Contributors xxv

Part I Hplc Theory and Practice 1

1 Introduction 3
Yuri Kazakevich and Rosario LoBrutto

1.1 Chromatography in the Pharmaceutical World, 3

1.2 Chromatographic Process, 4

1.3 Classification, 4

1.4 History of Discovery and Early Development (1903–1933), 6

1.5 General Separation Process, 8

1.5.1 Modern HPLC Column, 9

1.5.2 HPLC System, 9

1.6 Types of HPLC, 10

1.6.1 Normal-Phase Chromatography (NP HPLC), 10

1.6.2 Reversed-Phase HPLC (RP HPLC or RPLC), 11

1.6.3 Ion-Exchange Chromatography (IEX), 13

1.6.4 Size-Exclusion Chromatography (SEC), 14

1.7 HPLC Descriptors (Vr, k, N, etc.), 15

1.7.1 Retention Volume, 15

1.7.2 Void Volume, 16

1.7.3 Retention Factor, 17

1.7.4 Selectivity, 18

1.7.5 Efficiency, 19

1.7.6 Resolution, 22

References, 23

2 HPLC Theory 25
Yuri Kazakevich

2.1 Introduction, 25

2.2 Basic Chromatographic Descriptors, 26

2.3 Efficiency, 27

2.4 Resolution, 32

2.5 HPLC Retention, 34

2.6 Retention Mechanism, 35

2.7 General Column Mass Balance, 37

2.8 Partitioning Model, 39

2.9 Adsorption Model, 40

2.10 Total and Excess Adsorption, 41

2.11 Mass Balance in Adsorption Model, 42

2.12 Adsorption of the Eluent Components, 43

2.13 Void Volume Considerations, 47

2.14 Thermodynamic Relationships, 49

2.14.1 Effect of the Eluent Composition, 53

2.15 Adsorption-Partitioning Retention Mechanism, 54

2.16 Secondary Equilibria, 57

2.16.1 Inclusion of Secondary Equilibria in the Mass Balance, 58

2.16.2 Salt Effect, 62

2.17 Gradient Elution Principles, 67

2.18 Types of Analyte Interactions with the Stationary Phase, 69

2.19 Conclusion, 70

References, 71

3 Stationary Phases 75
Yuri Kazakevich and Rosario LoBrutto

3.1 Introduction, 75

3.2 Type of Packing Material (Porous, Nonporous, Monolithic), 77

3.3 Base Material (Silica, Zirconia, Alumina, Polymers), 77

3.4 Geometry, 80

3.4.1 Shape (Spherical/Irregular), 80

3.4.2 Particle Size Distribution, 80

3.4.3 Surface Area, 81

3.4.4 Pore Volume, 82

3.4.5 Surface Geometry, 84

3.5 Adsorbent Surface Chemistry, 85

3.5.1 Surface Chemistry of the Base Material, 85

3.5.2 Silica, 86

3.5.3 Silica Hybrid, 88

3.5.4 Polymeric Packings, 89

3.5.5 Zirconia (Metal Oxides), 90

3.5.6 Porous Carbon (or Carbon-Coated Phases), 90

3.6 Surface of Chemically Modified Material, 91

3.6.1 Limits of Surface Modification, 93

3.6.2 Chemical Modification, 93

3.6.3 Types of Bonded Phases, 101

3.6.4 Structure of the Bonded Layer, 103

3.6.5 Density of Bonded Ligands, 105

3.6.6 Residual Silanoles, 110

3.6.7 Surface Area of Modified Adsorbent, 110

3.7 Polymer-Based Adsorbents, 113

3.8 Stationary Phases for Chiral Separations, 115

3.8.1 Polysaccharide-Coated Phases, 115

3.8.2 Pirkle-Type Phases, 116

3.8.3 Protein Phases, 116

3.8.4 Molecular Imprinted Polymers for Chiral Separations, 117

