HPLC Made to Measure

A Practical Handbook for Optimization

Edited byStavros Kromidas

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InnodataFile Attachment9783527611997.jpg

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HPLC Made to Measure

Edited byStavros Kromidas

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Related Titles

S. Kromidas

Practical Problem Solving in HPLC

2000

ISBN 3-527-29842-8

S. Kromidas

More Practical Problem Solving in HPLC

2004

ISBN 3-527-31113-0

V. R. Meyer

Practical High-Performance Liquid Chromatography

2004

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Troubleshooting HPLC SystemsA Bench Manual

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HPLC Made to Measure

A Practical Handbook for Optimization

Edited byStavros Kromidas

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The Editor

Dr. Stavros KromidasRosenstrasse 1666125 SaarbrückenGermany

All books published by Wiley-VCH are carefullyproduced. Nevertheless, authors, editors, andpublisher do not warrant the information containedin these books, including this book, to be free oferrors. Readers are advised to keep in mind thatstatements, data, illustrations, procedural details orother items may inadvertently be inaccurate.

Library of Congress Card No.: applied for

British Library Cataloguing-in-Publication DataA catalogue record for this book is available fromthe British Library

Bibliographic information published byDie Deutsche BibliothekDie Deutsche Bibliothek lists this publicationin the Deutsche Nationalbibliografie; detailedbibliographic data is available in the Internet at

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

All rights reserved (including those of translationinto other languages). No part of this book may bereproduced in any form – by photoprinting,microfilm, or any other means – nor transmitted ortranslated into a machine language without writtenpermission from the publishers. Registered names,trademarks, etc. used in this book, even when notspecifically marked as such, are not to be consideredunprotected by law.

Printed in the Federal Republic of GermanyPrinted on acid-free paper

Cover Design SCHULZ Grafik-Design,FußgönheimTypesetting Manuela Treindl, LaaberPrinting betz-druck GmbH, DarmstadtBinding J. Schäffer GmbH, Grünstadt

ISBN-13: 978-3-527-31377-8ISBN-10: 3-527-31377-X

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V

Foreword

HPLC has become the analytical method against which all others are measured andcompared. It is perhaps the most widely employed method of analysis of all thoseinstrumental approaches that have ever been or are now in vogue. Having beeninvolved with HPLC for perhaps the past 35 years, since the early 1970s, I have seenthe technique and field grow and prosper, academically and commercially. It hasbecome an incredible commercial success, and the cornerstone of many academiccareers in analytical and other fields of chemistry. Annual, dedicated meetings, aswell as major parts of ACS, ASMS, AAPS and AAAS meetings, are routinelydevoted to talks and discussions on or involving HPLC. Though it has not quitedisplaced GC or flat-bed electrophoresis, it has surely been highly competitive forvolatiles and biological macromolecules, respectively. Indeed, one could argue thatit is the very first technique that most analysts, biologists or biochemists wouldconsider investigating and applying for virtually any class of analytes, regardlessof molecular weight, size, volatility, ionic charges, polarity, hydrophobicity, or otherphysical or chemical properties. HPLC has become a technique that can be appliedto virtually any analyte or class of analytes, almost without regard to the propertiesthereof. There are very few other analytical methods for which this can be claimed.HPLC has truly become the “800 pound gorilla”, and it may be virtually impossiblefor any other technique to displace it from this niche in the analytical world, noteven CEC or 2DE or multidimensional CE.

Why then another book dealing with this same topic? I have read some othertexts by Stavros Kromidas, and was thus eager to preview this current one. Thistext is really an edited book, though Stavros Kromidas has contributed severalexcellent chapters of his own. The other contributions come from an internationalgroup of invited authors, mainly from the US, Canada, and Western Europe.Virtually all of these individuals are well known in the HPLC community, such asUwe Neue, Michael McBrien, Lloyd Snyder, John Dolan, Klaus Unger, and so forth.Most, if not all, have been heavily involved in HPLC matters for decades, andhave, in their own right, become well-regarded and recognized experts in theirvarious fields. The book is heavily practice and practicality oriented, in that itaims to help the readers become more knowledgeable and better adept at usingvarious forms of and approaches in HPLC. However, it is not a “Methods”-typetext, such as those published by Humana Press; it is not just a compilation ofpractice-oriented HPLC methods for various analytes.

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VI

Rather, the text is quite detailed, with scientific discussions and theory, lots ofequations and principles, reference to a variety of practical software, and with anemphasis on understanding the fundamentals in each and every chapter. This isnot an introductory text; it is not meant as a text for a graduate AnalyticalSeparations type course. Rather, it is quite an advanced text, dealing with manyrecent and contemporary aspects of HPLC. It deals with approaches for methodoptimization, currently available software and practices, chemometrics, principalcomponent analysis, the selection of ideal stationary phases, and tools for columncharacterization and method optimization. Of course, it deals extensively withreversed-phase HPLC, but it also covers many other areas, including GPC/SEC,affinity chromatography, chiral separations, microLC, nanoLC, and even micro-chip-based LC instrumentation and techniques. It also deals with immuno-chromatographic methods, two-dimensional HPLC (MDLC), LC-MS, LC-NMR,and even how magic-angle spinning NMR spectroscopy can be used to betterunderstand the selectivity of stationary phases in HPLC. All in all, there are overtwo-dozen individual chapters, some authored by the same author(s), but mostnot. It is to the Editor’s credit that he has not written most or even close to 50% ofthe total chapters, but rather that he has invited the most highly regarded andbest-known authors, young and old, to contribute in areas of their unique expertise.He has made an exceptionally good selection of such authors, each of whom hasdone an admirable job in their final writings and efforts.

