HPLC made to measure a practical handbook for optimization

1.4 Selecting the Correct pH Value for HPLC 891.4.5 Correction of pH Based on Organic Content 93 1.4.6 Optimization of Mobile Phase pH Without Chemical Structures 94 1.4.7 A Systematic A

Edited by Stavros Kromidas

V R Meyer

Practical High-Performance Liquid Chromatography

2004 ISBN 0-470-09378-1

P C Sadek

Troubleshooting HPLC Systems

A Bench Manual

2000 ISBN 0-471-17834-9

U D Neue

HPLC Columns

Theory, Technology, and Practice

1997 ISBN 0-471-19037-3

L R Snyder, J J Kirkland, J L Glajch

Practical HPLC Method Development

1997 ISBN 0-471-00703-X

HPLC Made to Measure

A Practical Handbook for Optimization

Edited by

Stavros Kromidas

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

All rights reserved (including those of translation into other languages) No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers Registered names, trademarks, etc used in this book, even when not specifically marked as such, are not to be considered unprotected by law.

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

Cover Design SCHULZ Grafik-Design,

Fußgönheim

Typesetting Manuela Treindl, Laaber Printing betz-druck GmbH, Darmstadt Binding J Schäffer GmbH, Grünstadt ISBN-13: 978-3-527-31377-8 ISBN-10: 3-527-31377-X

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 that

it is the very first technique that most analysts, biologists or biochemists wouldconsider investigating and applying for virtually any class of analytes, regardless

of molecular weight, size, volatility, ionic charges, polarity, hydrophobicity, or otherphysical or chemical properties HPLC has become a technique that can be applied

to 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 other

texts by Stavros Kromidas, and was thus eager to preview this current one This text is really an edited book, though Stavros Kromidas has contributed several

excellent 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 as

Uwe 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

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 portion

of our careers to researching and developing HPLC areas It is not an easy read; it

is not a trivial text Rather, it is clearly an advanced, involved, and detailed text It

is a book to be read slowly and carefully, because it contains an incredible amount

of 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 handbook

or “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 about

to 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

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 Krull

Associate ProfessorDepartment of Chemistry and Chemical BiologyNortheastern University

Boston, MA, USA

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 aim

is 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

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 by

RP-Chromatography 3

Stavros Kromidas

1.1.1 Before the First Steps of Optimization 3

1.1.2 What Exactly Do We Mean By “Optimization”? 5

1.1.3 Improvement of Resolution (“Separate Better”) 6

1.1.3.1 Principal Possibilities for Improving Resolution 8

1.1.3.2 What has the Greatest Effect on Resolution? 10

1.1.3.3 Which Sequence of Steps is Most Logical When Attempting

1.1.4 Testing of the Peak Homogeneity 22

1.1.5 Unknown Samples: “How Can I Start?”; Strategies and Concepts 35

1.1.5.1 The “Two Days Method” 36

1.1.5.2 “The 5-Step Model” 39

1.1.6 Shortening of the Run Time (“Faster Separation”) 48

1.1.7 Improvement of the Sensitivity

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

1.2.2.3 Optimal Operating Conditions and Limits of Currently Available

Technology 66

1.2.2.4 Problems and Solutions 67 1.2.2.4.1 Gradient Delay Volume 67 1.2.2.4.2 Detector Sampling Rate and Time Constant 68 1.2.2.4.3 Ion Suppression in Mass Spectrometry 69

References 70

1.3 pH and Selectivity in RP-Chromatography 71

Uwe D Neue, Alberto Méndez, KimVan Tran, and Diane M Diehl

Solvent 76

1.3.2.3 Buffers 78 1.3.2.3.1 Classical HPLC Buffers 78 1.3.2.3.2 MS-Compatible pH Control 79

1.3.2.4 Influence of the Samples 79 1.3.2.4.1 The Sample Type: Acids, Bases, Zwitterions 80

1.3.2.4.2 Influence of the Organic Solvent on the Ionization of the

Analytes 81

1.3.3 Application Example 81

1.3.4 Troubleshooting 85

1.3.4.1 Reproducibility Problems 85

1.3.4.2 Buffer Strength and Solubility 86

1.3.4.3 Constant Buffer Concentration 86

1.3.5 Summary 87

References 87

1.4 Selecting the Correct pH Value for HPLC 89

1.4.5 Correction of pH Based on Organic Content 93

1.4.6 Optimization of Mobile Phase pH Without Chemical Structures 94

1.4.7 A Systematic Approach to pH Selection 96

1.4.8 An Example – Separation of

1,4-Bis[(2-pyridin-2-ylethyl)thio]butane-2,3-diol from its Impurities 97

1.4.9 Troubleshooting Mobile Phase pH 102

1.6 Calibration Characteristics and Uncertainty – Indicating Starting

Points to Optimize Methods 117

Stefan Schömer

1.6.1 Optimizing Calibration – What is the Objective? 117

1.6.2 The Essential Performance Characteristic of Calibration 118

1.6.3 Examples 118

1.6.3.1 Does Enhanced Sensitivity Improve Methods? 118

1.6.3.2 A Constant Variation Coefficient – Is it Good, Poor or Just an

Inevitable Characteristic of Method Performance? 122

1.6.3.3 How to Prove Effects Due to Matrices – May the Recovery Function

be Replaced? 133

1.6.3.4 Having Established Matrix Effects – Does Spiking Prove Necessary

in Every Case? 136

2.1.1.2 Reasons for the Diversity of Commercially Available RP-Columns –

First Consequences 151 2.1.1.2.1 On Polar Interactions 156 2.1.1.2.2 First Consequences 156

