application thatthe separating power needed to resolve even the normal proteins in the body is sodemanding thatmaximumseparation power is needed to provide this capability.Manyaspects of separations in proteomics are discussed in Chapters 9–13, 15 and 16.Chapter 14 discusses enantiomeric compound separations by MDLC.

    Industrial applications of MDLC are discussed in Chapters 17 and 18. Thesechapters embody the types of applications used in polymer and surfactant analysisthat have become mainstream in the MDLC literature.

    Most life science applications are currently centered on proteomics. However,other systems biology approaches are also likely to need the resolving power ofMDLC. These approaches include metabolomics, the small-molecule metaboliteprofiles of cellular processes and glycomics, the study of oligosaccharides or chains ofsugars. The developments that are given in the life sciences applications in this bookset the stage for studying these other areas. Inmost cases, themixtures ofmolecules inmetabolomics and glycomics are too complex to study by any single chromatographiccolumn methods. MDLC is well suited for these types of studies, especially when thedetection is by mass spectrometry or some multidimensional version of massspectrometry such as MS–MS.

    The concepts in multidimensional separations are also well suited for otherbiological system separations such as cell separations. Cell separations are generallydone by techniques such as flow cytometry (cell sorting) magnetic encoding,sedimentation, and isopycnic (density gradient) separation. However, techniquessuch as field-flow fractionation (FFF) and dielectrophoresis (separation by nonuni-formACelectric fields)may augment themain techniques in the context of the seconddimension. Having learned a number of experimental and theoretical aspects ofmultidimensional techniques from 2DLC, forming new, more advanced systems thatseparate cells may be expedited through the application of present MDLC research.

    REFERENCES

    Bushey, M.M., Jorgenson, J.W. (1990). Automated instrumentation for comprehensivetwo-dimensional high-performance liquid chromatography of proteins. Anal. Chem. 62,161–167.

    6 INTRODUCTION

  • Celis, J.E., Bravo, R. (1984). Two-Dimensional Gel Electrophoresis of Proteins. AcademicPress, New York.

    Consden, R., Gordon, A.H., Martin, A.J.P. (1944). Qualitative analysis of proteins: a partitionchromatographic method using paper. Biochemical J 38, 224–232.

    Cortes, H.J. (1990). Multidimensional Chromatography, Techniques and Applications, Chro-matographic Series Vol. 50. Marcel Dekker Publishing, New York.

    Dixon, S.P., Pitfield, I.D., Perrett, D. (2006). Comprehensive multi-dimensional liquid chro-matographic separation in biomedical and pharmaceutical analysis: a review. Biomed.Chromatogr. 20, 508–529.

    Erni, F., Frei, R.W. (1978). Two-dimensional column liquid chromatographic technique forresolution of complex mixtures. J. Chromatogr. 149, 561–569.

    Issaq, H.J., Chan, K.C., Janini, G.M., Conrads, T.P., Veenstra, T.D. (2005). Multidimensionalseparations of peptides for effective proteomics analysis. J. Chromatogr. B 817, 35–47.

    Jandera, P. (2006). Review: column selectivity or two-dimensional liquid chromatography. J.Sep. Sci. 29, 1763–1783.

    Mondello, L., Lewis, A.C., Bartle, K.D. (2002). Multidimensional Chromatography. JohnWiley & Sons, Inc., New York.

    Moore,A.W., Jorgenson, J.W. (1995). Comprehensive three-dimensional separation of peptidesusing size exclusion chromatography/reversed-phase liquid chromatography/opticallygated capillary zone electrophoresis. Anal. Chem. 67, 3456–3463.

    O’Farrell, P.H. (1975). High resolution two-dimensional electrophoresis of proteins. J. Biol.Chem. 250, 4007–4021.

    Provder, T., Barth, H.G., Urban, M.W., editors (1995). Chromatographic Characterization ofPolymers: Hyphenated and Multidimensional Techniques, Advance in Chemistry Series247. American Chemical Society,Washington, DC.

    Shalliker, R.A.,Gray,M.J. (2006). In:Grushka, E.,Grinberg,N., editors. Concepts andPracticeof Multidimensional High-Performance Liquid Chromatography Advances in Chromatog-raphy, Vol. 44. Taylor and Francis Group, New York.

    Schoenmakers, P.J., Marriott, P., Beens, J. (2003). Nomenclature and Conventions in Compre-hensive Multidimensional Chromatography. LC�GC Europe, June 2003, 1–4.

    Stroink, T., Ortiz, N.C., Bult, A., Lingeman,H., de Jong,G., Underberg,W.J.M. (2005). On-linemultidimensional liquid chromatography and capillary electrophoresis for peptides andproteins. J. Chromatogr. B 817, 49–66.

    Van der Horst, A., Schoenmakers, P.J. (2003). Comprehensive two-dimensional liquid chro-matography of polymers. J. Chromatogr. A 1000, 693–709.

    Yan, F., Subramanian, B., Nakeff, A., Barder, T.J., Parus, S.J., Lubman, D.M. (2003).A comparison of drug-treated and untreated HCT-116 human colon adenocarcinoma cellsusing a 2-D liquid separation mapping method based on chromatofocusing pI fractionation.Anal. Chem. 75, 2299–2308.

    REFERENCES 7