This is not a book that you will pick up and read in a single sitting; that wouldappear impossible, even for those of us who have already devoted a major portionof our careers to researching and developing HPLC areas. It is not an easy read; itis not a trivial text. Rather, it is clearly an advanced, involved, and detailed text. Itis a book to be read slowly and carefully, because it contains an incredible amountof useful and important practical knowledge. It also covers the very latestdevelopments in HPLC, not just the fundamentals, but where the field standstoday, and where it is going tomorrow. It is a practical handbook for the optimiza-tion of HPLC and its ultimate application, but it is far from being just a handbookor “how to do it” text. It is really more of a summary of where HPLC stands today,what can be done with its various techniques and instrumentation, and what isimportant to know about its future developments and applications. It is anincredibly useful and practical tome, collated by experts pooling their expertise,and it will make better chromatographers out of those of us who take up thebook, study it carefully, and then apply its lessons to our own future needs. It isnot a simplistic methods development type book, though it does aim to help usoptimize and improve our methods development approaches. It is far more than“just” a Practical Handbook for Optimization, though the subtitle might makethat suggestion.

It is my hope that those of you thinking of purchasing this particular, newertext on HPLC, and those who have already made this wise decision and are aboutto pursue the text itself, will benefit from these choices. They were and are wisechoices; now it is up to you to make the most of the book, which means not justreading the text and studying the figures and tables, but making every effort

Foreword

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VII

possible to really understand what the authors are trying to impart to the readers.This may require re-reading of the same chapter more than once; I did – actuallyseveral times, as these are not easy chapters or contributions. However, in thelong run, the time will be well spent and such efforts will be rewarded, for thebook is truly a wealth of useful and practical information. Obviously, I highlyrecommend the book to those contemplating purchase and study, for it is reallyone of the better texts to have come along in many years dealing with this, one ofour very favorite subjects, HPLC.

January 2006 Ira S. KrullAssociate ProfessorDepartment of Chemistry and Chemical BiologyNortheastern UniversityBoston, MA, USA

Foreword

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IX

Preface

The optimizing of practices and processes constitutes an essential prerequisitefor long-term success. The objective and the motives may be very different: self-preservation among living things, “saving lives” among volunteers in Africa,maximizing profits among marketing strategists, new discoveries among scien-tists. This principle is of course also valid in chemistry and in analytics.

This book deals exclusively with the subject of optimization in HPLC. The aimis to examine this important aspect of HPLC from diverse perspectives. First, wehave set out the fundamental aspects, encompassing the principal considerationsand background information. At the same time, we have endeavored to presentand discuss as many practical examples, ideas, and suggestions as possible forthe everyday application of HPLC. The implementation of concepts for rapidoptimization should equally aid and support the planning of effective methoddevelopment strategies as in daily practice at the laboratory bench. The aim of thebook is to contribute to purposeful, affordable, forward-looking method develop-ment and optimization in HPLC.

To this end, internationally renowned experts have offered their knowledge andexperience. I extend my sincere thanks to these colleagues. I also thank Wiley-VCH,in particular Steffen Pauly, for their valued collaboration and good cooperation.

Saarbrücken, January 2006 Stavros Kromidas

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XI

Contents

Foreword V

Preface IX

List of Contributors XXV

Structure of the Book XXXI

1 Fundamentals of Optimization 1

1.1 Principles of the Optimization of HPLC Illustrated byRP-Chromatography 3Stavros Kromidas

1.1.1 Before the First Steps of Optimization 31.1.2 What Exactly Do We Mean By “Optimization”? 51.1.3 Improvement of Resolution (“Separate Better”) 61.1.3.1 Principal Possibilities for Improving Resolution 81.1.3.2 What has the Greatest Effect on Resolution? 101.1.3.3 Which Sequence of Steps is Most Logical When Attempting

an Optimization? 111.1.3.4 How to Change k, α, and N 171.1.3.4.1 Isocratic Mode 171.1.3.4.2 Gradient Mode 181.1.3.4.3 Acetonitrile or Methanol? 191.1.4 Testing of the Peak Homogeneity 221.1.5 Unknown Samples: “How Can I Start?”; Strategies and Concepts 351.1.5.1 The “Two Days Method” 361.1.5.2 “The 5-Step Model” 391.1.6 Shortening of the Run Time (“Faster Separation”) 481.1.7 Improvement of the Sensitivity

(“To See More”, i.e. Lowering of the Detection Limit) 48

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1.1.8 Economics in HPLC (“Cheaper Separation”) 481.1.9 Final Remarks and Outlook 51

References 57

1.2 Fast Gradient Separations 59Uwe D. Neue, Yung-Fong Cheng, and Ziling Lu

1.2.1 Introduction 591.2.2 Main Part 591.2.2.1 Theory 591.2.2.2 Results 611.2.2.2.1 General Relationships 611.2.2.2.2 Short Columns, Small Particles 621.2.2.2.3 An Actual Example 641.2.2.3 Optimal Operating Conditions and Limits of Currently Available