3.9 Columns, 118

3.9.1 Capillary/Monolithic/Packed Columns, 118

3.9.2 Column Cleaning, 126

3.9.3 Column Void Volume, 128

3.9.4 Mass of Adsorbent in the Column, 130

References, 132

4 Reversed-Phase HPLC 139
Rosario LoBrutto and Yuri Kazakevich

4.1 Introduction, 139

4.2 Retention in Reversed-Phase HPLC, 140

4.3 Stationary Phases for RPLC, 142

4.4 Mobile Phases for RPLC, 145

4.4.1 Eluent Composition and Solvent Strength of the Mobile Phase, 146

4.4.2 Type of Organic Modifier, 151

4.4.3 Selectivity as a Function of Type and Concentration of Organic Composition, 153

4.5 pH Effect on HPLC Separations, 158

4.5.1 Mobile-Phase pH. Practical Considerations, 158

4.5.2 Analyte Ionization (Acids, Bases, Zwitterions), 160

4.5.3 pK a and pK b Relationship, 161

4.5.4 Retention of Ionizible Analytes in Reversed-Phase Hplc, 161

4.5.5 Case Studies: Effects of pH on Ionizable Analyte Retention, 166

4.5.6 Mobile-Phase pH, 171

4.5.7 Analyte Dissociation Constants, 179

4.5.8 Determination of Chromatographic pK a , 180

4.6 Effect of Organic Eluent Composition on Analyte Ionization, 182

4.6.1 Effect of Organic Modifier on Basic Analyte pK a Shift, 182

4.6.2 Effect of Organic Modifier on Acidic Analyte pK a Shift, 186

4.7 Synergistic Effect of pH, Organic Eluent, and Temperature on Ionizable Analyte Retention and Selectivity, 189

4.8 Examples of Applying pH Shift and Analyte pK a Shift Rules, 191

4.9 Effect of Temperature on Analyte Ionization, 195

4.10 Ion-Interaction Chromatography, 197

4.10.1 Introduction, 197

4.10.2 Double Layer Theory, 198

4.10.3 Ion Pairs, 200

4.10.4 Chaotropic Effect, 206

4.11 Concluding Remarks, 227

References, 228

5 Normal-Phase HPLC 241
Yong Liu and Anant Vailaya

5.1 Introduction, 241

5.2 Theory of Retention in Normal-Phase Chromatography, 241

5.3 Effect of Mobile Phase on Retention, 245

5.4 Selectivity, 248

5.4.1 Effect of Analyte Structure, 248

5.4.2 Types of Stationary Phases, 249

5.5 Applications, 251

5.5.1 Analytes Prone to Hydrolysis, 251

5.5.2 Extremely Hydrophobic Compounds, 252

5.5.3 Separation of Isomers, 253

5.5.4 Carbohydrates, 256

5.5.5 Separation of Saturated/Unsaturated Compounds, 257

5.6 Conclusions, 257

References, 257

6 Size-Exclusion Chromatography 263
Yuri Kazakevich and Rosario LoBrutto

6.1 Separation of the Analyte Molecules by Their Size, 263

6.2 Molecular Size and Molecular Weight, 266

6.3 Separation Mechanism, 267

6.4 Calibration, 268

6.5 Columns, 271

6.6 Molecular Weight Distribution, 273

6.7 Effect of Eluent, 274

6.8 Effect of Temperature, 274

6.9 Detectors, 275

6.10 Solving Mass Balance Issues, 275

6.11 Aqueous SEC Applications, 276

References, 278

7 LC/MS: Theory, Instrumentation, and Applications to Small Molecules 281
Guodong Chen, Li-Kang Zhang, and Birendra N. Pramanik

