Practical High-Performance Liquid Chromatography E-Book

Veronika R. Meyer

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch
• Veröffentlichungsjahr: 2013

Seitenzahl: 556

Beschreibung

Jump into the HPLC adventure! Three decades on from publication of the 1st German edition of Veronika Meyer's book on HPLC, this classic text remains one of the few titles available on general HPLC aimed at practitioners. New sections on the following topics have been included in this fifth edition: * Comparison of HPLC with capillary electrophoresis * How to obtain peak capacity * van Deemter curves and other coherences * Hydrophilic interaction chromatography * Method transfer * Comprehensive two-dimensional HPLC * Fast separations at 1000 bar * HPLC with superheated water In addition, two chapters on the instrument test and troubleshooting in the appendix have been updated and expanded by Bruno E. Lendi, and many details have been improved and numerous references added. A completely new chapter is presented on quality assurance covering: * Is it worth the effort? * Verification with a second method * Method validation * Standard operating procedures * Measurement uncertainty * Qualifications, instrument test, and system suitability test * The quest for quality Reviews of earlier editions "That this text is written by an expert in both the practice and teaching of HPLC is evident from the first paragraph....not only an enjoyable, fascinating and easy read, but a truly excellent text that has and will serve many teachers, students and practitioners very well." --The Analyst "...provides essential information on HPLC for LC practitioners in academia, industry, government, and research laboratories...a valuable introduction." - American Journal of Therapeutics

