Introduction to Modern Liquid Chromatography E-Book

Lloyd R. Snyder

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 1663

Veröffentlichungsjahr: 2011

Beschreibung

The latest edition of the authoritative reference to HPLC High-performance liquid chromatography (HPLC) is today the leading technique for chemical analysis and related applications, with an ability to separate, analyze, and/or purify virtually any sample. Snyder and Kirkland's Introduction to Modern Liquid Chromatography has long represented the premier reference to HPLC. This Third Edition, with John Dolan as added coauthor, addresses important improvements in columns and equipment, as well as major advances in our understanding of HPLC separation, our ability to solve problems that were troublesome in the past, and the application of HPLC for new kinds of samples. This carefully considered Third Edition maintains the strengths of the previous edition while significantly modifying its organization in light of recent research and experience. The text begins by introducing the reader to HPLC, its use in relation to other modern separation techniques, and its history, then leads into such specific topics as: * The basis of HPLC separation and the general effects of different experimental conditions * Equipment and detection * The column--the "heart" of the HPLC system * Reversed-phase separation, normal-phase chromatography, gradient elution, two-dimensional separation, and other techniques * Computer simulation, qualitative and quantitative analysis, and method validation and quality control * The separation of large molecules, including both biological and synthetic polymers * Chiral separations, preparative separations, and sample preparation * Systematic development of HPLC separations--new to this edition * Troubleshooting tricks, techniques, and case studies for both equipment and chromatograms Designed to fulfill the needs of the full range of HPLC users, from novices to experts, Introduction to Modern Liquid Chromatography, Third Edition offers the most up-to-date, comprehensive, and accessible survey of HPLC methods and applications available.

Leseprobe

CONTENTS

PREFACE

GLOSSARY OF SYMBOLS AND ABBREVIATIONS

1 INTRODUCTION

1.1 Background Information

1.2 A Short History of HPLC

1.3 Some Alternatives to HPLC

1.4 Other Sources of HPLC Information

References

2 BASIC CONCEPTS AND THE CONTROL OF SEPARATION

2.1 Introduction

2.2 The Chromatographic Process

2.3 Retention

2.4 Peak Width and the Column Plate Number N

2.5 Resolution and Method Development

2.6 Sample Size Effects

2.7 RELATED TOPICS

References

3 EQUIPMENT

3.1 Introduction

3.2 Reservoirs and Solvent Filtration

3.3 Mobile-Phase Degassing

3.4 Tubing and Fittings

3.5 Pumping Systems

3.6 Autosamplers

3.7 Column Ovens

3.8 Data Systems

3.9 Extra-Column Effects

3.10 Maintenance

References

4 DETECTION

4.1 Introduction

4.2 Detector Characteristics

4.3 Introduction to Individual Detectors

4.4 UV-Visible Detectors

4.5 Fluorescence Detectors

4.6 Electrochemical (Amperometric) Detectors

4.7 Radioactivity Detectors

4.8 Conductivity Detectors

4.9 Chemiluminescent Nitrogen Detector

4.10 Chiral Detectors

4.11 Refractive Index Detectors

4.12 Light-Scattering Detectors

4.13 Corona-Discharge Detector (CAD)

4.14 Mass Spectral Detectors (MS)

4.15 Other Hyphenated Detectors

4.16 Sample Derivatization and Reaction Detectors

References

5 THE COLUMN

5.1 Introduction

5.2 Column Supports

5.3 Stationary Phases

5.4 Column Selectivity

5.5 Column Hardware

5.6 Column-Packing Methods

5.7 Column Specifications

5.8 Column Handling

References

6 REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL SAMPLES

6.1 Introduction

6.2 Retention

6.3 Selectivity

6.4 Method Development and Strategies for Optimizing Selectivity

6.5 Nonaqueous Reversed-Phase Chromatography (NARP)

6.6 Special Problems

References

7 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND IONEXCHANGE CHROMATOGRAPHY