7.1 Introduction, 281

7.2 Ionization Methods and LC/MS Interfaces, 282

7.2.1 Ionization Methods, 282

7.2.2 Historical View of Interfaces, 286

7.2.3 Common Interfaces, 288

7.2.4 Special Interfaces, 290

7.3 Mass Analyzers, 291

7.3.1 Magnetic Sector, 291

7.3.2 Quadrupole, 292

7.3.3 Ion Trap, 293

7.3.4 Time-of-Flight, 294

7.3.5 Ft-icr, 295

7.3.6 Tandem MS, 296

7.4 Role of Instrumental Parameters on Ionization Efficiency in LC/MS, 299

7.4.1 Optimization of Ionization Settings, 299

7.4.2 Effect of Flow Rate, 302

7.5 Effect of Mobile-Phase Composition on Ionization Efficiency in LC/MS, 303

7.5.1 Choice of Solvents, 303

7.5.2 Choice of Mobile-Phase Additives, 303

7.5.3 Adduct Formation, 304

7.5.4 Effect of Analyte Concentration, 304

7.5.5 Selected Ion Monitoring and Multiple Reaction Monitoring, 305

7.6 MS Interpretation, 305

7.6.1 Molecular Weight and Empirical Formula Determination, 305

7.6.2 Fragmentation Pattern, 313

7.7 Practical Applications, 315

7.7.1 High-Throughput LC/MS for Combinatorial Chemistry, 315

7.7.2 Characterization of Impurities and Decomposition Products in Bulk Drug Substances, 317

7.7.3 Pharmacokinetic Studies of Drugs, 325

7.7.4 Identification of Drug Metabolites, 332

7.8 Conclusions, 336

Acknowledgment, 338

References, 338

8 Method Development 347
Rosario LoBrutto

8.1 Introduction, 347

8.2 Types of Methods, 348

8.2.1 Key Raw Materials, 348

8.2.2 Drug Substance (Active Pharmaceutical Ingredient), 352

8.2.3 Drug Product, 355

8.2.4 Achiral Versus Chiral Methods, 359

8.3 Defining the Method, 360

8.4 Method Development Considerations, 361

8.4.1 Sample Properties, 361

8.4.2 Detector Considerations, 367

8.4.3 Solution Stability and Sample Preparation, 369

8.4.4 Choice of Stationary Phase, 373

8.4.5 Mobile-Phase Considerations, 375

8.4.6 Gradient Separations, 381

8.5 Method Development Approaches, 385

8.5.1 If Analyte Structure Is Known, 385

8.5.2 If Method Is Being Developed for Separation of Active and Unknown Component, 387

8.5.3 Defining System Suitability, 389

8.5.4 Case Study 1: Method Development for a Zwitterionic Compound, 391

8.5.5 Case Study 2: Influence of pH, Temperature, and Type and Concentration of Solvent on the Retention and Selectivity of Acidic (Phenolic) Compounds, 396