2.1.1.3 Criteria for Comparing RP-Phases 174 2.1.1.3.1 Similarity According to Physico-chemical Properties 174

2.1.1.3.2 Similarity Based on Chromatographic Behavior;

Expressiveness of Retention and Selectivity Factors 175 2.1.1.3.3 Tests for the Comparison of Columns and Their Expressiveness 181

2.1.1.4 Similarity of RP-Phases 195 2.1.1.4.1 Selectivity Maps 196 2.1.1.4.2 Selectivity Plots 200 2.1.1.4.3 Selectivity Hexagons 205 2.1.1.4.4 Chemometric Analysis of Chromatographic Data 229

2.1.1.5 Suitability of RP-Phases for Special Types of Analytes and Proposals

for the Choice of Columns 233 2.1.1.5.1 Polar and Hydrophobic RP-Phases 233 2.1.1.5.2 Suitability of RP-Phases for Different Classes of Substances 237 2.1.1.5.3 Procedure for the Choice of an RP-Column 248

References 253

2.1.2 Column Selectivity in RP-Chromatography 254

Uwe D Neue, Bonnie A Alden, and Pamela C Iraneta

2.1.2.1 Introduction 254

2.1.2.2 Main Section 255 2.1.2.2.1 Hydrophobicity and Silanol Activity (Ion Exchange) 255 2.1.2.2.2 Polar Interactions (Hydrogen Bonding) 259

2.1.2.2.3 Reproducibility of the Selectivity 261

References 263

2.1.3 The Use of Principal Component Analysis for the Characterization

of Reversed-Phase Liquid Chromatographic Stationary Phases 264 Melvin R Euerby and Patrik Petersson

2.1.3.1 Introduction 264

2.1.3.2 Theory of Principal Component Analysis 265

2.1.3.3 PCA of the Database of RP Silica Materials 267

2.1.3.3.1 PCA of Polar Embedded, Enhanced Polar Selectivity, and AQ/Aqua

Phases 269 2.1.3.3.2 PCA of Perfluorinated Phases 270

2.1.3.4 Use of PCA in the Identification of Column/Phase Equivalency 271

2.1.3.5 Use of PCA in the Rational Selection of Stationary Phases for

Method Development 277 2.1.3.5.1 Proposed Solvent/Stationary Phase Optimization Strategy 278

References 279

2.1.4 Chemometrics – A Powerful Tool for Handling a Large Number

of Data 280 Cinzia Stella and Jean-Luc Veuthey

2.1.4.1 Introduction 280

2.1.4.2 Chromatographic Tests and Their Importance in Column

Selection 280

2.1.4.3 Use of Principal Component Analysis (PCA) in the Evaluation

and Selection of Test Compounds 281 2.1.4.3.1 Physicochemical Properties of Test Compounds 281

2.1.4.3.2 Chromatographic Properties of Test Compounds 284

2.1.4.4 Use of PCA for the Evaluation of Chromatographic Supports 285

2.1.4.4.1 Evaluation of Chromatographic Supports in Mobile Phases

Composed of pH 7.0 Phosphate Buffer 286

2.1.4.4.2 Evaluation of Chromatographic Supports in Mobile Phases

Composed of pH 3.0 Phosphate Buffer 289

2.1.4.5 How a Chromatographic Test can be Optimized by

Chemometrics 291 2.1.4.5.1 Test Compounds 291

Parameters (Descriptors) from HPLC Data 310

2.1.5.5.1 How does this Strategy Differ from the Use of Predetermined

Solute Descriptors 310 2.1.5.5.2 The Experimental Plan 311

2.1.5.5.3 Determination of the Five LFER Parameters – A Procedure in

Eight Steps 312 2.1.5.5.4 Variation of the Eluent Conditions 315

2.1.5.5.5 Stationary Phase Characterization with Empirical LFER

Parameters 317

2.1.5.6 Concluding Remarks on LFER Applications in HPLC 319

References 320

2.1.6 Column Selectivity in Reversed-Phase Liquid Chromatography 321

Lloyd R Snyder and John W Dolan

2.1.6.1 Introduction 321

2.1.6.2 The “Subtraction” Model of Reversed-Phase Column Selectivity 323

2.1.6.3 Applications 326 2.1.6.3.1 Selecting “Equivalent” Columns 326 2.1.6.3.2 Selecting Columns of Very Different Selectivity 330