7.1 Introduction

7.2 Acid-Base Equilibria and Reversed-Phase Retention

7.3 Separation of Ionic Samples by Reversed-Phase Chromatography (RPC)

7.4 Ion-Pair Chromatography (IPC)

7.5 Ion-Exchange Chromatography (IEC)

References

8 NORMAL-PHASE CHROMATOGRAPHY

8.1 Introduction

8.2 Retention

8.3 Selectivity

8.4 Method-Development Summary

8.5 Problems in the Use of NPC

8.6 Hydrophilic Interaction Chromatography (HILIC)

References

9 GRADIENT ELUTION

9.1 INTRODUCTION

9.2 Experimental Conditions and Their Effects on Separation

9.3 Method Development

9.4 Large-Molecule Separations

9.5 Other Separation Modes

9.6 Problems

References

10 COMPUTER-ASSISTED METHOD DEVELOPMENT

10.1 Introduction

10.2 Computer-Simulation Software

10.3 Other Method-Development Software

10.4 Computer Simulation and Method Development

References

11 QUALITATIVE AND QUANTITATIVE ANALYSIS

11.1 Introduction

11.2 Signal Measurement

11.3 Qualitative Analysis

11.4 Quantitative Analysis

11.5 Summary

References

12 METHOD VALIDATION with Michael Swartz

12.1 Introduction

12.2 Terms and Definitions

12.3 System Suitability

12.4 Documentation

12.5 Validation for Different Pharmaceutical-Method Types

12.6 Bioanalytical Methods

12.7 Analytical Method Transfer (AMT)

12.8 Method Adjustment or Method Modification

12.9 quality Control and Quality Assurance

12.10 Summary

References

13 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS with Timothy Wehr, Carl Scandella, and Peter Schoenmakers

13.1 Biomacromolecules

13.2 Molecular Structure and Conformation

13.3 Special Considerations for Biomolecule HPLC

13.4 Separation of Peptides and Proteins

13.5 Separation of Nucleic Acids

13.6 Separation of Carbohydrates

13.7 Separation of Viruses

13.8 Size-Exclusion Chromatography (SEC)

13.9 Large-Scale Purification of Large Biomolecules

13.10 Synthetic Polymers

References

14 ENANTIOMER SEPARATIONS with Michael Lämmerhofer, Norbert M. Maier and Wolfgang Lindner

14.1 Introduction

14.2 Background and Definitions

14.3 Indirect Method

14.4 Direct Method

14.5 Peak Dispersion and Tailing

14.6 Chiral Stationary Phases and Their Characteristics

14.7 Thermodynamic Considerations

References

15 PREPARATIVE SEPARATIONS with Geoff Cox

15.1 Introduction

15.2 Equipment for Prep-LC Separation

15.3 Isocratic Elution

15.4 Severely Overloaded Separation

15.5 Gradient Elution

15.6 Production-Scale Separation

References

16 SAMPLE PREPARATION with Ronald Majors

16.1 Introduction

16.2 Types of Samples

16.3 Preliminary Processing of Solid and Semi-Solid Samples

16.4 Sample Preparation for Liquid Samples

16.5 Liquid-Liquid Extraction

16.6 Solid-Phase Extraction (SPE)

16.7 Membrane Techniques in Sample Preparation

16.8 Sample Preparation Methods for Solid Samples

16.9 Column-Switching

16.10 Sample Preparation for Biochromatography

16.11 Sample Preparation for LC-MS

16.12 Derivatization in HPLC

References

17 TROUBLESHOOTING Quick Fix

17.1 Introduction

17.2 Prevention of Problems

17.3 Problem-Isolation Strategies

17.4 Common Symptoms of HPLC Problems

17.5 Troubleshooting Tables

References

APPENDIX I PROPERTIES OF HPLC SOLVENTS

I.1 Solvent-Detector Compatibility

I.2 Solvent Polarity and Selectivity

I.3 Solvent Safety

Reference

APPENDIX II. PREPARINGBUFFERED MOBILE PHASES

II.1 Sequence of Operations

II.2 Recipes for Some Commonly Used Buffers

Reference

Index

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Library of Congress Cataloging-in-Publication Data:

Snyder, Lloyd R.

Introduction to modern liquid chromatography / Lloyd R. Snyder, Joseph J. Kirkland. – 3rd ed. / John W. Dolan.

p. cm.

Includes index.

ISBN 978-0-470-16754-0 (cloth)

1. Liquid chromatography. I. Kirkland, J. J. (Joseph Jack), 1925- II. Dolan, John W. III. Title.

QD79.C454S58 2009

543'.84–dc22

2009005626

PREFACE

High-performance liquid chromatography (HPLC) is today the premier technique for chemical analysis and related applications, with an ability to separate, analyze, and/or purify virtually any sample. The second edition of this book appeared in 1979, and for tens of thousands of readers it eventually became their choice of an HPLC reference book. The remarkable staying power of the second edition (with significant sales into the first decade of the present century) can be attributed to certain features which continue to be true for the present book. First, all three editions have been closely tied to short courses presented by the three authors over the past four decades, to an audience of more than 10,000 industrial, governmental, and academic chromatographers. Teaching allows different approaches to a subject to be tried and evaluated, and a pragmatic emphasis is essential when dealing with practicing chromatographers as students. Second, all three editions have tried to combine practical suggestions (''how to?'') with a theoretical background (''why?''). Both theory and practice continue to be emphasized so that the reader can better understand and evaluate the various recommendations presented here. Finally, each of the three authors has been an active participant in HPLC research, development, and/or routine application throughout most of their careers.