Technology 661.2.2.4 Problems and Solutions 671.2.2.4.1 Gradient Delay Volume 671.2.2.4.2 Detector Sampling Rate and Time Constant 681.2.2.4.3 Ion Suppression in Mass Spectrometry 69

References 70

1.3 pH and Selectivity in RP-Chromatography 71Uwe D. Neue, Alberto Méndez, KimVan Tran, and Diane M. Diehl

1.3.1 Introduction 711.3.2 Main Section 711.3.2.1 Ionization and pH 711.3.2.2 Mobile Phase and pH 731.3.2.2.1 Buffer Capacity 741.3.2.2.2 Changes of pK and pH Value in the Presence of an Organic

Solvent 761.3.2.3 Buffers 781.3.2.3.1 Classical HPLC Buffers 781.3.2.3.2 MS-Compatible pH Control 791.3.2.4 Influence of the Samples 791.3.2.4.1 The Sample Type: Acids, Bases, Zwitterions 801.3.2.4.2 Influence of the Organic Solvent on the Ionization of the

Analytes 811.3.3 Application Example 811.3.4 Troubleshooting 851.3.4.1 Reproducibility Problems 851.3.4.2 Buffer Strength and Solubility 861.3.4.3 Constant Buffer Concentration 861.3.5 Summary 87

References 87

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XIIIContents

1.4 Selecting the Correct pH Value for HPLC 89Michael McBrien

1.4.1 Introduction 891.4.2 Typical Approaches to pH Selection 901.4.3 Initial pH Selection 911.4.4 Basis of pKa Prediction 921.4.5 Correction of pH Based on Organic Content 931.4.6 Optimization of Mobile Phase pH Without Chemical Structures 941.4.7 A Systematic Approach to pH Selection 961.4.8 An Example – Separation of 1,4-Bis[(2-pyridin-2-ylethyl)thio]butane-

2,3-diol from its Impurities 971.4.9 Troubleshooting Mobile Phase pH 1021.4.10 The Future 1021.4.11 Conclusion 103

References 103

1.5 Optimization of the Evaluation in Chromatography 105Hans-Joachim Kuss

1.5.1 Evaluation of Chromatographic Data – An Introduction 1051.5.2 Working Range 1051.5.3 Internal Standard 1061.5.4 Calibration 1071.5.5 Linear Regression 1071.5.6 Weighting Exponent 1101.5.7 In Real Practise 1111.5.8 Drug Analysis 1111.5.9 Measurement Uncertainty 1121.5.10 Calibration Line Through the Origin 115

References 115

1.6 Calibration Characteristics and Uncertainty – Indicating StartingPoints to Optimize Methods 117Stefan Schömer

1.6.1 Optimizing Calibration – What is the Objective? 1171.6.2 The Essential Performance Characteristic of Calibration 1181.6.3 Examples 1181.6.3.1 Does Enhanced Sensitivity Improve Methods? 1181.6.3.2 A Constant Variation Coefficient – Is it Good, Poor or Just an

Inevitable Characteristic of Method Performance? 1221.6.3.3 How to Prove Effects Due to Matrices – May the Recovery Function

be Replaced? 1331.6.3.4 Having Established Matrix Effects – Does Spiking Prove Necessary

in Every Case? 136

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1.6.3.5 Testing Linearity – Does a Calibration Really Need to Fit a StraightLine? 139

1.6.3.6 Enhancing Accuracy – Obtaining ‘Robust’ Calibration Functionswith Weighting 143References 147

2 Characteristics of Optimization in Individual HPLC Modes 149

2.1 RP-HPLC 151

2.1.1 Comparison and Selection of Commercial RP-Columns 151Stavros Kromidas

2.1.1.1 Introduction 1512.1.1.2 Reasons for the Diversity of Commercially Available RP-Columns –

First Consequences 1512.1.1.2.1 On Polar Interactions 1562.1.1.2.2 First Consequences 1562.1.1.3 Criteria for Comparing RP-Phases 1742.1.1.3.1 Similarity According to Physico-chemical Properties 1742.1.1.3.2 Similarity Based on Chromatographic Behavior;

Expressiveness of Retention and Selectivity Factors 1752.1.1.3.3 Tests for the Comparison of Columns and Their Expressiveness 1812.1.1.4 Similarity of RP-Phases 1952.1.1.4.1 Selectivity Maps 1962.1.1.4.2 Selectivity Plots 2002.1.1.4.3 Selectivity Hexagons 2052.1.1.4.4 Chemometric Analysis of Chromatographic Data 2292.1.1.5 Suitability of RP-Phases for Special Types of Analytes and Proposals

for the Choice of Columns 2332.1.1.5.1 Polar and Hydrophobic RP-Phases 2332.1.1.5.2 Suitability of RP-Phases for Different Classes of Substances 2372.1.1.5.3 Procedure for the Choice of an RP-Column 248