2.1.6.4 Conclusions 332

References 333

2.1.7 Understanding Selectivity by the Use of Suspended-State

High-Resolution Magic-Angle Spinning NMR Spectroscopy 334 Urban Skogsberg, Heidi Händel, Norbert Welsch, and Klaus Albert

2.1.7.1 Introduction 334

2.1.7.2 Is the Comparison Between NMR and HPLC Valid? 337

2.1.7.3 The Transferred Nuclear Overhauser Effect (trNOE) 340

2.1.7.4 Suspended-State 1H HR/MAS T1 Relaxation Measurements 343

2.1.7.5 Where do the Interactions Take Place? 345

2.3 Optimization of GPC/SEC Separations by Appropriate Selection

of the Stationary Phase and Detection Mode 359

Peter Kilz

2.3.1 Introduction 359

2.3.2 Fundamentals of GPC Separations 360

2.3.2.1 Chromatographic Modes of Column Separation 362

2.3.2.2 GPC Column Selection Criteria and Optimization of GPC

Separations 364 2.3.2.2.1 Selection of Pore Size and Separation Range 364

2.3.2.2.2 Advantages and Disadvantages of Linear or Mixed-Bed

2.3.3.2 Copolymer GPC Analysis by Multiple Detection 372

2.3.3.3 Simultaneous Separation and Identification by GPC-FTIR 375

2.3.3.4 Application of Molar Mass-Sensitive Detectors in GPC 377

2.3.3.4.1 Light-Scattering Detection 377

2.3.3.4.2 Viscometry Detection 379

2.3.4 Summary 380

References 381

2.4 Gel Filtration/Size-Exclusion Chromatography (SEC) of Biopolymers –

Optimization Strategies and Troubleshooting 383

Milena Quaglia, Egidijus Machtejevas, Tom Hennessy, and Klaus K Unger

2.4.1 Where Are We Now and Where Are We Going? 383

2.4.2 Theory in Brief 384

2.4.3 SEC vs HPLC Variants 387

(Downstream Processing) 399

2.4.5.5 SEC Columns Based on the Principle of Restricted Access

and Their Use in Proteome Analysis 400 References 403

2.5 Optimization in Affinity Chromatography 405

2.6.2 Basic Principles of Enantioselective HPLC 427

2.6.2.1 Thermodynamic Fundamentals of Enantioselective HPLC 429

2.6.2.2 Adsorption and Chiral Recognition 430

2.6.2.3 Differences to Reversed-Phase and Normal-Phase HPLC 433

2.6.2.4 Principles for Optimization of Enantioselective HPLC

Separations 433

2.6.3 Selectors and Stationary Phases 433

2.6.4 Method Selection and Optimization 440

2.6.4.1 Cellulose and Amylose Derivatives 441

2.6.4.2 Immobilized Cellulose and Amylose Derivatives 443

2.6.4.3 Stationary Phases Derived from Tartaric Acid 444

2.6.4.4 π-Acidic and π-Basic Stationary Phases 444

2.6.4.5 Macrocyclic Selectors, Cyclodextrins, and Antibiotics 446

2.6.4.6 Proteins and Peptides 450

2.6.4.7 Ruthenium Complexes 450

2.6.4.8 Synthetic and Imprinted Polymers 450

2.6.4.9 Metal Complexation and Ligand-Exchange Phases 451

2.6.4.10 Chiral Ion Exchangers 451

2.6.5 Avoiding Errors and Troubleshooting 452

2.6.5.1 Equipment and Columns – Practical Tips 452

2.6.5.2 Detection 454

2.6.5.3 Mistakes Originating from the Analyte 454

2.6.6 Preparative Enantioselective HPLC 454

2.6.6.1 Determination of the Loading Capacity 455

2.6.6.2 Determination of Elution Volumes and Flow Rates 456

2.6.6.3 Enantiomer Separation using Simulated Moving Bed (SMB)

Chromatography 458 2.6.6.3.1 Principles of Simulated Moving Bed Chromatography 458

2.6.6.3.2 Separation of Commercial Active Pharmaceutical Ingredients by

SMB 459

2.6.7 Enantioselective Chromatography by the Addition of

Chiral Additives to the Mobile Phase in HPLC and Capillary

Electrophoresis 461

2.6.8 Determination of Enantiomeric Purity Through the Formation of

Diastereomers 462

2.6.9 Indirect Enantiomer Separation on a Preparative Scale 462

2.6.10 Enantiomer Separations Under Supercritical Fluid Chromatographic

2.7.1.2.1 Influence of Column Length 467

2.7.1.2.2 Influence of Column Internal Diameter 467

2.7.1.2.3 Influence of Stationary Phase 469

2.7.1.3 Robustness 469

Jörg P Kutter

2.7.2.1 Introduction 487

2.7.2.2 Techniques 488 2.7.2.2.1 Pressure-Driven Liquid Chromatography (LC) 488 2.7.2.2.2 Open-Channel Electrochromatography (OCEC) 488 2.7.2.2.3 Packed-Bed Electrochromatography 488