8.5.6 Case Study 3: Method Development for a Diprotic Basic Compound, 405

8.5.7 Case Study 4: Structural Elucidation Employing a Deuterated Eluent, 426

8.6 Effect of pH on UV Absorbance, 429

8.7 Analyte pK a —From an Analytical Chemist’s Perspective, 432

8.7.1 Aromatic Acids, 432

8.7.2 Amines, 434

8.8 Reversed-Phase Versus Normal-Phase Separations, 435

8.9 Instrument/System Considerations, 438

8.9.1 Column/System Backpressure, 438

8.9.2 Column Inlet and Outlet Frits, 439

8.9.3 Seals, 440

8.9.4 Mobile-Phase Preparation, 440

8.9.5 Guard Columns, 441

8.9.6 Instrument/System Considerations (Concluding Remarks), 442

8.10 Column Testing (Stability and Selectivity), 442

8.10.1 Column Selectivity Testing, 442

8.10.2 Column Stability Testing, 445

8.10.3 Choice of Buffer Related to Bonded-Phase Stability, 448

8.11 Concluding Remarks, 451

References, 452

9 Method Validation 455
Rosario LoBrutto and Tarun Patel

9.1 Introduction, 455

9.2 Validation Report, 457

9.3 Revalidation, 458

9.4 Assignment of Validation Parameters, 459

9.4.1 Accuracy, 460

9.4.2 Precision, 470

9.4.3 Linearity, 471

9.4.4 Lod/loq, 481

9.4.5 Relative Response Factors, 484

9.4.6 Stability of Solution, 485

9.4.7 Ruggedness/Robustness, 486

9.4.8 Specificity, 490

9.4.9 Forced Degradation Studies (Solid State and Solution)— Drug Substance and Drug Product, 491

9.5 Distinguishing Drug-Related and Non-Drug-Related Degradation Products, 495

9.5.1 Drug Product Stress, 497

9.6 Concluding Remarks, 499

References, 499

10 Computer-Assisted HPLC and Knowledge Management 503
Yuri Kazakevich, Michael McBrien, and Rosario LoBrutto

10.1 Introduction, 503

10.2 Prediction of Retention and Simulation of Profiles, 504

10.2.1 General Thermodynamic Basis, 505

10.2.2 Structure–Retention Relationships, 506

10.3 Optimization of HPLC Methods, 507

10.3.1 Off-Line Optimization, 507

10.3.2 On-Line Optimization, 510

10.3.3 Method Screening, 511

10.3.4 Method Optimization, 512

10.4 Structure-Based Tools, 517

10.4.1 Knowledge Management, 517

10.4.2 Applications Databases, 519

10.4.3 Structure-Based Prediction, 521

10.5 Conclusion, 528

References, 529

Part II Hplc in the Pharmaceutical Industry 533

11 The Expanding Role of HPLC in Drug Discovery 535
Daniel B. Kassel

11.1 Introduction, 535

11.2 Applications of HPLC/MS for Protein Identification and Characterization, 536

11.3 Applications of HPLC/MS/MS in Support of Protein Chemistry, 538

11.4 Applications of HPLC/MS/MS in Support of Assay Development and Screening, 539

11.5 Sources of Compounds for Biological Screening, 540

11.6 HPLC/MS Analysis to Support Compound Characterization, 542

11.6.1 Purity Assessment of Compound Libraries, 544

11.7 Purification Technologies for Drug Discovery, 547

11.7.1 UV-Directed Purification, 548

11.7.2 Mass-Directed Preparative Purification, 549

11.8 Higher-Throughput Purification Strategies, 552

11.8.1 Fluorous Split-Mix Library Synthesis and Prepartive LC/MS De-Mixing, 552

11.8.2 Parallel Analysis and Parallel Purification, 553

11.8.3 Streamlining the Purification Process, 558

11.9 ADME Applications, 559

11.10 Fast Serial ADME Analyses Incorporating LC-MS and Lc-ms/ms, 561

11.10.1 Automated Data Processing Is Instrumental to Achieving High-Throughput ADME, 561

11.10.2 Enhancing Throughput by Incorporating Pooling Strategies, 563

11.11 Parallel Approaches to Speeding ADME Analyses, 563

11.11.1 Nonindexed Parallel Mass Spectrometry, 563

11.11.2 Indexed (“MUX”) Parallel Mass Spectrometry, 564

11.12 Automated “Intelligent” Metabolic Stability and Metabolite Id, 565

11.13 Conclusions, 568

References, 569

12 Role of HPLC in Preformulation 577
Irina Kazakevich

12.1 Introduction, 577

12.2 Initial Physicochemical Characterization (Discovery Support), 579

12.2.1 Ionization Constant, pK a , 580

12.2.2 Partition and Distribution Coefficients, 582

12.2.3 Solubility and Solubilization, 586

12.3 Chemical Stability, 590

12.4 Salt Selection, 594

12.5 Polymorphism, 594

12.6 Preformulation Late Stage (Development Support), 596

12.7 Conclusions, 599

References, 600

13 The Role of Liquid Chromatography–Mass Spectrometry in Pharmacokinetics and Drug Metabolism 605
Ray Bakhtiar, Tapan K. Majumdar, and Francis L. S. Tse

