Practical High-Performance Liquid Chromatography / Edition 5

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Overview

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

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Editorial Reviews

From the Publisher
"This book is very much ‘‘an aid to this end'' and should be number one on every young chromatographer's book list. The publishers too have played their part in producing a book that is pleasing on the eye and a pleasure to read." (Chromatographia, 1 December 2010)
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Product Details

  • ISBN-13: 9780470682173
  • Publisher: Wiley
  • Publication date: 6/8/2010
  • Edition description: New Edition
  • Edition number: 5
  • Pages: 426
  • Sales rank: 901,294
  • Product dimensions: 5.90 (w) x 8.90 (h) x 1.00 (d)

Table of Contents

Preface to the Fifth Edition xiii

Important and Useful Equations for HPLC 1

1 Introduction 5

1.1 HPLC: A powerful separation method 5

1.2 A first HPLC experiment 5

1.3 Liquid chromatographic separation modes 8

1.4 The HPLC instrument 9

1.5 Safety in the HPLC laboratory 10

1.6 Comparison between high-performance liquid chromatography and gas chromatography 11

1.7 Comparison between high-performance liquid chromatography and capillary electrophoresis 12

1.8 Units for pressure, length and viscosity 13

1.9 Scientific journals 14

1.10 Recommended books 15

2 Theoretical Principles 17

2.1 The chromatographic process 17

2.2 Band broadening 19

2.3 The chromatogram and its purport 23

2.4 Graphical representation of peak pairs with different degree of resolution 30

2.5 Factors affecting resolution 35

2.6 Extra-column volumes (dead volumes) 40

2.7 Tailing 41

2.8 Peak capacity and statistical resolution probability 46

2.9 Effects of temperature in HPLC 49

2.10 The limits of HPLC 51

2.11 How to obtain peak capacity 55

3 Pumps 59

3.1 General requirements 59

3.2 The short-stroke piston pump 59

3.3 Maintenance and repair 62

3.4 Other pump designs 63

4 Preparation of Equipment up to Sample Injection 65

4.1 Selection of the mobile phase 65

4.2 Preparation of the mobile phase 67

4.3 Gradient systems 68

4.4 Capillary tubing 70

4.5 Fittings 72

4.6 Sample injectors 74

4.7 Sample solution and sample volume 78

5 Solvent Properties 81

5.1 Table of organic solvents 81

5.2 Solvent selectivity 83

5.3 Miscibility 83

5.4 Buffers 84

5.5 Shelf life of mobile phases 87

5.6 The mixing cross 88

6 Detectors 91

6.1 General 91

6.2 UV detectors 96

6.3 Refractive index detectors 99

6.4 Fluorescence detectors 101

6.5 Electrochemical (amperometric) detectors 103

6.6 Light-scattering detectors 104

6.7 Other detectors 106

6.8 Multiple detection 107

6.9 Indirect detection 108

6.10 Coupling with spectroscopy 109

7 Columns and Stationary Phases 117

7.1 Columns for HPLC 117

7.2 Precolumns 119

7.3 General properties of stationary phases 120

7.4 Silica 125

7.5 Chemically modified silica 126

7.6 Styrene-divinylbenzene 129

7.7 Some other stationary phases 133

7.8 Column care and regeneration 136

8 HPLC Column Tests 141

8.1 Simple tests for HPLC columns 141

8.2 Determination of particle size 143

8.3 Determination of breakthrough time 144

8.4 The test mixture 146

8.5 Dimensionless parameters for HPLC column characterization 148

8.6 The van Deemter equation from reduced parameters and its use in column diagnosis 152

8.7 van Deemter curves and other coherences 153

8.8 Diffusion coefficients 155

9 Adsorption Chromatography: Normal-Phase Chromatography 159

9.1 What is adsorption? 159

9.2 The eluotropic series 162

9.3 Selectivity properties of the mobile phase 165

9.4 Choice and optimization of the mobile phase 166

9.5 Applications 168

10 Reversed-Phase Chromatography 173

10.1 Principle 173

10.2 Mobile phases in reversed-phase chromatography 174

