HPLC made to measure a practical handbook for optimization

HPLC Made to Measure
Edited by
Stavros 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
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
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1997
ISBN 0-471-19037-3

L. R. Snyder, J. J. Kirkland, J. L. Glajch

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1997
ISBN 0-471-00703-X

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HPLC Made to Measure
A Practical Handbook for Optimization

Edited by
Stavros Kromidas

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The Editor
Dr. Stavros Kromidas
Rosenstrasse 16
66125 Saarbrücken
Germany

All books published by Wiley-VCH are carefully
produced. Nevertheless, authors, editors, and
publisher do not warrant the information contained
in these books, including this book, to be free of
errors. Readers are advised to keep in mind that
statements, data, illustrations, procedural details or
other items may inadvertently be inaccurate.
Library of Congress Card No.: applied for
British Library Cataloguing-in-Publication Data
A catalogue record for this book is available from
the British Library
Bibliographic information published by
Die Deutsche Bibliothek
Die Deutsche Bibliothek lists this publication

in the Deutsche Nationalbibliografie; detailed
bibliographic data is available in the Internet at
<>
© 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

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V

Foreword
HPLC has become the analytical method against which all others are measured and
compared. It is perhaps the most widely employed method of analysis of all those
instrumental approaches that have ever been or are now in vogue. Having been
involved with HPLC for perhaps the past 35 years, since the early 1970s, I have seen
the technique and field grow and prosper, academically and commercially. It has
become an incredible commercial success, and the cornerstone of many academic
careers in analytical and other fields of chemistry. Annual, dedicated meetings, as
well as major parts of ACS, ASMS, AAPS and AAAS meetings, are routinely
devoted to talks and discussions on or involving HPLC. Though it has not quite
displaced GC or flat-bed electrophoresis, it has surely been highly competitive for
volatiles and biological macromolecules, respectively. Indeed, one could argue that
it is the very first technique that most analysts, biologists or biochemists would
consider investigating and applying for virtually any class of analytes, regardless
of molecular weight, size, volatility, ionic charges, polarity, hydrophobicity, or other
physical 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 properties
thereof. 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 impossible
for any other technique to displace it from this niche in the analytical world, not
even 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 international
group 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, and
have, in their own right, become well-regarded and recognized experts in their
various fields. The book is heavily practice and practicality oriented, in that it
aims to help the readers become more knowledgeable and better adept at using
various forms of and approaches in HPLC. However, it is not a “Methods”-type
text, such as those published by Humana Press; it is not just a compilation of
practice-oriented HPLC methods for various analytes.
HPLC Made to Measure: A Practical Handbook for Optimization. Edited by Stavros Kromidas
Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 3-527-31377-X

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VI

Foreword

Rather, the text is quite detailed, with scientific discussions and theory, lots of
equations and principles, reference to a variety of practical software, and with an
emphasis on understanding the fundamentals in each and every chapter. This is
not an introductory text; it is not meant as a text for a graduate Analytical
Separations type course. Rather, it is quite an advanced text, dealing with many
recent and contemporary aspects of HPLC. It deals with approaches for method
optimization, currently available software and practices, chemometrics, principal

component analysis, the selection of ideal stationary phases, and tools for column
characterization and method optimization. Of course, it deals extensively with
reversed-phase HPLC, but it also covers many other areas, including GPC/SEC,
affinity chromatography, chiral separations, microLC, nanoLC, and even microchip-based LC instrumentation and techniques. It also deals with immunochromatographic methods, two-dimensional HPLC (MDLC), LC-MS, LC-NMR,
and even how magic-angle spinning NMR spectroscopy can be used to better
understand the selectivity of stationary phases in HPLC. All in all, there are over
two-dozen individual chapters, some authored by the same author(s), but most
not. It is to the Editor’s credit that he has not written most or even close to 50% of
the total chapters, but rather that he has invited the most highly regarded and
best-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 has
done 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 would
appear 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 latest
developments in HPLC, not just the fundamentals, but where the field stands
today, and where it is going tomorrow. It is a practical handbook for the optimization 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 is
important to know about its future developments and applications. It is an
incredibly useful and practical tome, collated by experts pooling their expertise,
and it will make better chromatographers out of those of us who take up the
book, study it carefully, and then apply its lessons to our own future needs. It is
not a simplistic methods development type book, though it does aim to help us
optimize and improve our methods development approaches. It is far more than
“just” a Practical Handbook for Optimization, though the subtitle might make
that suggestion.