References 253

2.1.2 Column Selectivity in RP-Chromatography 254Uwe D. Neue, Bonnie A. Alden, and Pamela C. Iraneta

2.1.2.1 Introduction 2542.1.2.2 Main Section 2552.1.2.2.1 Hydrophobicity and Silanol Activity (Ion Exchange) 2552.1.2.2.2 Polar Interactions (Hydrogen Bonding) 2592.1.2.2.3 Reproducibility of the Selectivity 261

References 263

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2.1.3 The Use of Principal Component Analysis for the Characterizationof Reversed-Phase Liquid Chromatographic Stationary Phases 264Melvin R. Euerby and Patrik Petersson

2.1.3.1 Introduction 2642.1.3.2 Theory of Principal Component Analysis 2652.1.3.3 PCA of the Database of RP Silica Materials 2672.1.3.3.1 PCA of Polar Embedded, Enhanced Polar Selectivity, and AQ/Aqua

Phases 2692.1.3.3.2 PCA of Perfluorinated Phases 2702.1.3.4 Use of PCA in the Identification of Column/Phase Equivalency 2712.1.3.5 Use of PCA in the Rational Selection of Stationary Phases for

Method Development 2772.1.3.5.1 Proposed Solvent/Stationary Phase Optimization Strategy 278

References 279

2.1.4 Chemometrics – A Powerful Tool for Handling a Large Numberof Data 280Cinzia Stella and Jean-Luc Veuthey

2.1.4.1 Introduction 2802.1.4.2 Chromatographic Tests and Their Importance in Column

Selection 2802.1.4.3 Use of Principal Component Analysis (PCA) in the Evaluation

and Selection of Test Compounds 2812.1.4.3.1 Physicochemical Properties of Test Compounds 2812.1.4.3.2 Chromatographic Properties of Test Compounds 2842.1.4.4 Use of PCA for the Evaluation of Chromatographic Supports 2852.1.4.4.1 Evaluation of Chromatographic Supports in Mobile Phases

Composed of pH 7.0 Phosphate Buffer 2862.1.4.4.2 Evaluation of Chromatographic Supports in Mobile Phases

Composed of pH 3.0 Phosphate Buffer 2892.1.4.5 How a Chromatographic Test can be Optimized by

Chemometrics 2912.1.4.5.1 Test Compounds 2912.1.4.5.2 Mobile Phases 2922.1.4.5.3 Chromatographic Parameters and Batch (Column)

Reproducibility 2922.1.4.6 Conclusion and Perspectives 295

References 295

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2.1.5 Linear Free Energy Relationships (LFER) – Tools for ColumnCharacterization and Method Optimization in HPLC? 296Frank Steiner

2.1.5.1 Characterization and Selection of Stationary Phases for HPLC 2962.1.5.2 What are LFERs and Why can they be Profitable in HPLC? 2972.1.5.3 How to Obtain Analyte Descriptors for the Multivariate

Regression 3002.1.5.4 LFER Procedure Using the Solvation Equation 3012.1.5.4.1 Comparing Stationary Phases on the Basis of LFER Data 3012.1.5.4.2 The Influence of the Mobile Phase Expressed in LFER

Parameters 3072.1.5.4.3 The Prediction of Chromatographic Selectivity from LFER Data 3082.1.5.5 An Empirical Approach to the Determination of LFER Solute

Parameters (Descriptors) from HPLC Data 3102.1.5.5.1 How does this Strategy Differ from the Use of Predetermined

Solute Descriptors 3102.1.5.5.2 The Experimental Plan 3112.1.5.5.3 Determination of the Five LFER Parameters – A Procedure in

Eight Steps 3122.1.5.5.4 Variation of the Eluent Conditions 3152.1.5.5.5 Stationary Phase Characterization with Empirical LFER

Parameters 3172.1.5.6 Concluding Remarks on LFER Applications in HPLC 319

References 320

2.1.6 Column Selectivity in Reversed-Phase Liquid Chromatography 321Lloyd R. Snyder and John W. Dolan

2.1.6.1 Introduction 3212.1.6.2 The “Subtraction” Model of Reversed-Phase Column Selectivity 3232.1.6.3 Applications 3262.1.6.3.1 Selecting “Equivalent” Columns 3262.1.6.3.2 Selecting Columns of Very Different Selectivity 3302.1.6.4 Conclusions 332

References 333

2.1.7 Understanding Selectivity by the Use of Suspended-StateHigh-Resolution Magic-Angle Spinning NMR Spectroscopy 334Urban Skogsberg, Heidi Händel, Norbert Welsch, and Klaus Albert

2.1.7.1 Introduction 3342.1.7.2 Is the Comparison Between NMR and HPLC Valid? 3372.1.7.3 The Transferred Nuclear Overhauser Effect (trNOE) 3402.1.7.4 Suspended-State 1H HR/MAS T1 Relaxation Measurements 3432.1.7.5 Where do the Interactions Take Place? 345

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2.1.7.6 Hydrogen Bonding 3452.1.7.7 Some Practical Considerations 3452.1.7.8 Future Aspects 347

References 347

2.2 Optimization in Normal-Phase HPLC 349Veronika R. Meyer

2.2.1 Introduction 3492.2.2 Mobile Phases in NP-HPLC 3502.2.3 Stationary Phases in NP-HPLC 3542.2.4 Troubleshooting in Normal-Phase HPLC 356