2.7.2.2.4 Microfabricated Chromatographic Beds (Pillar Arrays) 489 2.7.2.2.5 In Situ Polymerized Monolithic Stationary Phases 489

2.7.2.3 Optimization and Possibilities 490 2.7.2.3.1 Separation Performance 490 2.7.2.3.2 Isocratic and Gradient Elution 491 2.7.2.3.3 Tailor-Made Stationary Phases 492 2.7.2.3.4 Sample Pretreatment and More-Dimensional Separations 492 2.7.2.3.5 Issues and Challenges 492

2.7.2.4 Application Examples 493

2.7.2.5 Conclusions and Outlook 496

References 496

2.7.3 Ultra-Performance Liquid Chromatography 498

Uwe D Neue, Eric S Grumbach, Marianna Kele, Jeffrey R Mazzeo, and Dirk Sievers

2.7.3.1 Introduction 498

2.7.3.2 Isocratic Separations 499

2.7.3.3 Gradient Separations 502

References 505

3.1.4.1 Affinity Enrichment (Affinity SPE) 513

3.1.4.2 “Weak Affinity Chromatography”

(True Affinity Chromatography) 519

3.1.4.3 Biochemical Detectors 520

3.1.5 Examples 522

3.1.5.1 Example 1: Affinity Extraction (Affinity SPE) 522

3.1.5.2 Example 2: “Weak Affinity Chromatography” (WAC) 523

3.1.5.3 Example 3: Biochemical Detection 525

3.2.2 How Can I Take Advantage? – Experimental Aspects 529

3.2.3 2D Data Presentation and Analysis 533

3.2.4 The State-of-the-Art in 2D Chromatography 535

3.3.2.1 Mobile Phase pH at the Edge of the Optimum Range 543

3.3.2.2 Ion-Pairing Agents in the HPLC System 543

3.3.2.3 Ion Suppression by the Sample Matrix or Sample

Contaminants 544

3.3.3 How Clean Should an LC/MS Ion Source Be? 544

3.3.4 Ion Suppression 545

References 549

4.2.3 Experimental Set-Up for On-Line Mode 588

4.2.4 Method Development with ChromSword® 588

4.2.4.1 Off-Line Mode (Computer-Assisted Method Development) 588

4.2.4.2 On-Line Mode – Fully Automated Optimization of Isocratic

and Gradient Separations 592 4.2.4.2.1 Software Functions for Automation 597

4.2.4.2.2 How Does the System Optimize Separations? 597

4.3.1 Introduction and Factorial Viewpoint 601

4.3.2 Strategy for Partially Automated Method Development 603

4.3.3 Comparison of Commercially Available Software Packages with

Regard to Their Contribution to Factorial Method Development 608

4.3.4 Development of a New System for Multifactorial Method

Development 609

4.3.4.1 Selection of Stationary Phases 611

4.3.4.2 Optimizing Methods with HEUREKA 612

4.3.4.3 Evaluation of Data with HEUREKA 618

4.3.5 Conclusion and Outlook 623

5.1.4 On-Line LC-ESI-MS/MS Coupling 633

5.1.5 Off-Line LC-MALDI-MS/MS Coupling 635

5.2.2 Testing Robustness in Analytical RP-HPLC by Means of

Systematic Method Development 643

5.2.3 Robustness Test in Analytical RP-HPLC by Means of Statistical

Experimental Design (DoE) 652

5.2.4 Conclusion 665

References 666

5.4 Evaluation of an Integrated Procedure for the Characterization of

Chemical Libraries on the Basis of HPLC-UV/MS/CLND 685

Mario Arangio, Federico R Sirtori, Katia Marcucci, Giuseppe Razzano, Maristella Colombo, Roberto Biancardi, and Vincenzo Rizzo