It is my hope that those of you thinking of purchasing this particular, newer
text 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 wise
choices; now it is up to you to make the most of the book, which means not just
reading the text and studying the figures and tables, but making every effort

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Foreword

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 – actually
several times, as these are not easy chapters or contributions. However, in the
long run, the time will be well spent and such efforts will be rewarded, for the
book is truly a wealth of useful and practical information. Obviously, I highly
recommend the book to those contemplating purchase and study, for it is really
one of the better texts to have come along in many years dealing with this, one of
our very favorite subjects, HPLC.
January 2006

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Ira S. Krull
Associate Professor
Department of Chemistry and Chemical Biology

Northeastern University
Boston, MA, USA

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VII


IX

Preface
The optimizing of practices and processes constitutes an essential prerequisite
for long-term success. The objective and the motives may be very different: selfpreservation among living things, “saving lives” among volunteers in Africa,
maximizing profits among marketing strategists, new discoveries among scientists. 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, we
have set out the fundamental aspects, encompassing the principal considerations
and background information. At the same time, we have endeavored to present
and discuss as many practical examples, ideas, and suggestions as possible for
the everyday application of HPLC. The implementation of concepts for rapid
optimization should equally aid and support the planning of effective method
development strategies as in daily practice at the laboratory bench. The aim of the
book is to contribute to purposeful, affordable, forward-looking method development and optimization in HPLC.
To this end, internationally renowned experts have offered their knowledge and
experience. 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

HPLC Made to Measure: A Practical Handbook for Optimization. Edited by Stavros Kromidas
Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 3-527-31377-X

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XI

Contents

Foreword
Preface

V
IX

List of Contributors
Structure of the Book

XXV
XXXI

1

Fundamentals of Optimization

1

1.1

Principles of the Optimization of HPLC Illustrated by
RP-Chromatography 3
Stavros Kromidas

1.1.1
1.1.2
1.1.3
1.1.3.1
1.1.3.2
1.1.3.3

Before the First Steps of Optimization 3
What Exactly Do We Mean By “Optimization”? 5
Improvement of Resolution (“Separate Better”) 6
Principal Possibilities for Improving Resolution 8
What has the Greatest Effect on Resolution? 10
Which Sequence of Steps is Most Logical When Attempting
an Optimization? 11
1.1.3.4 How to Change k, α, and N 17
1.1.3.4.1 Isocratic Mode 17
1.1.3.4.2 Gradient Mode 18
1.1.3.4.3 Acetonitrile or Methanol? 19
1.1.4

Testing of the Peak Homogeneity 22
1.1.5
Unknown Samples: “How Can I Start?”; Strategies and Concepts
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

HPLC Made to Measure: A Practical Handbook for Optimization. Edited by Stavros Kromidas
Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 3-527-31377-X

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XII

Contents

1.1.8
1.1.9

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

1.2

48

57

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

1.2.1
1.2.2
1.2.2.1
1.2.2.2
1.2.2.2.1
1.2.2.2.2
1.2.2.2.3
1.2.2.3

Introduction 59
Main Part 59
Theory 59
Results 61
General Relationships 61
Short Columns, Small Particles 62
An Actual Example 64

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

1.3

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

1.3.1
1.3.2
1.3.2.1
1.3.2.2
1.3.2.2.1
1.3.2.2.2

Introduction 71
Main Section 71
Ionization and pH 71
Mobile Phase and pH 73
Buffer Capacity 74
Changes of pK and pH Value in the Presence of an Organic
Solvent 76
Buffers 78
Classical HPLC Buffers 78
MS-Compatible pH Control 79

Influence of the Samples 79
The Sample Type: Acids, Bases, Zwitterions 80
Influence of the Organic Solvent on the Ionization of the
Analytes 81
Application Example 81
Troubleshooting 85
Reproducibility Problems 85
Buffer Strength and Solubility 86
Constant Buffer Concentration 86
Summary 87