References/Further Reading 357

2.3 Optimization of GPC/SEC Separations by Appropriate Selectionof the Stationary Phase and Detection Mode 359Peter Kilz

2.3.1 Introduction 3592.3.2 Fundamentals of GPC Separations 3602.3.2.1 Chromatographic Modes of Column Separation 3622.3.2.2 GPC Column Selection Criteria and Optimization of GPC

Separations 3642.3.2.2.1 Selection of Pore Size and Separation Range 3642.3.2.2.2 Advantages and Disadvantages of Linear or Mixed-Bed

Columns 3652.3.2.3 HighSpeed GPC Separations 3672.3.3 The Role of Comprehensive Detection in the Investigation

of Macromolecular Materials 3692.3.3.1 Coupling of Liquid Chromatography with Information-Rich

Detectors 3712.3.3.2 Copolymer GPC Analysis by Multiple Detection 3722.3.3.3 Simultaneous Separation and Identification by GPC-FTIR 3752.3.3.4 Application of Molar Mass-Sensitive Detectors in GPC 3772.3.3.4.1 Light-Scattering Detection 3772.3.3.4.2 Viscometry Detection 3792.3.4 Summary 380

References 381

2.4 Gel Filtration/Size-Exclusion Chromatography (SEC) of Biopolymers –Optimization Strategies and Troubleshooting 383Milena Quaglia, Egidijus Machtejevas, Tom Hennessy, and Klaus K. Unger

2.4.1 Where Are We Now and Where Are We Going? 3832.4.2 Theory in Brief 3842.4.3 SEC vs. HPLC Variants 387

Contents

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2.4.4 Optimization Aspects in SEC of Biopolymers 3882.4.4.1 Column Selection and Optimal Flow Rate 3882.4.4.2 Optimization of the Mobile Phase 3922.4.4.3 Sample Preparation 3942.4.4.4 Sample Viscosity and Sample Volume – Two Critical Parameters

at Injection 3952.4.4.5 Detection Methods 3962.4.5 Applications 3972.4.5.1 High-Performance SEC 3972.4.5.2 Determination of Molecular Weight 3982.4.5.3 Gel Filtration as a Tool to Study Conformational Changes of

Proteins 3982.4.5.4 Gel Filtration in Preparative and Process Separations

(Downstream Processing) 3992.4.5.5 SEC Columns Based on the Principle of Restricted Access

and Their Use in Proteome Analysis 400References 403

2.5 Optimization in Affinity Chromatography 405Egbert Müller

2.5.1 Introduction to Resin Design and Method Development inAffinity Chromatography 405

2.5.2 Base Matrix 4082.5.3 Immobilization Methods 4092.5.4 Activation Methods 4092.5.5 Spacer 4122.5.6 Site-Directed Immobilization 4152.5.7 Non-Particulate Affinity Matrices 4162.5.8 Affinity Purification 4172.5.9 Factorial Design for the Preparation of Affinity Resins 4192.5.10 Summary of Immobilization 423

References 423

2.6 Optimization of Enantiomer Separations in HPLC 427Markus Juza

2.6.1 Introduction 4272.6.2 Basic Principles of Enantioselective HPLC 4272.6.2.1 Thermodynamic Fundamentals of Enantioselective HPLC 4292.6.2.2 Adsorption and Chiral Recognition 4302.6.2.3 Differences to Reversed-Phase and Normal-Phase HPLC 4332.6.2.4 Principles for Optimization of Enantioselective HPLC

Separations 4332.6.3 Selectors and Stationary Phases 4332.6.4 Method Selection and Optimization 440

Contents

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2.6.4.1 Cellulose and Amylose Derivatives 4412.6.4.2 Immobilized Cellulose and Amylose Derivatives 4432.6.4.3 Stationary Phases Derived from Tartaric Acid 4442.6.4.4 π-Acidic and π-Basic Stationary Phases 4442.6.4.5 Macrocyclic Selectors, Cyclodextrins, and Antibiotics 4462.6.4.6 Proteins and Peptides 4502.6.4.7 Ruthenium Complexes 4502.6.4.8 Synthetic and Imprinted Polymers 4502.6.4.9 Metal Complexation and Ligand-Exchange Phases 4512.6.4.10 Chiral Ion Exchangers 4512.6.5 Avoiding Errors and Troubleshooting 4522.6.5.1 Equipment and Columns – Practical Tips 4522.6.5.2 Detection 4542.6.5.3 Mistakes Originating from the Analyte 4542.6.6 Preparative Enantioselective HPLC 4542.6.6.1 Determination of the Loading Capacity 4552.6.6.2 Determination of Elution Volumes and Flow Rates 4562.6.6.3 Enantiomer Separation using Simulated Moving Bed (SMB)

Chromatography 4582.6.6.3.1 Principles of Simulated Moving Bed Chromatography 4582.6.6.3.2 Separation of Commercial Active Pharmaceutical Ingredients by

SMB 4592.6.7 Enantioselective Chromatography by the Addition of

Chiral Additives to the Mobile Phase in HPLC and CapillaryElectrophoresis 461

2.6.8 Determination of Enantiomeric Purity Through the Formation ofDiastereomers 462

2.6.9 Indirect Enantiomer Separation on a Preparative Scale 4622.6.10 Enantiomer Separations Under Supercritical Fluid Chromatographic