5.4.1 Introduction 685

5.4.2 Materials and Methods 686

5.4.2.1 Instrumentation 686

5.4.2.2 Chemicals and Consumables 686

5.4.2.3 High-Throughput Platform (HTP1) Method Set-up 688

5.4.2.4 Chromatographic Conditions 688

5.4.2.5 Mass Spectrometer and CLND Conditions 689

5.4.2.6 Data Processing and Reporting 689

5.4.2.7 Multilinear Regression Analysis for the Derivation of CLND

Response Factors 690

5.4.3 Results and Discussion 691

5.4.3.1 Liquid Chromatography and UV Detection 691

5.4.3.2 Mass Spectrometric Method Development 692

5.4.3.3 CLND Set-Up 693

5.4.3.4 Validation with Commercial Standards 693

5.4.3.5 Validation with Proprietary Compounds 695

5.4.4 Conclusions 699

References 700

Appendix 703 Subject Index 729

6250 KundlAustria

Yung-Fong Cheng

Cubist Pharmaceuticals

65 Hayden Ave

Lexington, MA 02421USA

Maristella Colombo

Oncology – Analytical ChemistryNerviano Medical SciencesVia le Pasteur, 10

20014 Nerviano (MI)Italy

Diane M Diehl

Waters Corporation, CAT

34 Maple StreetMilford, MA 01757USA

John W Dolan

BASi Northwest Laboratory

3138 NE RivergateBuilding 301CMcMinnville, OR 97128USA

Im Wiesengrund 49b

64367 MühltalGermany

Eric S Grumbach

Waters Corporation, CAT

34 Maple StreetMilford, MA 01757USA

Marc D Grynbaum

Institute of Organic ChemistryUniversity of TübingenAuf der Morgenstelle 18

72076 TübingenGermany

Heidi Händel

Institute of Organic ChemistryUniversity of TübingenAuf der Morgenstelle 18

72076 TübingenGermany

Tom Hennessy

BiopolisBiomedical Science Group

20 Biopolis WaySingapore 1 38668Singapore

Waters Corporation, CRD

34 Maple StreetMilford, MA 01757USA

Peter Kilz

PSS Polymer Standard Service GmbHPOB 3368

55023 MainzGermany

Stavros Kromidas

Rosenstrasse 16

66125 SaarbrückenGermany

Manfred Krucker

Institute of Organic ChemistryUniversity of TübingenAuf der Morgenstelle 18

72076 TübingenGermany

Institute for Anorganic Chemistry

and Analytical Chemistry

53100 SienaItaly

Katrin Marcus

Medical Proteom-CenterCenter for Clinical ResearchRuhr-University of BochumUniversitätsstrasse 150

44780 BochumGermany

Jeffrey R Mazzeo

Waters Corporation, CAT

34 Maple StreetMilford, MA 01757USA

Michael McBrien

Advanced Chemistry DevelopmentInc

110 Yonge StreetToronto, Ontario M5C 1T4Canada

Alberto Méndez

Waters Cromatografia S.A

Parc Tecnològic del Vallès

08290 Cerdanyola del VallèsBarcelona

Spain

Helmut E Meyer

Medical Proteom-CenterCenter for Clinical ResearchRuhr-University of BochumUniversitätsstrasse 150

44780 BochumGermany

Uwe D Neue

Waters Corporation, CRD

34 Maple StreetMilford, MA 01757USA

Patrik Petersson

AstraZeneca R&D LundAnalytical DevelopmentPharmaceutical and Analytical R&DCharnwood/Lund

22187 LundSweden

Michael Pfeffer

Schering AGIn-Process-Control

13342 BerlinGermany

Karsten Putzbach

Institute of Organic ChemistryUniversity of TübingenAuf der Morgenstelle 18

72076 TübingenGermany

United Kingdom

Giuseppe Razzano

Via D Manin, 18Magenta (Cap 20013)Milano

Heike Schäfer

Medical Proteom-CenterCenter for Clinical ResearchRuhr-University of BochumUniversitätsstrasse 150

44780 BochumGermany

Stefan Schömer

pro-isomehrAltenkesseler Strasse 17

66115 SaarbrückenGermany

Irina Shishkina

Institute of Bioorganic Chemistry of

Ukrainian National Academy of

Nerviano Medical Sciences

Oncology – Analytical Chemistry

United Kingdom

Vsevolod Tanchuk

Institute of Bioorganic Chemistry ofUkrainian National Academy ofSciences

Murmanskaja str., 1

02660 Kiev-94, MCP-600Ukraine

KimVan Tran

Waters Corporation, CAT

34 Maple StreetMilford, MA 01757USA

Klaus K Unger

Institute for Anorganic Chemistryand Analytical Chemistry

Johannes-Gutenberg-UniversityDuesbergweg 10–14

55099 MainzGermany

Jean-Luc Veuthey

Faculty of SciencesSchool of Pharmaceutical SciencesUniversity of Geneva

20, Bd d’Yvoy

1211 Genève 4Switzerland

Structure of the Book

The book consists of five parts:

Part 1 Fundamentals of Optimization

The aim of Part 1 is to provide an overview of important aspects of optimization

in HPLC from various viewpoints In Chapter 1.1 (Stavros Kromidas), the

prin-ciples of optimization are illustrated using RP-HPLC as an example, and mendations for method development are made Fast gradients on short columnslead more often than one might think to sufficient resolution in the shortest

recom-of analysis times, and this topic is discussed in Chapter 1.2 (Uwe D Neue) For

the separation of polar/ionic substances, pH is by far the most important factor

in optimization procedures The next two chapters (1.3 Uwe D Neue, 1.4 Michael McBrien) are devoted to this aspect Optimization means more than merely the

“correct” choice of method parameters Efforts to obtain as much information

as possible, or at least the necessary information, are also a part of optimization

In this context, the evaluation of chromatographic data and calibration take on

a special significance These topics are dealt with in Chapter 1.5 (Hans-Joachim Kuss) and Chapter 1.6 (Stefan Schömer).