1.3.2.3
1.3.2.3.1
1.3.2.3.2
1.3.2.4
1.3.2.4.1
1.3.2.4.2
1.3.3
1.3.4
1.3.4.1
1.3.4.2
1.3.4.3
1.3.5

References

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Contents

1.4

Selecting the Correct pH Value for HPLC
Michael McBrien

1.4.1
1.4.2
1.4.3
1.4.4
1.4.5
1.4.6
1.4.7
1.4.8

Introduction 89
Typical Approaches to pH Selection 90
Initial pH Selection 91
Basis of pKa Prediction 92
Correction of pH Based on Organic Content 93
Optimization of Mobile Phase pH Without Chemical Structures 94
A Systematic Approach to pH Selection 96

An Example – Separation of 1,4-Bis[(2-pyridin-2-ylethyl)thio]butane2,3-diol from its Impurities 97
Troubleshooting Mobile Phase pH 102
The Future 102
Conclusion 103

1.4.9
1.4.10
1.4.11

References

103

1.5

Optimization of the Evaluation in Chromatography
Hans-Joachim Kuss

1.5.1
1.5.2
1.5.3
1.5.4
1.5.5
1.5.6
1.5.7
1.5.8
1.5.9
1.5.10

Evaluation of Chromatographic Data – An Introduction

Working Range 105
Internal Standard 106
Calibration 107
Linear Regression 107
Weighting Exponent 110
In Real Practise 111
Drug Analysis 111
Measurement Uncertainty 112
Calibration Line Through the Origin 115
References

105
105

115

1.6

Calibration Characteristics and Uncertainty – Indicating Starting
Points to Optimize Methods 117
Stefan Schömer

1.6.1
1.6.2
1.6.3
1.6.3.1
1.6.3.2

Optimizing Calibration – What is the Objective? 117
The Essential Performance Characteristic of Calibration 118

Examples 118
Does Enhanced Sensitivity Improve Methods? 118
A Constant Variation Coefficient – Is it Good, Poor or Just an
Inevitable Characteristic of Method Performance? 122
How to Prove Effects Due to Matrices – May the Recovery Function
be Replaced? 133
Having Established Matrix Effects – Does Spiking Prove Necessary
in Every Case? 136

1.6.3.3
1.6.3.4

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XIV

Contents

1.6.3.5
1.6.3.6

Testing Linearity – Does a Calibration Really Need to Fit a Straight
Line? 139
Enhancing Accuracy – Obtaining ‘Robust’ Calibration Functions
with Weighting 143
References

2

Characteristics of Optimization in Individual HPLC Modes

2.1

RP-HPLC

2.1.1

Comparison and Selection of Commercial RP-Columns
Stavros Kromidas

2.1.1.1
2.1.1.2

Introduction 151
Reasons for the Diversity of Commercially Available RP-Columns –
First Consequences 151
On Polar Interactions 156
First Consequences 156
Criteria for Comparing RP-Phases 174
Similarity According to Physico-chemical Properties 174
Similarity Based on Chromatographic Behavior;

Expressiveness of Retention and Selectivity Factors 175
Tests for the Comparison of Columns and Their Expressiveness 181
Similarity of RP-Phases 195
Selectivity Maps 196
Selectivity Plots 200
Selectivity Hexagons 205
Chemometric Analysis of Chromatographic Data 229
Suitability of RP-Phases for Special Types of Analytes and Proposals
for the Choice of Columns 233
Polar and Hydrophobic RP-Phases 233
Suitability of RP-Phases for Different Classes of Substances 237
Procedure for the Choice of an RP-Column 248

2.1.1.2.1
2.1.1.2.2
2.1.1.3
2.1.1.3.1
2.1.1.3.2
2.1.1.3.3
2.1.1.4
2.1.1.4.1
2.1.1.4.2
2.1.1.4.3
2.1.1.4.4
2.1.1.5
2.1.1.5.1
2.1.1.5.2
2.1.1.5.3