13.1 Introduction, 605

13.2 Ionization Processes, 606

13.3 Tandem-Mass Spectrometry (MS/MS), 610

13.4 Sample Preparation Using an Off-Line Approach, 611

13.4.1 Spe, 612

13.4.2 Ppt, 613

13.4.3 Lle, 615

13.5 Automated Sample Transfer, 615

13.6 Sample Processing Using an On-Line Approach, 616

13.7 Matrix Effect and Ion Suppression, 619

13.8 Regulatory Requirements for LC/MS Method Validation, 620

13.9 Ritalin ® : An Application of Enantioselective LC-MS/MS, 624

13.10 Gleevec TM (STI571), 626

13.11 Biomarkers, 629

13.12 Conclusions, 633

Acknowledgments, 633

References, 633

14 Role of HPLC in Process Development 641
Richard Thompson and Rosario LoBrutto

14.1 Responsibilities of the Analytical Chemist During Process Development, 641

14.2 HPLC Separation Modes, 643

14.2.1 Reversed-Phase Liquid Chromatography, 643

14.2.2 Normal-Phase Chromatography, 644

14.2.3 Sub-/Supercritical Chromatography, 645

14.2.4 Hydrophilic Interaction Chromatography, 647

14.2.5 Ion-Exchange Chromatography, 649

14.2.6 Chiral Chromatography, 650

14.3 Sample Preparation, 653

14.4 HPLC Detectors, 654

14.5 Method Development, 657

14.6 In-Process Monitoring, 660

14.7 Impurity Identification, 663

14.8 Establishment of HPLC Selectivity by Stress Studies, 665

14.8.1 Stability in Solution and Forced Degradation Studies (Process Intermediate Compound A), 666

14.9 HPLC Method Validation, 670

14.9.1 Prevalidation and System Suitability, 671

14.9.2 Validation, 672

14.10 Technology Transfer, 673

14.11 Concluding Remarks, 674

References, 674

15 Role of HPLC During Formulation Development 679
Tarun S. Patel and Rosario LoBrutto

15.1 Introduction, 679

15.2 Prerequisite for Analytical Chemists During Formulation Development, 681

15.2.1 Major Degradation Pathways in Pharmaceuticals, 681

15.3 Properties of Drug Substance, 682

15.3.1 Solubility of Drug Substance in Presence of Formulation, 682

15.3.2 Solution Stability, 683

15.4 Properties of Excipients, 683

15.5 Impact of Excipients on Degradation of API(s), 684

15.6 Test Methods for Most Common Dosage Forms in which HPLC Is the Primary Technique, 686

15.6.1 Assay and Related Substances, 687

15.6.2 Stability-Indicating Method (SIM), 688

15.7 Forced Decomposition, 691

15.8 Compatibility of Excipients with API(s) (Type and Ratio), 695

15.9 Mass Balance, 698

15.9.1 Case Study 1, 698

15.9.2 Case Study 2, 702

15.9.3 Detection Considerations, 706

15.9.4 Mass Balance Concluding Remarks, 707

15.10 Summary of Assay and Related Substances, 707

15.11 Uniformity of Dosage Units, 707

15.12 Blend Uniformity (BU), 708

15.13 Cleaning Verification, 709

15.14 Extractables/Leachables, 710

15.15 Dissolution, 713

15.16 Method Development, 713

15.16.1 Sample Preparation Solvent, 714

15.17 Method Validation, 714

15.17.1 Completeness of Extraction, 714

15.18 Testing of Samples, 715

15.18.1 Clinical Release, 715

15.18.2 Stability, 715

15.19 Automation Opportunities, 718

15.20 Implementation of Alternative Technologies, 719

15.21 Challenges and Future Trends, 720

References, 720

A15.1 Addendum (Common Functional Groups), 723

A15.1.1 Carbonyls, 724

A15.1.2 Nitrogen Functional Groups, 728

A15.1.3 Ethers, Thioethers, 730

A15.1.4 Alkyl/Aryl Halides, 730

A15.1.5 Hydroxyls, 731

A15.1.6 Thiols, 731

A15.1.7 Phenols, 731

A15.1.8 Olefins, 731

A15.1.9 Dimerization, 732

A15.1.10 Ring Transformations, 733

Addendum References, 733

16 The Role of HPLC in Technical Transfer and Manufacturing 735
Joseph Etse

16.1 Introduction, 735

16.2 Prerequisites for Transfer of HPLC Methods, 736

16.2.1 Availability of Either Fully or Partially Validated Methods, 736

16.2.2 Availability of the Finalized Pharmaceutical Active Ingredient (API), Known Degradation Products, By-products and Reference Standards, 738