(SFC) Conditions 4622.6.11 New Chiral Stationary Phases and Information Management

Software 4632.6.12 Summary 463

References 464

2.7 Miniaturization 467

2.7.1 mLC/NanoLC – Optimization and Troubleshooting 467Jürgen Maier-Rosenkranz

2.7.1.1 Introduction 4672.7.1.2 Sensitivity 4672.7.1.2.1 Influence of Column Length 4672.7.1.2.2 Influence of Column Internal Diameter 4672.7.1.2.3 Influence of Stationary Phase 4692.7.1.3 Robustness 469

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2.7.1.3.1 System Choice 4692.7.1.3.2 Capillary Connections 4722.7.1.3.3 Precautions Against Blocking 4772.7.1.3.4 Testing for Leakages 4782.7.1.3.5 Guard Column Switching and Sample Loading Strategies 4782.7.1.4 Sensitivity/Resolution 4832.7.1.4.1 Column Dimensions 4832.7.1.4.2 Packing Materials/Surface Covering 4842.7.1.4.3 Detectors 484

References 486

2.7.2 Microchip-Based Liquid Chromatography – Techniques andPossibilities 487Jörg P. Kutter

2.7.2.1 Introduction 4872.7.2.2 Techniques 4882.7.2.2.1 Pressure-Driven Liquid Chromatography (LC) 4882.7.2.2.2 Open-Channel Electrochromatography (OCEC) 4882.7.2.2.3 Packed-Bed Electrochromatography 4882.7.2.2.4 Microfabricated Chromatographic Beds (Pillar Arrays) 4892.7.2.2.5 In Situ Polymerized Monolithic Stationary Phases 4892.7.2.3 Optimization and Possibilities 4902.7.2.3.1 Separation Performance 4902.7.2.3.2 Isocratic and Gradient Elution 4912.7.2.3.3 Tailor-Made Stationary Phases 4922.7.2.3.4 Sample Pretreatment and More-Dimensional Separations 4922.7.2.3.5 Issues and Challenges 4922.7.2.4 Application Examples 4932.7.2.5 Conclusions and Outlook 496

References 496

2.7.3 Ultra-Performance Liquid Chromatography 498Uwe D. Neue, Eric S. Grumbach, Marianna Kele,Jeffrey R. Mazzeo, and Dirk Sievers

2.7.3.1 Introduction 4982.7.3.2 Isocratic Separations 4992.7.3.3 Gradient Separations 502

References 505

Contents

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3 Coupling Techniques 507

3.1 Immunochromatographic Techniques 509Michael G. Weller

3.1.1 Introduction 5093.1.2 Binding Molecules 5093.1.3 Immunoassays 5113.1.4 Immunochromatographic Techniques 5113.1.4.1 Affinity Enrichment (Affinity SPE) 5133.1.4.2 “Weak Affinity Chromatography”

(True Affinity Chromatography) 5193.1.4.3 Biochemical Detectors 5203.1.5 Examples 5223.1.5.1 Example 1: Affinity Extraction (Affinity SPE) 5223.1.5.2 Example 2: “Weak Affinity Chromatography” (WAC) 5233.1.5.3 Example 3: Biochemical Detection 525

References 525

3.2 Enhanced Characterization and Comprehensive Analysesby Two-Dimensional Chromatography 527Peter Kilz

3.2.1 Introduction 5273.2.2 How Can I Take Advantage? – Experimental Aspects 5293.2.3 2D Data Presentation and Analysis 5333.2.4 The State-of-the-Art in 2D Chromatography 5353.2.5 Summary 539

References 540

3.3 LC/MS – Hints and Recommendations on Optimization andTroubleshooting 541Friedrich Mandel

3.3.1 Optimization of the Ionization Process 5413.3.2 Lost LC/MS Peaks 5423.3.2.1 Mobile Phase pH at the Edge of the Optimum Range 5433.3.2.2 Ion-Pairing Agents in the HPLC System 5433.3.2.3 Ion Suppression by the Sample Matrix or Sample

Contaminants 5443.3.3 How Clean Should an LC/MS Ion Source Be? 5443.3.4 Ion Suppression 545

References 549

Contents

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3.4 LC-NMR Coupling 551Klaus Albert, Manfred Krucker, Karsten Putzbach, and Marc D. Grynbaum

3.4.1 NMR Basics 5513.4.2 Sensitivity of the NMR Experiment 5523.4.3 NMR Spectroscopy in Flowing Systems 5533.4.4 NMR Probes for LC-NMR 5533.4.5 Practical Realization of Analytical HPLC-NMR and

Capillary-HPLC-NMR 5543.4.6 Continuous-Flow Measurements 5553.4.7 Stopped-Flow Measurements 5573.4.8 Capillary Separations 5593.4.9 Outlook 560

References 563

4 Computer-Aided Optimization 565

4.1 Computer-Facilitated HPLC Method Development Using DryLab®

Software 567Lloyd R. Snyder and Loren Wrisley

4.1.1 Introduction 5674.1.1.1 History 5694.1.1.2 Theory 5704.1.2 DryLab Capabilities 5704.1.2.1 DryLab Operation 5704.1.2.2 Mode Choices 5714.1.3 Practical Applications of DryLab® in the Laboratory 5724.1.4 Conclusions 584