References

253

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

2.1.2.1
2.1.2.2
2.1.2.2.1
2.1.2.2.2
2.1.2.2.3

Introduction 254
Main Section 255
Hydrophobicity and Silanol Activity (Ion Exchange)
Polar Interactions (Hydrogen Bonding) 259
Reproducibility of the Selectivity 261

14

149

151

2.1.2

References

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255

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Contents

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
2.1.3.2
2.1.3.3
2.1.3.3.1

Introduction 264
Theory of Principal Component Analysis 265
PCA of the Database of RP Silica Materials 267
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

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
2.1.4.2

Introduction 280
Chromatographic Tests and Their Importance in Column
Selection 280
Use of Principal Component Analysis (PCA) in the Evaluation
and Selection of Test Compounds 281
Physicochemical Properties of Test Compounds 281
Chromatographic Properties of Test Compounds 284
Use of PCA for the Evaluation of Chromatographic Supports 285
Evaluation of Chromatographic Supports in Mobile Phases
Composed of pH 7.0 Phosphate Buffer 286
Evaluation of Chromatographic Supports in Mobile Phases
Composed of pH 3.0 Phosphate Buffer 289
How a Chromatographic Test can be Optimized by
Chemometrics 291
Test Compounds 291

Mobile Phases 292
Chromatographic Parameters and Batch (Column)
Reproducibility 292
Conclusion and Perspectives 295

2.1.4.3
2.1.4.3.1
2.1.4.3.2
2.1.4.4
2.1.4.4.1
2.1.4.4.2
2.1.4.5
2.1.4.5.1
2.1.4.5.2
2.1.4.5.3
2.1.4.6

References

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295

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XV

XVI

Contents

2.1.5

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

2.1.5.1
2.1.5.2
2.1.5.3

Characterization and Selection of Stationary Phases for HPLC 296
What are LFERs and Why can they be Profitable in HPLC? 297
How to Obtain Analyte Descriptors for the Multivariate
Regression 300
LFER Procedure Using the Solvation Equation 301
Comparing Stationary Phases on the Basis of LFER Data 301
The Influence of the Mobile Phase Expressed in LFER
Parameters 307
The Prediction of Chromatographic Selectivity from LFER Data 308
An Empirical Approach to the Determination of LFER Solute
Parameters (Descriptors) from HPLC Data 310
How does this Strategy Differ from the Use of Predetermined
Solute Descriptors 310
The Experimental Plan 311

Determination of the Five LFER Parameters – A Procedure in
Eight Steps 312
Variation of the Eluent Conditions 315
Stationary Phase Characterization with Empirical LFER
Parameters 317
Concluding Remarks on LFER Applications in HPLC 319

2.1.5.4
2.1.5.4.1
2.1.5.4.2
2.1.5.4.3
2.1.5.5
2.1.5.5.1
2.1.5.5.2
2.1.5.5.3
2.1.5.5.4
2.1.5.5.5
2.1.5.6

References

2.1.6

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

2.1.6.1
2.1.6.2
2.1.6.3
2.1.6.3.1

2.1.6.3.2
2.1.6.4

Introduction 321
The “Subtraction” Model of Reversed-Phase Column Selectivity
Applications 326
Selecting “Equivalent” Columns 326
Selecting Columns of Very Different Selectivity 330
Conclusions 332
References

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321

323

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
2.1.7.2
2.1.7.3

2.1.7.4
2.1.7.5

Introduction 334
Is the Comparison Between NMR and HPLC Valid? 337
The Transferred Nuclear Overhauser Effect (trNOE) 340
Suspended-State 1H HR/MAS T1 Relaxation Measurements
Where do the Interactions Take Place? 345

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343


Contents

2.1.7.6
2.1.7.7
2.1.7.8

Hydrogen Bonding 345
Some Practical Considerations
Future Aspects 347
References

347

2.2

Optimization in Normal-Phase HPLC
Veronika R. Meyer

2.2.1
2.2.2
2.2.3
2.2.4

Introduction 349
Mobile Phases in NP-HPLC 350
Stationary Phases in NP-HPLC 354
Troubleshooting in Normal-Phase HPLC
References/Further Reading