Part 2 Characteristics of Optimization in Individual HPLC Modes

In Part 2, specific aspects of optimization in individual techniques are considered

In RP chromatography (Section 2.1), besides the choice of eluents (for this, seealso Chapters 1.1 to 1.4), above all the choice of column represents a difficult andtime-consuming task The subject of RP columns is covered by a total of six authors:

two authors (2.1.1 Stavros Kromidas, 2.1.2 Uwe D Neue) focus on the more tical aspects of this issue, while Frank Steiner (Chapter 2.1.5) and Lloyd R Snyder

prac-(Chapter 2.1.6) present more fundamental, theoretical considerations, withnevertheless real practical relevance, on the questions of column characterizationand column selection Naturally, the meaningfulness of results increases withthe number of experimental data, and so the handling of figures, and above allthe identification and interpretation of correlations, is only possible with the aid

of mathematical tools Chemometrics is a suitable tool, for example, for ing the similarity of columns on the basis of chromatographic data The application

establish-of chemometrics from a practical viewpoint is briefly described in Chapter 2.1.1

(Stavros Kromidas) and extensively detailed in Chapters 2.1.3 (Melvin R Euerby)

Part 3 Coupling Techniques

Part 3 is exclusively devoted to coupling techniques The more demanding theanalytical problem to be addressed (complexity and number of sample compo-nents, greater chemical similarity of the analytes to be separated, etc.), the morenecessary coupling techniques become A coupling of different separationtechniques can lead to an improved chromatographic resolution, as, for example,

in immunochromatography (3.1 Michael G Weller) and LC-GPC coupling (3.2 Peter Kilz) In other cases, at a given resolution, LC-spectroscopy couplings can

yield specific information The most popular coupling techniques are LC-MS (3.3

Friedrich Mandel) and LC-NMR (3.4 Klaus Albert).

Part 4 Computer-Aided Optimization

Automation can generally lead to the elimination of errors and to time saving.Meanwhile, fully automated computer-aided method development and semi-automated optimization in HPLC have reached a remarkable level of maturity

and sophistication Through several real examples, Lloyd R Snyder (Chapter 4.1) and Sergey Galushko (Chapter 4.2) describe the possibilities offered by the software

packages DryLab® and ChromSword®, respectively Michael Pfeffer (Chapter 4.3)

compares the two software concepts from the point of view of the user, and presents

a new software tool that also incorporates automatic column selection

Part 5 User Reports

In the final part, users are able to express their opinions In four different cases,rather sophisticated and/or new techniques/concepts are presented for the solution

of particular chromatographic problems, although equally from the point of view

of the user and with practical relevance One or other of the presented solutions

to these problems may be of interest to some readers Katrin Marcus (Chapter 5.1) presents the application of LC-MS/MS coupling in proteomics, Hans Bilke (Chapter 5.2) demonstrates ways of testing for robustness in RP-HPLC, Knut Wagner (Chapter 5.3) describes a hardware solution for the separation of complex

mixtures, and Mario Arangio (Chapter 5.4) considers the potential of multiple

detection (UV, MS, CLND) in the characterization of libraries of newly synthesizedsubstances

The five parts describe discrete units of subject matter, but nevertheless the bookdoes not necessarily have to be read in a linear fashion from beginning to end.The individual chapters have been written so that they constitute self-containedmodules, and so one can always be skipped In this way, we have tried to makethe character of the book meet the criteria of a reference work Differentinterpretations of a topic by different authors have been accepted, as has somerepetition, so as not to disrupt the flow of the writing Finally, some importantareas have been covered by several authors, who have naturally placed more

emphasis on certain aspects This applies, for example, to pH (Uwe D Neue, Michael McBrien), weighted regression (Hans-Joachim Kuss, Stefan Schömer), the selectivity of stationary RP phases (Stavros Kromidas, Uwe D Neue, Melvin R Euerby, Cinzia Stella, Lloyd R Snyder), chemometrics (Stavros Kromidas, Melvin R Euerby, Cinzia Stella), and LC-MS (Friedrich Mandel, Katrin Markus) The reader may benefit

from the different descriptions of the topics and from the individual evaluations

of the authors

a method and checking peak homogeneity.

The last section will show ways to achieve other aims than “better separation”:

“make it faster”, “raise sensitivity” or “save money” The chapter ends with aconclusion and an outlook

1.1.1

Before the First Steps of Optimization

For economic reasons, one really ought to address the following questions prior

to commencing the development of a method or the optimization of a givenseparation

• What do I want? In other words, what is the true intention of the separation?

• What do I have? That is to say, what relevant information about the analyticalpurpose and the samples is available?

• How should I do it? Do I have all what I need, and is what I want to do reallypossible?

At first glance, these questions might appear too theoretical or even over-critical.Nevertheless, careful consideration of the actual aims and realistic possibilitiesfor solving an analytical problem would seem to be important at the outset Anearly discussion with my boss, a colleague or my client – if you are short, evenwith yourself – can later prevent a good deal of trouble, time expenditure, and lastbut not least costs This time-saving can be considered a good investment

As regards the first question: “What do I want?”

If it is at all possible, at the outset the following or similar questions should beanswered:

• Do I need a method for the accurate quantification of this toxic metabolite, or is

the aim that the authorities just accept my method?

• What is most important in this case: short analysis times, durable columns,

robust conditions, or simply optimal specificity?