16.2.3 Availability of Drug Products Made by the Definitive Manufacturing Process, 739

16.2.4 Availability of Suitable Instruments and Personnel, 739

16.2.5 Availability of a Protocol Containing Predetermined Acceptance Criteria, 740

16.3 Types of Technical Transfer, 745

16.3.1 From Analytical Research and Development (AR&D) to Quality Control (QC) Lab of the Commercial Organization, 745

16.3.2 Transfer from AR&D to Another AR&D Organization, 747

16.3.3 Transfer from AR&D to Contract Research Organization (CRO), 748

16.4 Different Approaches for Technical Transfer and Manufacturing, 748

16.4.1 Comparative Testing, 748

16.4.2 Co-validation of Methods, 750

16.4.3 Revalidation of Methods, 750

16.4.4 Waiver of Transfer, 752

16.5 Potential Pitfalls During Technical Transfer and Manufacturing, 753

16.5.1 Sample Handling, 753

16.5.2 Sample Type and Number of Replicate Determination, 755

16.5.3 Time of Testing, 756

16.5.4 Instrumental Issues, 757

16.5.5 Column and Instrumental Issues, 757

16.5.6 Differences in Chromatographic Data Acquisition Systems, 759

16.6 Conclusion, 760

References, 760

Part III Hyphenated Techniques and Specialized Hplc Separations 763

17 Development of Fast HPLC Methods 765
Anton D. Jerkovich and Richard V. Vivilecchia

17.1 Introduction, 765

17.2 Basic Theory, 766

17.2.1 Resolution and Analysis Time, 767

17.2.2 Plate Height and Band-Broadening, 769

17.2.3 Flow Velocity and Column Backpressure, 773

17.3 Monolithic Columns, 775

17.3.1 Physical Properties and Preparation of Monolithic Columns, 775

17.3.2 Chromatographic Properties and Applications of Monolithic Columns, 776

17.4 Ultra-High-Pressure Liquid Chromatography, 777

17.4.1 Instrument Considerations when Using Ultra-High Pressures, 779

17.4.2 Chromatographic Effects of Ultra-High Pressures, 781

17.4.3 UHPLC Applications, 783

17.4.4 Method Transfer Considerations, 785

17.5 Separations on Chips, 786

17.6 Optimizing Gradient Separations for Speed, 788

17.6.1 Advantages of Gradient Chromatography, 788

17.6.2 Optimizing Instrumental Factors, 788

17.6.3 Basic Parameters Controlling Speed and Resolution, 790

17.7 Instrumental Requirements for Operating High-Efficiency Columns, 798

17.7.1 Extra-column Band-Broadening, 798

17.7.2 Detector Requirements, 802

17.7.3 Injection Considerations, 804

17.7.4 Geometric Scaling Relationships, 806

17.8 Conclusions, 807

References, 807

18 Temperature as a Variable in Pharmaceutical Applications 811
Roger M. Smith

18.1 The Influence of Temperature on Chromatography, 811

18.2 Effects on Method Transferability and Reproducibility, 812

18.3 Elevated Temperature and Pharmaceutical Separations, 813

18.3.1 Effect of Temperature on Selectivity, 814

18.3.2 Effect of Temperature on Separation Efficiency, 815

18.3.3 Other Temperature Effects, 817

18.3.4 Applications of Elevated Temperatures, 817

18.4 Superheated Water Chromatography, 821

18.4.1 Columns for Superheated Water Chromatography, 823

18.4.2 Detectors in Superheated Water Chromatography, 824

18.4.3 Pharmaceutical Applications of Superheated Water Chromatography, 824

18.6 Subambient Separations, 826

18.7 Conclusion, 830

References, 830

19 LC/MS Analysis of Proteins and Peptides in Drug Discovery 837
Guodong Chen, Yan-Hui Liu, and Birendra N. Pramanik