349

356

357

2.3

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

2.3.1
2.3.2
2.3.2.1

2.3.2.2

Introduction 359
Fundamentals of GPC Separations 360
Chromatographic Modes of Column Separation 362
GPC Column Selection Criteria and Optimization of GPC
Separations 364
Selection of Pore Size and Separation Range 364
Advantages and Disadvantages of Linear or Mixed-Bed
Columns 365
HighSpeed GPC Separations 367
The Role of Comprehensive Detection in the Investigation
of Macromolecular Materials 369
Coupling of Liquid Chromatography with Information-Rich
Detectors 371
Copolymer GPC Analysis by Multiple Detection 372
Simultaneous Separation and Identification by GPC-FTIR 375
Application of Molar Mass-Sensitive Detectors in GPC 377
Light-Scattering Detection 377
Viscometry Detection 379
Summary 380

2.3.2.2.1
2.3.2.2.2
2.3.2.3
2.3.3
2.3.3.1
2.3.3.2
2.3.3.3
2.3.3.4

2.3.3.4.1
2.3.3.4.2
2.3.4

References

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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
2.4.2
2.4.3

Where Are We Now and Where Are We Going?
Theory in Brief 384
SEC vs. HPLC Variants 387

17

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XVII


XVIII

Contents

2.4.4
2.4.4.1
2.4.4.2
2.4.4.3
2.4.4.4
2.4.4.5
2.4.5
2.4.5.1
2.4.5.2
2.4.5.3
2.4.5.4
2.4.5.5

Optimization Aspects in SEC of Biopolymers 388
Column Selection and Optimal Flow Rate 388
Optimization of the Mobile Phase 392
Sample Preparation 394
Sample Viscosity and Sample Volume – Two Critical Parameters
at Injection 395
Detection Methods 396
Applications 397

High-Performance SEC 397
Determination of Molecular Weight 398
Gel Filtration as a Tool to Study Conformational Changes of
Proteins 398
Gel Filtration in Preparative and Process Separations
(Downstream Processing) 399
SEC Columns Based on the Principle of Restricted Access
and Their Use in Proteome Analysis 400
References

2.5

Optimization in Affinity Chromatography
Egbert Müller

2.5.1

Introduction to Resin Design and Method Development in
Affinity Chromatography 405
Base Matrix 408
Immobilization Methods 409
Activation Methods 409
Spacer 412
Site-Directed Immobilization 415
Non-Particulate Affinity Matrices 416
Affinity Purification 417
Factorial Design for the Preparation of Affinity Resins 419
Summary of Immobilization 423

2.5.2

2.5.3
2.5.4
2.5.5
2.5.6
2.5.7
2.5.8
2.5.9
2.5.10

References

405

423

2.6

Optimization of Enantiomer Separations in HPLC
Markus Juza

2.6.1
2.6.2
2.6.2.1
2.6.2.2
2.6.2.3
2.6.2.4

Introduction 427
Basic Principles of Enantioselective HPLC 427
Thermodynamic Fundamentals of Enantioselective HPLC 429

Adsorption and Chiral Recognition 430
Differences to Reversed-Phase and Normal-Phase HPLC 433
Principles for Optimization of Enantioselective HPLC
Separations 433
Selectors and Stationary Phases 433
Method Selection and Optimization 440

2.6.3
2.6.4

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18

427

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Contents

2.6.4.1
2.6.4.2
2.6.4.3
2.6.4.4
2.6.4.5
2.6.4.6
2.6.4.7

2.6.4.8
2.6.4.9
2.6.4.10
2.6.5
2.6.5.1
2.6.5.2
2.6.5.3
2.6.6
2.6.6.1
2.6.6.2
2.6.6.3

Cellulose and Amylose Derivatives 441
Immobilized Cellulose and Amylose Derivatives 443
Stationary Phases Derived from Tartaric Acid 444
π-Acidic and π-Basic Stationary Phases 444
Macrocyclic Selectors, Cyclodextrins, and Antibiotics 446
Proteins and Peptides 450
Ruthenium Complexes 450
Synthetic and Imprinted Polymers 450
Metal Complexation and Ligand-Exchange Phases 451
Chiral Ion Exchangers 451
Avoiding Errors and Troubleshooting 452
Equipment and Columns – Practical Tips 452
Detection 454
Mistakes Originating from the Analyte 454
Preparative Enantioselective HPLC 454
Determination of the Loading Capacity 455
Determination of Elution Volumes and Flow Rates 456
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
(SFC) Conditions 462
2.6.11
New Chiral Stationary Phases and Information Management
Software 463
2.6.12
Summary 463
References