• Must the relative standard deviation Srel be no higher than 2%? What loss of

qua-lity would be incurred if Srel were to be 2.5%? Is there actually a correlation tween the cost of the analysis and real improvement in the quality of the product?

be-department on the light sensitivity and the sorption properties on glass surfaces

• May I quickly calculate the pKa value of the known main component in thesample with appropriate software (see Chapter 1.4)?

• Has a colleague in a neighboring department worked in the past with similarcompounds and might therefore be able to provide valuable insights?

As far as possible, all means of communication with colleagues should be pursued

to gather information At times it may be helpful not to make this public

As regards the third question: “How should I do it?”

One should assess the feasibility of the proposed work absolutely unconditionally.Some examples:

• Can I convince my boss that it is useful from the overall company point of view

to discuss in advance with the later routine users the design of the method andadditional details? If fear of loss of know-how or questions of budget or otherpsychological and social barriers make impossible de facto a discussion with

“the others”, it is a bitter reality that one must accept

• On the other hand, is it worth fighting for a change of the following well-knownand accepted situation? A deadline is fixed and therefore a validation must befinished in two weeks Later, the burden of subsequent, substantial costs for arepeat of the measurements, complaints, out-of-spec situations, etc., whichinevitably result because an analytical method can hardly be validated withintwo weeks under real conditions, is not placed on “us” but on quality control,and as testing costs they have been accepted since decades in the absence ofoverall considerations The reader may imagine the consequences, or viewedmore positively, the possibilities for improvement

• Is it really worthwhile in the case of the development of a routine method,which shall be applied all over the world, to opt for a polar RP-phase because ofthe frequently observed higher selectivity, even if one has to expect problemswith the charge-to-charge reproducibility? Might a hydrophobic, more ruggedcolumn with a lower but still sufficient selectivity the better choice?

• Is it useful to demonstrate my analytical “knowledge” by further trimming therelative standard deviation of a method being used in diverse plant laboratories

to a value of 0.7%?

Realities – and opinions are also realities – which determine the success or failure

of an analytical activity should, wherever possible, influence the design of themethod It is useful if the number of meetings can be reduced to “a cup of coffee”

or “lunch often together” The point is to improve the communication and thiscan turn out to be easier in a less formal situation

In conclusion, two basic preconditions for successful method development may

be noted:

1 Expert knowledge exists or can be loaned or sold

2 The analytical possibilities correspond with the requirements, and it is possible

to talk about them

In the author’s opinion, a clear definition of requirements, unequivocally lated, understandable goals for all involved persons, shortcuts to information, and

formu-a criticformu-al estimformu-ation of possibilities/risks formu-are more importformu-ant, not only in formu-anformu-alytics,than obtaining exemplary results such as low detection limit, correlation factors

around 0.999, Srel smaller than 1%, or 30% less expensive equipment

1.1.2

What Exactly Do We Mean By “Optimization”?

Optimization of a separation is principally directed by the following goals:

• to separate better (higher resolution),

• to separate faster (shorter retention time),

• to see more (lower detection limit),

• to separate at lower cost (economic effort),

• to separate more (higher throughput)

The three first-named goals may be most important, and of these the improvement

of the resolution is the prime concern Therefore, we will treat this topic before

we start to deal with the other aspects Preparative HPLC is not the subject of thisbook

Preliminary Remarks

The theory of chromatography is fundamentally valid for all chromatogaphictechniques Therefore, basically the same principles are pursued However, it is

Resolution (R), in simplest terms, is the distance between two neighboring peaks

at the base of the peaks An increase in this distance is what every chromatographerroutinely strives for

The corresponding equation is:

α −

2 2

the column

The number of plates is in effect a measure of the widening of the substanceband because of diffusion effects The basic question is whether the molecules ofthe analyte that reach the detector are contained in a small or a large (peak) volume,i.e., will one get sharp or broad peaks?

Strictly speaking, one should distinguish between the theoretical and theeffective plate number The theoretical plate number is the number of plates of

an inert component (see below) and therefore a characteristic and constant valuefor a column under defined conditions The effective plate number is the number

of plates of a specified retained component, and the retention factor (see below)enters into the calculation Today, however, this distinction is not made everytime;one speaks only about plate number In the most cases, the theoretical platenumber is calculated, but of retained substances In this context, it should bemade clear that the plate number depends on a lot of factors, e.g the injectionvolume, the temperature, the composition of the eluent, the flow rate, theretention time, the analyte, and last but not least the equation used for thecalculation, i.e peak width at the peak base, at 10% or at 50% peak height.Therefore, the comparison of literature values of plate numbers is inherentlydifficult

α: Separation factor, formerly selectivity factor.

α is a measure of the capability of a chromatographic system (chromatographicsystem: the actual combination of the stationary phase, the mobile phase, and thetemperature) to distinguish two given compounds

The α value is the quotient of two net retention times, i.e the quotient of thedwell times of the two components within the stationary phase.