References 585

4.2 ChromSword® Software for Automated and Computer-AssistedDevelopment of HPLC Methods 587Sergey Galushko, Vsevolod Tanchuk, Irina Shishkina, Oleg Pylypchenko,and Wolf-Dieter Beinert

4.2.1 Introduction 5874.2.1.1 Off-Line Mode 5874.2.1.2 On-Line Mode 5874.2.2 ChromSword® Versions 5874.2.3 Experimental Set-Up for On-Line Mode 5884.2.4 Method Development with ChromSword® 5884.2.4.1 Off-Line Mode (Computer-Assisted Method Development) 5884.2.4.2 On-Line Mode – Fully Automated Optimization of Isocratic

and Gradient Separations 5924.2.4.2.1 Software Functions for Automation 597

Contents

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4.2.4.2.2 How Does the System Optimize Separations? 5974.2.5 Conclusion 600

References 600

4.3 Multifactorial Systematic Method Development and Optimizationin Reversed-Phase HPLC 601Michael Pfeffer

4.3.1 Introduction and Factorial Viewpoint 6014.3.2 Strategy for Partially Automated Method Development 6034.3.3 Comparison of Commercially Available Software Packages with

Regard to Their Contribution to Factorial Method Development 6084.3.4 Development of a New System for Multifactorial Method

Development 6094.3.4.1 Selection of Stationary Phases 6114.3.4.2 Optimizing Methods with HEUREKA 6124.3.4.3 Evaluation of Data with HEUREKA 6184.3.5 Conclusion and Outlook 623

References 623

5 User Reports 625

5.1 Nano-LC-MS/MS in Proteomics 627Heike Schäfer, Christiane Lohaus, Helmut E. Meyer, and Katrin Marcus

5.1.1 Proteomics – An Introduction 6275.1.2 Sample Preparation for Nano-LC 6285.1.3 Nano-LC 6295.1.4 On-Line LC-ESI-MS/MS Coupling 6335.1.5 Off-Line LC-MALDI-MS/MS Coupling 6355.1.5.1 Sample Fractionation 6355.1.5.2 MALDI-TOF-MS/MS Analyses 6355.1.6 Data Analysis 6375.1.7 Application in Practice: Analysis of α-Crystallin A in Mice Lenses 637

References 640

5.2 Verification Methods for Robustness in RP-HPLC 643Hans Bilke

5.2.1 Introduction 6435.2.2 Testing Robustness in Analytical RP-HPLC by Means of

Systematic Method Development 6435.2.3 Robustness Test in Analytical RP-HPLC by Means of Statistical

Experimental Design (DoE) 6525.2.4 Conclusion 665

References 666

Contents

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5.3 Separation of Complex Sample Mixtures 669Knut Wagner

5.3.1 Introduction 6695.3.2 Multidimensional HPLC 6705.3.3 Techniques for Multidimensional Separations 6725.3.3.1 Off-Line Technique 6725.3.3.2 On-Line Technique 6725.3.4 On-Line Sample Preparation as a Previous Stage of

Multidimensional HPLC 6745.3.5 Fields of Application of Multidimensional HPLC 6755.3.5.1 What can be Realized? – A Practical Example 6765.3.6 Critical Parameters of Multidimensional HPLC 682

References 683

5.4 Evaluation of an Integrated Procedure for the Characterization ofChemical Libraries on the Basis of HPLC-UV/MS/CLND 685Mario Arangio, Federico R. Sirtori, Katia Marcucci,Giuseppe Razzano, Maristella Colombo, Roberto Biancardi, andVincenzo Rizzo

5.4.1 Introduction 6855.4.2 Materials and Methods 6865.4.2.1 Instrumentation 6865.4.2.2 Chemicals and Consumables 6865.4.2.3 High-Throughput Platform (HTP1) Method Set-up 6885.4.2.4 Chromatographic Conditions 6885.4.2.5 Mass Spectrometer and CLND Conditions 6895.4.2.6 Data Processing and Reporting 6895.4.2.7 Multilinear Regression Analysis for the Derivation of CLND

Response Factors 6905.4.3 Results and Discussion 6915.4.3.1 Liquid Chromatography and UV Detection 6915.4.3.2 Mass Spectrometric Method Development 6925.4.3.3 CLND Set-Up 6935.4.3.4 Validation with Commercial Standards 6935.4.3.5 Validation with Proprietary Compounds 6955.4.4 Conclusions 699

References 700

Appendix 703

Subject Index 729

Contents

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Klaus AlbertInstitute of Organic ChemistryUniversity of TübingenAuf der Morgenstelle 1872076 TübingenGermany

Bonnie A. AldenWaters Corporation, CRD34 Maple StreetMilford, MA 01757USA

Mario ArangioCarboGen AGSchachenallee 295001 AarauSwitzerland

Wolf-Dieter BeinertVWR International GmbHScientific InstrumentsHilpertstrasse 20A64295 DarmstadtGermany

Roberto BiancardiSolvay Solexis SpAViale Lombardia, 2020021 Bollate (MI)Italy

List of Contributors

Hans BilkeSandoz GmbHBiochemiestrasse 106250 KundlAustria

Yung-Fong ChengCubist Pharmaceuticals65 Hayden Ave.Lexington, MA 02421USA

Maristella ColomboOncology – Analytical ChemistryNerviano Medical SciencesVia le Pasteur, 1020014 Nerviano (MI)Italy