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2.7

Miniaturization

2.7.1

mLC/NanoLC – Optimization and Troubleshooting
Jürgen Maier-Rosenkranz

2.7.1.1
2.7.1.2
2.7.1.2.1
2.7.1.2.2
2.7.1.2.3
2.7.1.3

Introduction 467
Sensitivity 467
Influence of Column Length 467
Influence of Column Internal Diameter
Influence of Stationary Phase 469
Robustness 469

19

467
467

467

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XIX

XX

Contents

2.7.1.3.1
2.7.1.3.2
2.7.1.3.3
2.7.1.3.4
2.7.1.3.5
2.7.1.4
2.7.1.4.1
2.7.1.4.2
2.7.1.4.3

System Choice 469
Capillary Connections 472
Precautions Against Blocking 477
Testing for Leakages 478
Guard Column Switching and Sample Loading Strategies
Sensitivity/Resolution 483
Column Dimensions 483
Packing Materials/Surface Covering 484
Detectors 484
References

486

2.7.2

Microchip-Based Liquid Chromatography – Techniques and
Possibilities 487
Jörg P. Kutter

2.7.2.1
2.7.2.2
2.7.2.2.1
2.7.2.2.2
2.7.2.2.3
2.7.2.2.4
2.7.2.2.5
2.7.2.3
2.7.2.3.1
2.7.2.3.2
2.7.2.3.3
2.7.2.3.4
2.7.2.3.5
2.7.2.4
2.7.2.5

Introduction 487
Techniques 488
Pressure-Driven Liquid Chromatography (LC) 488
Open-Channel Electrochromatography (OCEC) 488
Packed-Bed Electrochromatography 488
Microfabricated Chromatographic Beds (Pillar Arrays) 489
In Situ Polymerized Monolithic Stationary Phases 489
Optimization and Possibilities 490
Separation Performance 490

Isocratic and Gradient Elution 491
Tailor-Made Stationary Phases 492
Sample Pretreatment and More-Dimensional Separations 492
Issues and Challenges 492
Application Examples 493
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
2.7.3.2
2.7.3.3

Introduction 498
Isocratic Separations 499
Gradient Separations 502
References

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20

505

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Contents

3

Coupling Techniques

3.1

Immunochromatographic Techniques
Michael G. Weller

3.1.1
3.1.2
3.1.3
3.1.4
3.1.4.1
3.1.4.2

Introduction 509
Binding Molecules 509
Immunoassays 511
Immunochromatographic Techniques 511
Affinity Enrichment (Affinity SPE) 513
“Weak Affinity Chromatography”

(True Affinity Chromatography) 519
Biochemical Detectors 520
Examples 522
Example 1: Affinity Extraction (Affinity SPE) 522
Example 2: “Weak Affinity Chromatography” (WAC)
Example 3: Biochemical Detection 525

3.1.4.3
3.1.5
3.1.5.1
3.1.5.2
3.1.5.3

References

509

523

525

3.2

Enhanced Characterization and Comprehensive Analyses
by Two-Dimensional Chromatography 527
Peter Kilz

3.2.1
3.2.2
3.2.3

3.2.4
3.2.5

Introduction 527
How Can I Take Advantage? – Experimental Aspects
2D Data Presentation and Analysis 533
The State-of-the-Art in 2D Chromatography 535
Summary 539
References

529

540

3.3

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

3.3.1
3.3.2
3.3.2.1
3.3.2.2
3.3.2.3

Optimization of the Ionization Process 541
Lost LC/MS Peaks 542
Mobile Phase pH at the Edge of the Optimum Range
Ion-Pairing Agents in the HPLC System 543

Ion Suppression by the Sample Matrix or Sample
Contaminants 544
How Clean Should an LC/MS Ion Source Be? 544
Ion Suppression 545