Since the preparation of the second edition in 1979, there have been major improvements in columns and equipment, as well as numerous advances in (1) our understanding of HPLC separation, (2) our ability to solve problems that were troublesome in the past, and (3) the application of HPLC for new kinds of samples. Whereas six different HPLC procedures received comparable attention in the second edition, today reversed-phase chromatography (RPC) accounts for about 80% of all HPLC applications—and therefore receives major (but not exclusive) attention in the present edition. Over the past three decades the use of HPLC for biological samples, enantiomeric (chiral) separations, and sample purification has expanded enormously, accompanied by a much better understanding of these and other HPLC applications.

Commercial HPLC columns continue to be improved, and many new kinds of columns have been introduced for specific applications, as well as for faster, trouble-free operation. Prior to 1990, HPLC method development was an uncertain process—often requiring several months for the acceptable separation of a sample. Since then it has become possible to greatly accelerate method development, especially with the help of appropriate software. At the same time HPLC practice is increasingly carried out in a regulatory environment that can slow the release of a final method. These various advances and changes in the way HPLC is carried out have mandated major changes in the present edition.

The organization of the present book, while similar to that of the second edition, has been significantly modified in light of subsequent research and experience. Chapter 1 provides a general background for HPLC, with a summary of how its use compares with other modern separation techniques. Chapter 1 also reviews some of the history of HPLC. Chapter 2 develops the basis of HPLC separation and the general effects of different experimental conditions. Chapters 3 and 4 deal with equipment and detection, respectively. In 1979 the detector was still the weak link in the use of HPLC, but today the widespread use of diode-array UV and mass-spectrometric detection—as well as the availability of several special-purpose detectors—has largely addressed this problem. Chapter 5 deals with the column: the ''heart'' of the HPLC system. In 1979, numerous problems were associated with the column: peak tailing—especially for basic samples, column instability at elevated temperatures or extremes in mobile-phase pH, and batch-to-batch column variability; today these problems are much less common. We also now know a good deal about how performance varies among different columns, allowing a better choice of column for specific applications. Finally, improvements in the column are largely responsible for our current ability to carry out ultra-fast separations (run times of a few minutes or less) and to better separate mixtures that contain hundreds or even thousands of components.

Chapter 6, which deals with the reversed-phase separation of non-ionic samples, extends the discussion of Chapter 2 for these important HPLC applications. A similar treatment for normal-phase chromatography (NPC) is given in Chapter 8, including special attention to hydrophilic interaction liquid chromatography (HILIC). In Chapter 7 the separation of ionized or ionizable samples is treated, whether by RPC, ion-pair chromatography, or ion-exchange chromatography. Gradient elution is introduced in Chapter 9 for small-molecule samples, and as an essential prerequisite for the separation of large biomolecules in Chapter 13; two-dimensional separation—another technique of growing importance—is also discussed. Chapter 10 covers the use of computer-facilitated method development (computer simulation). Other important, general topics are covered in Chapters 11 (Qualitative and Quantitative Analysis) and 12 (Method Validation).

Chapter 13 introduces the separation of large molecules, including both biological and synthetic polymers. HPLC procedures that are uniquely useful for these separations are emphasized: reversed-phase, ion-exchange, and size-exclusion, as well as related two-dimensional separations. Chapter 14 (Enantiomer Separations) marks a decisive shift in approach, as the resolution of enantiomers requires columns and conditions that are sample-specific—unlike most of the HPLC applications described in earlier chapters.

Chapter 15 deals with preparative separations ("prep-LC"), where much larger sample weights are introduced to the column. The big change since 1979 for prep-LC is that we now have a much better understanding of how such separations vary with conditions, in turn making method development much more systematic and efficient. Chapter 16 (Sample Preparation) provides a comprehensive coverage of this important supplement to HPLC separation. As in the case of other HPLC-related topics, the past 30 years have seen numerous developments that today make sample preparation a routine addition to many HPLC procedures. Finally, Chapter 17 deals with HPLC troubleshooting. Despite all our advances in equipment, columns, materials, technique, and understanding, trouble-free HPLC operation is still not guaranteed. Fortunately, our ability to anticipate, diagnose, and solve HPLC problems is now more informed and systematic. One of our three authors (JWD) has been especially active in this area.