Diane M. DiehlWaters Corporation, CAT34 Maple StreetMilford, MA 01757USA

John W. DolanBASi Northwest Laboratory3138 NE RivergateBuilding 301CMcMinnville, OR 97128USA

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Melvin R. EuerbyAstraZeneca R&D CharnwoodAnalytical DevelopmentPharmaceutical and Analytical R&DCharnwood/LundBakewell RoadLoughboroughLeicestershire LE11 5RHUnited Kingdom

Sergey GalushkoDr. S. Galushko SoftwareDevelopmentIm Wiesengrund 49b64367 MühltalGermany

Eric S. GrumbachWaters Corporation, CAT34 Maple StreetMilford, MA 01757USA

Marc D. GrynbaumInstitute of Organic ChemistryUniversity of TübingenAuf der Morgenstelle 1872076 TübingenGermany

Heidi HändelInstitute of Organic ChemistryUniversity of TübingenAuf der Morgenstelle 1872076 TübingenGermany

Tom HennessyBiopolisBiomedical Science Group20 Biopolis WaySingapore 1 38668Singapore

Pamela C. IranetaWaters Corporation, CRD34 Maple StreetMilford, MA 01757USA

Markus JuzaSiegfried Ltd.Untere Brühlstrasse 44800 ZofingenSwitzerland

Marianna KeleWaters Corporation, CRD34 Maple StreetMilford, MA 01757USA

Peter KilzPSS Polymer Standard Service GmbHPOB 336855023 MainzGermany

Stavros KromidasRosenstrasse 1666125 SaarbrückenGermany

Manfred KruckerInstitute of Organic ChemistryUniversity of TübingenAuf der Morgenstelle 1872076 TübingenGermany

Hans-Joachim KussPsychiatric Department (LMU)University of MunichNussbaumstrasse 780336 MunichGermany

List of Contributors

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XXVII

Jörg P. KutterMIC – Department of Micro andNanotechnologyTechnical University of Denmark2800 LyngbyDenmark

Christiane LohausMedical Proteom-CenterCenter for Clinical ResearchRuhr-University of BochumUniversitätsstrasse 15044780 BochumGermany

Ziling LuWaters Corporation, CAT34 Maple StreetMilford, MA 01757USA

Egidijus MachtejevasInstitute for Anorganic Chemistryand Analytical ChemistryJohannes-Gutenberg-UniversityDuesbergweg 10–1455099 MainzGermany

Jürgen Maier-RosenkranzGRACE Davison – Alltech GromGmbHDiscovery SciencesEtzwiesenstrasse 3772108 Rottenburg-HailfingenGermany

Friedrich MandelAgilent TechnologiesHewlett-Packard-Strasse 876337 WaldbronnGermany

Katia MarcucciSienabiotech SpAVia Fiorentina, 153100 SienaItaly

Katrin MarcusMedical Proteom-CenterCenter for Clinical ResearchRuhr-University of BochumUniversitätsstrasse 15044780 BochumGermany

Jeffrey R. MazzeoWaters Corporation, CAT34 Maple StreetMilford, MA 01757USA

Michael McBrienAdvanced Chemistry DevelopmentInc.110 Yonge StreetToronto, Ontario M5C 1T4Canada

Alberto MéndezWaters Cromatografia S.A.Parc Tecnològic del Vallès08290 Cerdanyola del VallèsBarcelonaSpain

Helmut E. MeyerMedical Proteom-CenterCenter for Clinical ResearchRuhr-University of BochumUniversitätsstrasse 15044780 BochumGermany

List of Contributors

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XXVIII

Veronika R. MeyerEMPA St. GallenMaterials Science and TechnologyLerchenfeldstrasse 59014 St. GallenSwitzerland

Egbert MüllerTosoh Bioscience GmbHZettachring 670567 StuttgartGermany

Uwe D. NeueWaters Corporation, CRD34 Maple StreetMilford, MA 01757USA

Patrik PeterssonAstraZeneca R&D LundAnalytical DevelopmentPharmaceutical and Analytical R&DCharnwood/Lund22187 LundSweden

Michael PfefferSchering AGIn-Process-Control13342 BerlinGermany

Karsten PutzbachInstitute of Organic ChemistryUniversity of TübingenAuf der Morgenstelle 1872076 TübingenGermany

Oleg PylypchenkoInstitute of Bioorganic Chemistryof Ukrainian National Academyof SciencesMurmanskaja str., 102660 Kiev-94, MCP-600Ukraine

Milena QuagliaLGCAnalytical TechnologyQueens RoadTeddington, Middlesex, TW11 OLYUnited Kingdom

Giuseppe RazzanoVia D. Manin, 18Magenta (Cap. 20013)MilanoItaly

Vincenzo RizzoCISI – University of MilanVia Fantoli, 16/1520138 MilanoItaly

Heike SchäferMedical Proteom-CenterCenter for Clinical ResearchRuhr-University of BochumUniversitätsstrasse 15044780 BochumGermany

Stefan Schömerpro-isomehrAltenkesseler Strasse 1766115 SaarbrückenGermany

List of Contributors

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