3.3.3
3.3.4

References

1293vch00.pmd

507

543

549

21

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XXII

Contents

3.4

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

3.4.1
3.4.2
3.4.3
3.4.4
3.4.5

NMR Basics 551
Sensitivity of the NMR Experiment 552
NMR Spectroscopy in Flowing Systems 553
NMR Probes for LC-NMR 553
Practical Realization of Analytical HPLC-NMR and
Capillary-HPLC-NMR 554
Continuous-Flow Measurements 555
Stopped-Flow Measurements 557
Capillary Separations 559
Outlook 560

3.4.6
3.4.7
3.4.8
3.4.9

References

563

4

Computer-Aided Optimization

4.1

Computer-Facilitated HPLC Method Development Using DryLab®
Software 567
Lloyd R. Snyder and Loren Wrisley

4.1.1
4.1.1.1
4.1.1.2
4.1.2
4.1.2.1
4.1.2.2
4.1.3
4.1.4

Introduction 567
History 569
Theory 570
DryLab Capabilities 570
DryLab Operation 570
Mode Choices 571
Practical Applications of DryLab® in the Laboratory
Conclusions 584
References

4.2

565

572

585

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

4.2.1
4.2.1.1
4.2.1.2
4.2.2
4.2.3
4.2.4
4.2.4.1
4.2.4.2

Introduction 587
Off-Line Mode 587
On-Line Mode 587
ChromSword® Versions 587
Experimental Set-Up for On-Line Mode 588
Method Development with ChromSword® 588
Off-Line Mode (Computer-Assisted Method Development) 588
On-Line Mode – Fully Automated Optimization of Isocratic
and Gradient Separations 592

4.2.4.2.1 Software Functions for Automation 597

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Contents

4.2.4.2.2 How Does the System Optimize Separations?
4.2.5
Conclusion 600
References

600

4.3

Multifactorial Systematic Method Development and Optimization
in Reversed-Phase HPLC 601
Michael Pfeffer

4.3.1
4.3.2
4.3.3

Introduction and Factorial Viewpoint 601
Strategy for Partially Automated Method Development 603

Comparison of Commercially Available Software Packages with
Regard to Their Contribution to Factorial Method Development 608
Development of a New System for Multifactorial Method
Development 609
Selection of Stationary Phases 611
Optimizing Methods with HEUREKA 612
Evaluation of Data with HEUREKA 618
Conclusion and Outlook 623

4.3.4
4.3.4.1
4.3.4.2
4.3.4.3
4.3.5

References

623

5

User Reports

5.1

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

5.1.1
5.1.2

5.1.3
5.1.4
5.1.5
5.1.5.1
5.1.5.2
5.1.6
5.1.7

Proteomics – An Introduction 627
Sample Preparation for Nano-LC 628
Nano-LC 629
On-Line LC-ESI-MS/MS Coupling 633
Off-Line LC-MALDI-MS/MS Coupling 635
Sample Fractionation 635
MALDI-TOF-MS/MS Analyses 635
Data Analysis 637
Application in Practice: Analysis of α-Crystallin A in Mice Lenses 637
References

625

640

5.2

Verification Methods for Robustness in RP-HPLC
Hans Bilke

5.2.1
5.2.2

Introduction 643
Testing Robustness in Analytical RP-HPLC by Means of
Systematic Method Development 643
Robustness Test in Analytical RP-HPLC by Means of Statistical
Experimental Design (DoE) 652
Conclusion 665

5.2.3
5.2.4

References

1293vch00.pmd

597

643

666

23

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XXIV

Contents

5.3

Separation of Complex Sample Mixtures
Knut Wagner

5.3.1
5.3.2
5.3.3
5.3.3.1
5.3.3.2
5.3.4

Introduction 669
Multidimensional HPLC 670
Techniques for Multidimensional Separations 672
Off-Line Technique 672
On-Line Technique 672
On-Line Sample Preparation as a Previous Stage of
Multidimensional HPLC 674
Fields of Application of Multidimensional HPLC 675
What can be Realized? – A Practical Example 676
Critical Parameters of Multidimensional HPLC 682