Leseprobe

Contents

Preface to the Fifth Edition

Important and Useful Equations for HPLC

1 Introduction

1.1 HPLC: A powerful separation method

1.2 A first HPLC experiment

1.3 Liquid chromatographic separation modes

1.4 The HPLC instrument

1.5 Safety in the HPLC laboratory

1.6 Comparison between high-performance liquid chromatography and gas chromatography

1.7 Comparison between high-performance liquid chromatography and capillary electrophoresis

1.8 Units for pressure, length and viscosity

1.9 Scientific journals

1.10 Recommended books

2 Theoretical Principles

2.1 The chromatographic process

2.2 Band broadening

2.3 The chromatogram and its purport

2.4 Graphical representation of peak pairs with different degree of resolution

2.5 Factors affecting resolution

2.6 Extra-column volumes (dead volumes)

2.7 Tailing

2.8 Peak capacity and statistical resolution probability

2.9 Effects of temperature in HPLC

2.10 The limits of HPLC

2.11 How to obtain peak capacity

3 Pumps

3.1 General requirements

3.2 The short-stroke piston pump

3.3 Maintenance and repair

3.4 Other pump designs

4 Preparation of Equipment up to Sample Injection

4.1 Selection of the mobile phase

4.2 Preparation of the mobile phase

4.3 Gradient systems

4.4 Capillary tubing

4.5 Fittings

4.6 Sample injectors

4.7 Sample solution and sample volume

5 Solvent Properties

5.1 Table of organic solvents

5.2 Solvent selectivity

5.3 Miscibility

5.4 Buffers

5.5 Shelf life of mobile phases

5.6 The mixing cross

6 Detectors

6.1 General

6.2 UV detectors

6.3 Refractive index detectors

6.4 Fluorescence detectors

6.5 Electrochemical (amperometric) detectors

6.6 Light-scattering detectors

6.7 Other detectors

6.8 Multiple detection

6.9 Indirect detection

6.10 Coupling with spectroscopy

7 Columns and Stationary Phases

7.1 Columns for HPLC

7.2 Precolumns

7.3 General properties of stationary phases

7.4 Silica

7.5 Chemically modified silica

7.6 Styrene-divinylbenzene

7.7 Some other stationary phases

7.8 Column care and regeneration

8 HPLC Column Tests

8.1 Simple tests for HPLC columns

8.2 Determination of particle size

8.3 Determination of breakthrough time

8.4 The test mixture

8.5 Dimensionless parameters for HPLC column characterization

8.6 The van Deemter equation from reduced parameters and its use in column diagnosis

8.7 van Deemter curves and other coherences

8.8 Diffusion coefficients

9 Adsorption Chromatography: Normal-Phase Chromatography

9.1 What is adsorption?

9.2 The eluotropic series

9.3 Selectivity properties of the mobile phase

9.4 Choice and optimization of the mobile phase

9.5 Applications

10 Reversed-Phase Chromatography

10.1 Principle

10.2 Mobile phases in reversed-phase chromatography

10.3 Solvent selectivity and strength

10.4 Stationary phases

10.5 Method development in reversed-phase chromatography

10.6 Applications

10.7 Hydrophobic interaction chromatography

11 Chromatography with Chemically Bonded Phases

11.1 Introduction

11.2 Properties of some stationary phases

11.3 Hydrophilic interaction chromatography

12 Ion-Exchange Chromatography

12.1 Introduction

12.2 Principle

12.3 Properties of ion exchangers

12.4 Influence of the mobile phase

12.5 Special possibilities of ion exchange

12.6 Practical hints

12.7 Applications

13 Ion-Pair Chromatography

13.1 Introduction

13.2 Ion-pair chromatography in practice

13.3 Applications

13.4 Appendix: UV detection using ion-pair reagents

14 Ion Chromatography

14.1 Principle

14.2 Suppression techniques

14.3 Phase systems

14.4 Applications

15 Size-Exclusion Chromatography

15.1 Principle

15.2 The calibration chromatogram

15.3 Molecular mass determination by means of size-exclusion chromatography

15.4 Coupled size-exclusion columns

15.5 Phase systems

15.6 Applications

16 Affinity Chromatography

16.1 Principle

16.2 Affinity chromatography as a special case of HPLC

16.3 Applications

17 Choice of Method

17.1 The various possibilities

17.2 Method transfer

18 Solving the Elution Problem

18.1 The elution problem

18.2 Solvent gradients

18.3 Column switching

18.4 Comprehensive two-dimensional HPLC

18.5 Optimization of an isocratic chromatogram using four solvents

18.6 Optimization of the other parameters

18.7 Mixed stationary phases

19 Analytical HPLC

19.1 Qualitative analysis

19.2 Trace analysis

19.3 Quantitative analysis

19.4 Recovery

19.5 Peak-height and peak-area determination for quantitative analysis

19.6 Integration errors

19.7 The detection wavelength

19.8 Derivatization

19.9 Unexpected peaks: Ghost and system peaks

20 Quality Assurance

20.1 Is it worth the effort?

20.2 Verification with a second method

20.3 Method validation

20.4 Standard operating procedures

20.5 Measurement uncertainty

20.6 Qualifications, instrument test and system suitability test

20.7 The quest for quality

21 Preparative HPLC

21.1 Problem

21.2 Preparative HPLC in practice

21.3 Overloading effects

21.4 Fraction collection

21.5 Recycling

21.6 Displacement chromatography

22 Separation of Enantiomers

22.1 Introduction

22.2 Chiral mobile phases

22.3 Chiral liquid stationary phases

22.4 Chiral solid stationary phases

22.5 Indirect separation of enantiomers

23 Special Possibilities

23.1 Micro, capillary and chip HPLC

23.2 High-speed and super-speed HPLC

23.3 Fast separations at 1000 bar: UHPLC

23.4 HPLC with supercritical mobile phases

23.5 HPLC with superheated water

23.6 Electrochromatography

24 Appendix 1: Applied HPLC Theory

25 Appendix 2: How to Perform the Instrument Test

25.1 Introduction

25.2 Test sequence

25.3 Preparations

25.4 Pump test

25.5 UV detector test

25.6 Autosampler test

25.7 Column oven test

25.8 Equations and calculations

25.9 Documentation

26 Appendix 3: Troubleshooting

26.1 Pressure problems

26.2 Leak in the pump system

26.3 Deviating retention times

26.4 Injection problems

26.5 Baseline problems

26.6 Peak shape problems

26.7 Problems with light-scattering detectors

26.8 Other causes

26.9 Instrument test

27 Appendix 4: Column Packing

Index of Separations

Subject Index

This edition first published 2010

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Library of Congress Cataloging-in-Publication Data

Meyer, Veronika.

[Praxis der Hochleistungs-Flüssigchromatographie. English]

Practical high-performance liquid chromatography / Veronika R. Meyer. – 5th ed.

p. cm.

Includes bibliographical references and index.

ISBN 978-0-470-68218-0 (cloth) – ISBN 978-0-470-68217-3 (pbk.)

1. High performance liquid chromatography. I. Title.

QD79.C454M4913 2010

543’.84–dc22

2009052143

A catalogue record for this book is available from the British Library.

ISBN H/bk 978-0470-682180 P/bk 978-0470-682173

To the memory of Otto Meyer

Alles ist einfacher, als man denken kann, zugleich verschränkter, als zu begreifen ist.

Goethe, Maximen

Everything is simpler than can be imagined, yet more intricate than can be comprehended.

Preface to the Fifth Edition

A small jubilee! This book started 30 years ago with the first German edition, with no idea that it could become a success story. Its content became younger with every edition, a fact which is not true concerning the author. In fact, I am sure that the latter cannot be a serious wish. No question: decades of experience are for the benefit of the book.