The point is that if this particular chromatographic system is selective for these

two compounds, then in principle they are separable Selectivity, in simplest terms,

is the distance between two peaks, from the top of one peak to the top of theother This is different from the resolution in that for the determination of theselectivity the form of the peak (plate number) is not considered, because α isonly the quotient of two (retention) times The separation factor depends only onthe chemistry; see below on the subject of retention factor

k: Retention factor, formerly capacity factor k′

k is a measure of the strength of the interaction of a given compound in a given

chromatographic system It expresses for how much longer a given compoundremains on the stationary phase compared to the mobile phase

The k value is an index like the α value, and is thus independent of instrumental

conditions such as the dimensions of the column or the flow rate The k value

changes only if parameters that have something to do with the interaction arechanged, i.e., the chemistry: stationary phase, mobile phase, temperature As long

as these parameters are kept constant, the k value also stays constant, irrespective,

e.g., of the flow rate or whether a 10 or 15 cm column is used

Although the dead time does not appear explicitly in the equation for the resolution,

it is useful for the following explanation to briefly deal with this term

tm: dead time, breakthrough time, front, “air peak”: This is the dwell duration of an

inert component in the HPLC equipment A component is designated as inert if

it is able to penetrate everywhere without steric hindrance, including, of course,

in the pores of the stationary phase, but is not retained there In other words, thedead time is the time of the presence of any not excluded component in the mobilephase – also in the standing mobile phase (i.e., within the pores), but again there

is “no” interaction with the stationary phase Therefore, the dead time only changes

if something “physically” or “mechanically” is altered, e.g a change in the length

or the inner diameter of the column, the packing density, or the flow rate The deadtime is a time, which is independent from the particular compound just analysed

As all components move equally quickly in the eluent, the time that the compoundsspend in the eluent makes no contribution to the separation A separation is onlypossible if the substances stay in the stationary phase for different lengths of time

The resolution R – the distance from peak base to peak base – depends only on

the following three factors:

• the strength of the interaction between the compound and the stationary phase

(if the peak comes soon or late), i.e on the k value,

• the ability of the chromatographic system to distinguish between the twocomponents of interest, i.e the α value,

• if the relevant peaks are sharp or wide, i.e., the plate number

component (increase of dead time, see above) elute later Because now the deadtime increases too a physical process must be responsible This could be a longercolumn, a larger inner diameter of the column, decrease of the flow rate (A largerinner diameter leads to peak broadening which rules it out for all practicalpurposes).

Possibility 2: The retention time stays largely constant, one seeks only a better

peak form Here, there are somewhat more possibilities: reduction of the deadvolume (e.g., thinner capillaries, smaller detector cell), reduction of the injectionvolume (remark: local overload of the column happens more frequently than one

Fig 1 Principle possibilities for improving resolution in HPLC; for comments, see text.

might imagine! Peak broadening caused by the injection is inversely proportional

to the injection volume.) at an elution composition with equal solvent strengthparameter, replacement of methanol with acetonitrile owing to the lower viscosity

of the latter (approximately constant retention time can be expected), using smallerparticles, or use of a newer, better packed column In this context, one shouldalso consider an optimization of the injection step as this also improves the peakform and consequently increases the plate number The solvent of the sampleshould be weaker than the eluent; to this end, one uses a little bit more water incomparison to the eluent composition when preparing the sample solution

In this way, it is possible to increase the concentration of the substance band atthe head of the column, and the result is a better peak form Finally, one shouldalso consider various settings, such as sample rate (sampling time, samplingperiod), bunching factor, peak width, slit width in the case of a diode-array detector,etc In this way, the peak form can also be improved measurably, without changingthe “real” method parameters such as column or eluent

Possibility 3a: This involves the increase of the interaction between the sample

and the stationary phase, and for this there are only three “chemical” possibilities,

as mentioned above: change of the eluent (e.g., increase of the water content),decrease of the temperature, and change of the stationary phase (e.g., use of amore hydrophobic phase)

The interactions increase for both/all components to be separated to the sameextent, and so the retention times will also increase equally

Possibility 3b: The same procedure as in Possibility 3a, but here it is possible to

change the interactions of the two components to different extents, i.e onecomponent responds more strongly to a change, e.g., a change in the pH, thanthe other one

Other possibilities do not exist in principle, because R = f(N,a,k) This means that

when trying to improve the resolution, one can only change consciously orintuitively these three factors

1 One may successfully increase the interaction of the components of interestwith the stationary phase per se, i.e “the whole” comes later (case 3a, increase

of k, e.g., by increasing the water content in the eluent) or one successfully

increases the interaction of the components with the stationary phase vidually, i.e one component responds more strongly to the change than theothers (case 3b, increase of α, e.g., change of the pH in the case of polar/ioniccomponents) Both cases relate to the “chemistry”: change of the temperature

indi-or change of the eluent (this includes the pH and other additives indi-or modifiers,

of course) or change of the stationary phase

2 One may increase the plate number, either at (theoretically) constant retentiontime, case 2, or can concomitantly increase the retention time, case 1

Other possibilities do not exist in principle