Introduction to Modern Liquid Chromatography

Preface xxxi

Glossary of Symbols and Abbreviations xxxv

1 Introduction 1

1.1 Background Information 2

1.2 A Short History of HPLC 6

1.3 Some Alternatives to HPLC 8

1.4 Other Sources of HPLC Information 12

References 15

2 Basic Concepts and the Control of Separation 19

2.1 Introduction 20

2.2 The Chromatographic Process 20

2.3 Retention 24

2.4 Peak Width and the Column Plate Number N 35

2.5 Resolution and Method Development 54

2.6 Sample Size Effects 69

2.7 Related Topics 74

References 83

3 Equipment 87

3.1 Introduction 88

3.2 Reservoirs and Solvent Filtration 89

3.3 Mobile-Phase Degassing 92

3.4 Tubing and Fittings 96

3.5 Pumping Systems 104

3.6 Autosamplers 113

3.7 Column Ovens 125

3.8 Data Systems 127

3.9 Extra-Column Effects 131

3.10 Maintenance 131

References 144

4 Detection 147

4.1 Introduction 148

4.2 Detector Characteristics 149

4.3 Introduction to Individual Detectors 160

4.4 UV-Visible Detectors 160

4.5 Fluorescence Detectors 167

4.6 Electrochemical (Amperometric) Detectors 170

4.7 Radioactivity Detectors 172

4.8 Conductivity Detectors 174

4.9 Chemiluminescent Nitrogen Detector 174

4.10 Chiral Detectors 175

4.11 Refractive Index Detectors 177

4.12 Light-Scattering Detectors 180

4.13 Corona-Discharge Detector (CAD) 184

4.14 Mass Spectral Detectors (MS) 185

4.15 Other Hyphenated Detectors 191

4.16 Sample Derivatization and Reaction Detectors 194

References 196

5 The Column 199

5.1 Introduction 200

5.2 Column Supports 200

5.3 Stationary Phases 217

5.4 Column Selectivity 227

5.5 Column Hardware 238

5.6 Column-Packing Methods 240

5.7 Column Specifications 244

5.8 Column Handling 246

References 250

6 Reversed-phase Chromatography for Neutral Sam- Ples 253

6.1 Introduction 254

6.2 Retention 256

6.3 Selectivity 263

6.4 Method Development and Strategies for Optimizing Selectivity 284

6.5 Nonaqueous Reversed-Phase Chromatography (narp) 295

6.6 Special Problems 297

References 298

7 Ionic Samples: Reversed-phase Ion-pair and Ion- Exchange Chromatography 303

7.1 Introduction 304

7.2 Acid-Base Equilibria and Reversed-Phase Retention 304

7.3 Separation of Ionic Samples by Reversed-Phase Chromatography (RPC) 319

7.4 Ion-Pair Chromatography (IPC) 331

7.5 Ion-Exchange Chromatography (IEC) 349

References 357

8 Normal-phase Chromatography 361

8.1 Introduction 362

8.2 Retention 363

8.3 Selectivity 376

8.4 Method-Development Summary 385

8.5 Problems in the Use of NPC 392

8.6 Hydrophilic Interaction Chromatography (HILIC) 395

References 401

9 Gradient Elution 403

9.1 Introduction 404

9.2 Experimental Conditions and Their Effects on Separation 412

9.3 Method Development 434

9.4 Large-Molecule Separations 464

9.5 Other Separation Modes 465

9.6 Problems 470

References 471

10 Computer-assisted Method Development 475

10.1 Introduction 475

10.2 Computer-Simulation Software 481

10.3 Other Method-Development Software 491

10.4 Computer Simulation and Method Development 492

References 497

11 Qualitative and Quantitative Analysis 499

11.1 Introduction 499

11.2 Signal Measurement 500

11.3 Qualitative Analysis 516

11.4 Quantitative Analysis 520

11.5 Summary 529

References 529

12 Method Validation 531 with Michael Swartz

12.1 Introduction 532

12.2 Terms and Definitions 534

12.3 System Suitability 542

12.4 Documentation 543

12.5 Validation for Different Pharmaceutical-Method Types 546

12.6 Bioanalytical Methods 548

12.7 Analytical Method Transfer (AMT) 554

12.8 Method Adjustment or Method Modification 561

12.9 Quality Control and Quality Assurance 564

12.10 Summary 565

References 566

13 Biochemical and Synthetic Polymer Separations 569 with Timothy Wehr, Carl Scandella, and Peter Schoenmakers

13.1 Biomacromolecules 570

13.2 Molecular Structure and Conformation 571

13.3 Special Considerations for Biomolecule HPLC 579

13.4 Separation of Peptides and Proteins 584

13.5 Separation of Nucleic Acids 618

13.6 Separation of Carbohydrates 625

13.7 Separation of Viruses 630

13.8 Size-Exclusion Chromatography (SEC) 631

13.9 Large-Scale Purification of Large Biomolecules 641

13.10 Synthetic Polymers 648

References 658

14 Enantiomer Separations 665 with Michael Lämmerhofer, Norbert M. Maier and Wolfgang Lindner

14.1 Introduction 666

14.2 Background and Definitions 666

14.3 Indirect Method 670

14.4 Direct Method 675

14.5 Peak Dispersion and Tailing 681

14.6 Chiral Stationary Phases and Their Characteristics 681

14.7 Thermodynamic Considerations 715

References 718

15 Preparative Separations 725 with Geoff Cox

15.1 Introduction 726

15.2 Equipment for Prep-LC Separation 730

15.3 Isocratic Elution 736

15.4 Severely Overloaded Separation 748

15.5 Gradient Elution 751

15.6 Production-Scale Separation 754

References 755

16 Sample Preparation 757 with Ronald Majors 757

16.1 Introduction 758

16.2 Types of Samples 759

16.3 Preliminary Processing of Solid and Semi-Solid Samples 760

16.4 Sample Preparation for Liquid Samples 764

16.5 Liquid-Liquid Extraction 764

16.6 Solid-Phase Extraction (SPE) 771

16.7 Membrane Techniques in Sample Preparation 790

16.8 Sample Preparation Methods for Solid Samples 791

16.9 Column-Switching 796

16.10 Sample Preparation for Biochromatography 797

16.11 Sample Preparation for LC-MS 800

16.12 Derivatization in HPLC 802

References 805

17 Troubleshooting 809

Quick Fix 809

17.1 Introduction 810

17.2 Prevention of Problems 811

17.3 Problem-Isolation Strategies 819

17.4 Common Symptoms of HPLC Problems 821

17.5 Troubleshooting Tables 865

References 876

Appendix I. Properties of HPLC Solvents 879

I.1 Solvent-Detector Compatibility 879

I. 2 Solvent Polarity and Selectivity 882

I. 3 Solvent Safety 885

References 886

Appendix II. Preparing Buffered Mobile Phases 887

II.1 Sequence of Operations 887

II.2 Recipes for Some Commonly Used Buffers 888

Reference 890

Index 891