A new topic is now included: Chapter 20 about quality assurance. Part of it could be found before in Chapter 19 but now the subject is presented much broadly and independent of ‘Analytical HPLC’. Two chapters in the appendix were updated and expanded by Bruno E. Lendi, namely the ones about the instrument test (now Chapter 25) and troubleshooting (now Chapter 26). Some new sections were created: 1.7, comparison of HPLC with capillary electrophoresis; 2.11, how to obtain peak capacity; 8.7, van Deemter curves and other coherences; 11.3, hydrophilic interaction chromatography; 17.2, method transfer; 18.4, comprehensive two-dimensional HPLC; 23.3, fast separations at 1000 bar; 23.5, HPLC with superheated water. In addition, many details were improved and numerous references added.

Jump into the HPLC adventure! It can be a pleasure if you know the craft and its theoretical background.

St. Gallen, July 2009

Veronika R. Meyer

Important and Useful Equations for HPLC

This is a synopsis. The equations are explained in Chapters 2 and 8.

Retention factor:

Separation factor, α value:

Resolution:

Number of theoretical plates:

Height of a theoretical plate:

Asymmetry, tailing:

Linear flow velocity of the mobile phase:

Porosity of the column packing:

Reduced height of a theoretical plate:

Reduced flow velocity of the mobile phase:

Reduced flow resistance:

Total analysis time:

Total solvent consumption:

Peak volume:

A

P

peak area

a

0.1

width of the leading half of the peak at 10% of height

b

0.1

width of the trailing half of the peak at 10% of height

d

c

inner diameter of the column

D

m

diffusion coefficient of the analyte in the mobile phase

d

p

particle diameter of the stationary phase

F

flow rate of the mobile phase

f

distance between peak front and peak maximum at 0.05

h

h

P

peak height

k

last

retention factor of the last peak

L

c

column length

t

R

retention time

t

0

breakthrough time

V

volume

w

peak width

w

1/2

peak width at half height

w

0.05

peak width at 0.05 h

η

viscosity of the mobile phase

Δ

p

pressure drop

1

Introduction

1.1 HPLC: A POWERFUL SEPARATION METHOD

A powerful separation method must be able to resolve mixtures with a large number of similar analytes. Figure 1.1 shows an example. Eight benzodiazepines can be separated within 70 seconds.

Such a chromatogram provides directly both qualitative and quantitative information: each compound in the mixture has its own elution time (the point at which the signal appears on the screen) under a given set of conditions; and both the area and height of each signal are proportional to the amount of the corresponding substance.

This example shows that high-performance liquid chromatography (HPLC) is very efficient, i.e. it yields excellent separations in a short time. The ‘inventors’ of modern chromatography, Martin and Synge,1 were aware as far back as 1941 that, in theory, the stationary phase requires very small particles and hence a high pressure is essential for forcing the mobile phase through the column. As a result, HPLC was sometimes referred to as high-pressure liquid chromatography.

1.2 A FIRST HPLC EXPERIMENT

Although this beginner’s experiment described here is simple, it is recommended that you ask an experienced chromatographer for assistance.

It is most convenient if a HPLC system with two solvent reservoirs can be used. Use water and acetonitrile; both solvents need to be filtered (filter with < 1 μm pores) and degassed. Flush the system with pure acetonitrile, then connect a so-called reversed-phase column (octadecyl ODS or C18, but an octyl or C8 column can be used as well) with the correct direction of flow (if indicated) and flush it for ca. 10 min with acetonitrile. The flow rate depends on the column diameter: 1–2 ml min−1 for 4.6 mm columns, 0.5–1 mlmin−1 for 3 mm and 0.3–0.5 mlmin−1 for 2 mm columns. Then switch to water–acetonitrile 8 : 2 and flush again for 10–20 min. The UV detector is set to 272 nm (although 254 nm will work too). Prepare a coffee (a ‘real’ one, not decaffeinated), take a small sample before you add milk, sugar or sweetener and filter it (< 1 μm). Alternatively you can use tea (again, without additives) or a soft drink with caffeine (preferably without sugar); these beverages must be filtered, too. Inject 10 μl of the sample. A chromatogram similar to the one shown in Figure 1.2 will appear. The caffeine signal is usually the last large peak. If it is too high, inject less sample and vice versa; the attenuation of the detector can also be adjusted. It is recommended to choose a sample volume which gives a caffeine peak not higher than one absorption unit as displayed on the detector. If the peak is eluted late, e.g. later than 10 min, the amount of acetonitrile in the mobile phase must be increased (try water–acetonitrile 6 : 4). If it is eluted too early and with poor resolution to the peak cluster at the beginning, decrease the acetonitrile content (e.g. 9 : 1).