10.3 Solvent selectivity and strength 177

10.4 Stationary phases 181

10.5 Method development in reversed-phase chromatography 185

10.6 Applications 188

10.7 Hydrophobic interaction chromatography 191

11 Chromatography with Chemically Bonded Phases 195

11.1 Introduction 195

11.2 Properties of some stationary phases 195

11.3 Hydrophilic interaction chromatography 200

12 Ion-Exchange Chromatography 203

12.1 Introduction 203

12.2 Principle 203

12.3 Properties of ion exchangers 204

12.4 Influence of the mobile phase 207

12.5 Special possibilities of ion exchange 208

12.6 Practical hints 210

12.7 Applications 213

13 Ion-Pair Chromatography 217

13.1 Introduction 217

13.2 Ion-pair chromatography in practice 218

13.3 Applications 220

13.4 Appendix: UV detection using ion-pair reagents 221

14 Ion Chromatography 225

14.1 Principle 225

14.2 Suppression techniques 226

14.3 Phase systems 226

14.4 Applications 230

15 Size-Exclusion Chromatography 231

15.1 Principle 231

15.2 The calibration chromatogram 234

15.3 Molecular mass determination by means of size-exclusion chromatography 238

15.4 Coupled size-exclusion columns 241

15.5 Phase systems 243

15.6 Applications 244

16 Affinity Chromatography 249

16.1 Principle 249

16.2 Affinity chromatography as a special case of HPLC 251

16.3 Applications 252

17 Choice of Method 255

17.1 The various possibilities 255

17.2 Method transfer 260

18 Solving the Elution Problem 263

18.1 The elution problem 263

18.2 Solvent gradients 264

18.3 Column switching 270

18.4 Comprehensive two-dimensional HPLC 272

18.5 Optimization of an isocratic chromatogram using four solvents 273

18.6 Optimization of the other parameters 276

18.7 Mixed stationary phases 284

19 Analytical HPLC 285

19.1 Qualitative analysis 285

19.2 Trace analysis 287

19.3 Quantitative analysis 291

19.4 Recovery 296

19.5 Peak-height and peak-area determination for quantitative analysis 299

19.6 Integration errors 303

19.7 The detection wavelength 304

19.8 Derivatization 306

19.9 Unexpected peaks: Ghost and system peaks 308

20 Quality Assurance 311

20.1 Is it worth the effort? 311

20.2 Verification with a second method 312

20.3 Method validation 312

20.4 Standard operating procedures 314

20.5 Measurement uncertainty 315

20.6 Qualifications, instrument test and system suitability test 317

20.7 The quest for quality 318

21 Preparative HPLC 321

21.1 Problem 321

21.2 Preparative HPLC in practice 322

21.3 Overloading effects 325

21.4 Fraction collection 328

21.5 Recycling 330

21.6 Displacement chromatography 331

22 Separation of Enantiomers 333

22.1 introduction 333

22.2 Chiral mobile phases 335

22.3 Chiral liquid stationary phases 336

22.4 Chiral solid stationary phases 337

22.5 Indirect separation of enantiomers 345

23 Special Possibilities 349

23.1 Micro, capillary and chip HPLC 349

23.2 High-speed and super-speed HPLC 352

23.3 Fast separations at 1000 bar: UHPLC 353

23.4 HPLC with supercritical mobile phases 355

23.5 HPLC with superheated water 359

23.6 Electrochromatography 361

24 Appendix 1 Applied HPLC Theory 363

25 Appendix 2 How to Perform the Instrument Test 373

25.1 Introduction 373

25.2 Test sequence 373

25.3 Preparations 374

25.4 Pump test 377

25.5 UV detector test 379

25.6 Autosampler test 383

25.7 Column oven test 383

25.8 Equations and calculations 384

25.9 Documentation 385

26 Appendix 3 Troubleshooting 387

26.1 Pressure problems 387

26.2 Leak in the pump system 389

26.3 Deviating retention times 389

26.4 Injection problems 390

26.5 Baseline problems 390

26.6 Peak shape problems 392

26.7 Problems with light-scattering detectors 393

26.8 Other causes 394

26.9 Instrument test 395

27 Appendix 4 Column Packing 397

Index of Separations 401

Subject Index 403

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