19.1 Introduction, 837

19.2 General Strategies for Analysis of Proteins/Peptides, 838

19.2.1 HPLC Methods in Proteins/Peptides, 838

19.2.2 MS Methods for Protein Characterization, 843

19.3 Applications for Biotechnology Products and Drug Targets, 845

19.3.1 Biotechnology Products Development, 845

19.3.2 Protein Glycosylation and Phosphorylation, 860

19.3.3 Microwave-Assisted Methods for Proteins/Peptides, 871

19.3.4 Drug–Protein Interaction by Affinity-Based Hplc/ms, 877

19.3.5 Multidimensional HPLC in Proteomics, 879

19.3.6 Characterization of Adenovirus Structural Proteins for Gene Therapy, 884

19.4 Conclusions, 890

Acknowledgment, 890

References, 890

20 LC-NMR Overview and Pharmaceutical Applications 901
Maria Victoria Silva Elipe

20.1 Introduction, 901

20.2 Historical Background of NMR, 902

20.2.1 Historical Development of NMR, 902

20.2.2 Historical Development of LC-NMR, 904

20.3 Lc-nmr, 905

20.3.1 Introduction, 905

20.3.2 Modes of Operation for LC-NMR, 908

20.3.3 Other Analytical Separation Techniques Hyphenated with NMR, 914

20.3.4 Applications of LC-NMR, 916

20.4 LC-MS-NMR (or LC-NMR-MS or LC-NMR/MS), 916

20.4.1 Introduction, 916

20.4.2 Modes of Operation for LC-MS-NMR, 917

20.4.3 Applications of LC-MS-NMR, 926

20.5 Conclusions, 926

Acknowledgments, 927

References, 927

21 Trends in Preparative HPLC 937
Ernst Kuesters

21.1 Introduction, 937

21.2 Method Development in Preparative HPLC, 939

21.2.1 Optimization of Selectivity, 940

21.2.2 Scale-Up of Analytical Methods, 941

21.2.3 Adsorption Isotherms and Their Determination, 946

21.3 Columns and Stationary Phases, 951

21.3.1 Stationary Phases, 951

21.3.2 Particle Size, Shape, and Distribution, 954

21.3.3 Columns and Packing Procedures, 954

21.4 Choice of Preparative LC Technology, 955

21.4.1 Classical Batch Elution, 956

21.4.2 Recycling Chromatography, 956

21.4.3 Displacement Chromatography, 959

21.4.4 Simulated Moving Bed Chromatography, 962

21.5 Detection Tools, 975

21.5.1 On-Line HPLC Detection, 975

21.5.2 Preparative HPLC-MS, 977

21.6 Conclusion, 978

Acknowledgments, 980

References, 980

22 Chiral Separations 987
Nelu Grinberg, Thomas Burakowski, and Apryll M. Stalcup

22.1 Introduction, 987

22.1.1 Enantiomers, Diastereomers, Racemates, 988

22.2 Separation of Enantiomers Through the Formation of Diastereomers, 989

22.2.1 Mechanism of Separation, 990

22.2.2 General Concepts for Derivatization of Functional Groups, 991

22.3 Molecular Interactions, 992

22.3.1 The Probability of Molecular Interactions, 992

22.3.2 The Types of Molecular Interactions, 995

22.3.3 Chiral Separation Through Hydrogen Bonding, 995

22.3.4 Chiral Separation Through Inclusion Compounds, 1002

22.3.5 Charge Transfer, 1011

22.4 Mixed Types of Interaction, 1018

22.4.1 Polysaccharide Phases, 1019

22.4.2 Antibiotic Phases, 1022

22.4.3 Protein Phases, 1026

22.5 Ligand Exchange, 1030

22.6 Chiral Mobile Phases, 1032

22.6.1 Chiral Mobile-Phase Retention Mechanisms, 1032

22.6.2 Selectivity with Chiral Mobile-Phase Additives, 1035

22.6.3 Chiral Additives with Chiral Stationary Phases, 1035

22.6.4 Interactions with Chiral Mobile Phases, 1037

22.7 Method Development for Chiral Separation, 1038

22.8 Concluding Remarks, 1040

References, 1041

Chemical and Drug Compound Index 1053

Subject Index 1061