Different readers will use this book in different ways. An experienced worker may wish to explore topics of his or her choice, or find an answer to specific problems. For this audience, the Index may be the best starting place. Beginning readers might first skim Chapters 1 through 7, followed by 9 through 10, all of which emphasize reversed-phase HPLC. The latter sequence is similar to the core of the basic HPLC short courses developed by the authors. After this introduction, the reader can jump to chapters or sections of special interest. Other readers may wish to begin with topics of interest from the Contents pages at the front of the book or at the beginning of individual chapters. The present book has been organized with these various options in mind.

This third edition is highly cross-referenced, so as to allow the reader to follow up on topics of special interest, or to clarify questions that may arise during reading. Because extensive cross-referencing represents a potential distraction, in most cases it is recommended that the reader simply ignore (or defer) these invitations to jump to other parts of the book. Some chapters include sections that are more advanced, detailed, and of less immediate interest; these sections are in each case clearly identified by an introductory advisory in italics, so that they can be bypassed at the option of the reader. We have also taken pains to provide definitions for all symbols used in this book (Glossary section), along with a comprehensive and detailed index. Finally, attention should be drawn to a ''best practices'' entry in the Index, which summarizes various recommendations for both method development and routine use.

We very much appreciate the participation of eight collaborators in the preparation of the present book: Peter Schoenmakers (Sections 9.3.10, 13.10), Mike Swartz (Chapter 12), Tim Wehr (Sections 13.1-13.8), Carl Scandella (Section 13.9), Wolfgang Lindner, Michael Lämmerhofer, and Norbert Maier (Chapter 14), Geoff Cox (Chapter 15), and Ron Majors (Chapter 16). Their affiliations are as follows:

Peter SchoenmakersUniversity of AmsterdamMike SwartzSynomics PharmaTim WehrBioRad Corp.Carl ScandellaCarl Scandella Consulting (4404 91st Avenue NE Bellevue, WA 98004)Wolfgang Lindner, Michael Lämmerhofer, and Norbert MaierUniversity of ViennaGeoff CoxChiral TechnologiesRon MajorsAgilent Technologies

We also are indebted to the following reviewers of various parts of the book: Peter Carr, Tom Chambers, Geoff Cox, Roy Eksteen, John Fetzer, Dick Henry, Vladimir Ioffe, Pavel Jandera, Peter Johnson, Tom Jupille, Ron Majors, Dan Marchand, David McCalley, Imre Molnar, Tom Mourey, Uwe Neue, Ravi Ravichandran, Karen Russo, Carl Scandella, Peter Schoenmakers, and Loren Wrisley. However, the authors accept responsibility for any errors or other shortcomings in this book.

LLOYD R. SNYDERJ. J. (JACK) KIRKLANDJOHN W. DOLAN

Orinda, CAWilmington, DEAmity, OR

CHAPTER ONEINTRODUCTION

High-performance liquid chromatography (HPLC) is one of several chromatographic methods for the separation and analysis of chemical mixtures (Section 1.3). Compared to these other separation procedures, HPLC is exceptional in terms of the following characteristics:

almost universal applicability; few samples are excluded from the possibility of HPLC separationremarkable assay precision (±0.5% or better in many cases)a wide range of equipment, columns, and other materials is commercially available, allowing the use of HPLC for almost every applicationmost laboratories that deal with a need for analyzing chemical mixtures are equipped for HPLC; it is often the first choice of technique

As a result, HPLC is today one of the most useful and widely applied analytical techniques. Mass spectrometry rivals and complements HPLC in many respects; the use of these two techniques in combination (LC-MS) is already substantial (Section 4.14), and will continue to grow in importance.

In the present chapter we will:

examine some general features of HPLCsummarize the history of HPLCvery briefly consider some alternatives to HPLC, with their preferred use for certain applicationslist other sources of information about HPLC

1.1 BACKGROUND INFORMATION

1.1.1 What Is HPLC?

Liquid chromatography began in the early 1900s, in the form illustrated in Figure 1.1a–e, known as ''classical column chromatography''. A glass cylinder was packed with a finely divided powder such as chalk (Fig. 1.1a), a sample was applied to the top of the column (Fig. 1.1b), and a solvent was poured onto the column (Fig. 1.1c). As the solvent flows down the column by gravity (Fig. 1.1d), the components of the sample (A, B, and C in this example) begins to move through the column at different speeds and became separated. In its initial form, colored samples were investigated so that the separation within the column could be observed visually. Then portions of the solvent leaving the column were collected, the solvent was evaporated, and the separated compounds were recovered for quantitative analysis or other use (). In those days a new column was required for each sample, and the entire process was carried out manually (no automation). Consequently the effort required for each separation could be tedious and time-consuming. Still, even at this stage of development, chromatography provided a unique capability compared to other methods for the analysis of chemical mixtures.