5.3.5
5.3.5.1
5.3.6

References

683

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
5.4.2
5.4.2.1
5.4.2.2
5.4.2.3
5.4.2.4
5.4.2.5
5.4.2.6
5.4.2.7

Introduction 685
Materials and Methods 686
Instrumentation 686
Chemicals and Consumables 686
High-Throughput Platform (HTP1) Method Set-up 688
Chromatographic Conditions 688
Mass Spectrometer and CLND Conditions 689
Data Processing and Reporting 689
Multilinear Regression Analysis for the Derivation of CLND

Response Factors 690
Results and Discussion 691
Liquid Chromatography and UV Detection 691
Mass Spectrometric Method Development 692
CLND Set-Up 693
Validation with Commercial Standards 693
Validation with Proprietary Compounds 695
Conclusions 699

5.4.3
5.4.3.1
5.4.3.2
5.4.3.3
5.4.3.4
5.4.3.5
5.4.4

References

700

Appendix

703

Subject Index

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669

24

729

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XXV

List of Contributors
Klaus Albert
Institute of Organic Chemistry
University of Tübingen
Auf der Morgenstelle 18
72076 Tübingen
Germany
Bonnie A. Alden
Waters Corporation, CRD
34 Maple Street
Milford, MA 01757
USA

Hans Bilke
Sandoz GmbH
Biochemiestrasse 10
6250 Kundl
Austria
Yung-Fong Cheng
Cubist Pharmaceuticals

65 Hayden Ave.
Lexington, MA 02421
USA
Maristella Colombo
Oncology – Analytical Chemistry
Nerviano Medical Sciences
Via le Pasteur, 10
20014 Nerviano (MI)
Italy

Mario Arangio
CarboGen AG
Schachenallee 29
5001 Aarau
Switzerland
Wolf-Dieter Beinert
VWR International GmbH
Scientific Instruments
Hilpertstrasse 20A
64295 Darmstadt
Germany
Roberto Biancardi
Solvay Solexis SpA
Viale Lombardia, 20
20021 Bollate (MI)
Italy

Diane M. Diehl
Waters Corporation, CAT
34 Maple Street

Milford, MA 01757
USA
John W. Dolan
BASi Northwest Laboratory
3138 NE Rivergate
Building 301C
McMinnville, OR 97128
USA

HPLC Made to Measure: A Practical Handbook for Optimization. Edited by Stavros Kromidas
Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 3-527-31377-X

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XXVI

List of Contributors

Melvin R. Euerby
AstraZeneca R&D Charnwood
Analytical Development
Pharmaceutical and Analytical R&D
Charnwood/Lund
Bakewell Road

Loughborough
Leicestershire LE11 5RH
United Kingdom
Sergey Galushko
Dr. S. Galushko Software
Development
Im Wiesengrund 49b
64367 Mühltal
Germany
Eric S. Grumbach
Waters Corporation, CAT
34 Maple Street
Milford, MA 01757
USA
Marc D. Grynbaum
Institute of Organic Chemistry
University of Tübingen
Auf der Morgenstelle 18
72076 Tübingen
Germany
Heidi Händel
Institute of Organic Chemistry
University of Tübingen
Auf der Morgenstelle 18
72076 Tübingen
Germany
Tom Hennessy
Biopolis
Biomedical Science Group
20 Biopolis Way

Singapore 1 38668
Singapore

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Pamela C. Iraneta
Waters Corporation, CRD
34 Maple Street
Milford, MA 01757
USA
Markus Juza
Siegfried Ltd.
Untere Brühlstrasse 4
4800 Zofingen
Switzerland
Marianna Kele
Waters Corporation, CRD
34 Maple Street
Milford, MA 01757
USA
Peter Kilz
PSS Polymer Standard Service GmbH
POB 3368
55023 Mainz
Germany
Stavros Kromidas
Rosenstrasse 16
66125 Saarbrücken

Germany
Manfred Krucker
Institute of Organic Chemistry
University of Tübingen
Auf der Morgenstelle 18
72076 Tübingen
Germany
Hans-Joachim Kuss
Psychiatric Department (LMU)
University of Munich
Nussbaumstrasse 7
80336 Munich
Germany

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