A Practical Approach to Chiral Separations by Liquid Chromatography

Edited by G. Subramanian

VCH Weinheim • New York Basel • Cambridge • Tokyo

Contents

1 An Introduction to Enantioseparation by Liquid Chromatography

Charles A. White and Ganapathy Subramanian

1.1 Introduction 1 1.1.1 What is Chirality? 1 1.1.2 What Causes Chirality? 2 1.1.3 Why is Chirality Important? 4 1.2 Industries which Require Enantioseparations 4 1.2.1 Pharmaceutical Industry 4 1.2.2 Agrochemical Industry 5 1.2.3 Food and Drink Industry 6 1.2.4 Petrochemical Industry 6 1.3 Chiral Liquid Chromatography 7 1.3.1 Type I Chiral Phases 8 1.3.2 Type II Chiral Stationary Phases 10 1.3.3 Type III Chiral Stationary Phases 10 1.3.3.1 Microcrystalline Cellulose Triacetate and Tribenzoate 10 1.3.3.2 Cyclodextrins 11 1.3.3.3 Crown Ethers 11 1.3.3.4 Synthetic Polymers •. 12 1.3.4 Type IV Chiral Stationary Phases 13 1.3.5 Type V Chiral Stationary Phases 13 1.3.6 Mobile Phase Additives 14 1.3.6.1 Metal Complexes 14 1.3.6.2 Ion-pair Formation 15 1.3.6.3 Uncharged Chiral Additives 15 1.4 The Future 16

2 Modeling Enantiodifferentiation in Chiral Chromatography

Kenny B. Lipkowitz

2.1 Introduction 19 2.2 Modeling 19

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VIII Contents

2.3 Computational Tools 22 2.3.1 Quantum Mechanics 22 2.3.2 Molecular Mechanics 23 2.3.3 Molecular Dynamics 24 2.3.4 Monte Carlo Simulations 24 2.3.5 Graphics 25 2.4 Modeling Enantioselective Binding in Chromatography 26 2.4.1 Type I CSPs 26* 2.4.2 Type II CSPs 41 2.4.3 Type III CSPs 44 2.5 Related Studies 49 2.6 Summary 50

3 Regulatory Implications and Chiral Separations

Jeffrey R. Brown

3.1 Introduction 57 3.2 Licensing of Medicinal Products 57 3.3 Implications of Chirality 58 3.3.1 Stereoisomerism 58 3.3.2 Biological Significance of Stereoisomerism 60 3.3.3 Chirality and Medicinal Products 63 3.4 Nomenclature of Mixtures of Isomers 64 3.5 Attitudes Towards Chirality 64 3.6 The Future 66

4 Enantiomer Separation Using Tailor-Made Phases Prepared by Molecular Imprinting

Börje Seilergren

4.1 Introduction 69 4.2 Historical Development 70 4.3 CSP Preparation and Evaluation 74 4.4 Polymer Enantioselectivity-Structural Correlations 74 4.5 Polymer Substrate Selectivity — For Better or for Worse? 78 4.5.1 Examples of High Substrate Selectivity - Antibody-Like

Recognition 78 4.5.2 Examples of Low Substrate Selectivity 80 4.6 Nature of the Sites 82 4.7 How to Increase Affinity and Selectivity ' 83 4.7.1 Stability of Template Assemblies 83 4.7.1.1 Concentration of MAA in Monomer Mixture 83 4.7.1.2 Polymerization Temperature 84

Contents IX

4.7.1.3 Solvent 84 4.7.2 Other Functional Monomers 86 4.8 Structure and Morphology of Imprinted Polymers 87 4.9 A Packing for Ion Exchange and Enantiomer Separation 87 4.10 Column Performance and Saturation Capacity 90 4.11 Conclusions and Outlook 92

5 Cyclodextrin Bonded Chiral Stationary Phases in Enantiomer Separations

Apryll M. Stalcup

5.1 Introduction 95 5.2 'Magic' Mobile Phase 96 5.3 Derivatized Cyclodextrin-Bonded Phases 98 5.4 Hydroxypropyl-/?-CD CSP 99 5.5 Normal Phase CSP 103 5.6 Naphthylethylcarbamoylated ß-CT) CSP 104 5.7 Applications to Biological Samples 111 5.8 Conclusions 113

6 Polysaccharide Phases in Enantioseparations

Jules Dingenen

6.1 Introduction 115 6.2 Cellulose Triacetate (CTA) 117 6.3 Coated Cellulose and Amylose Derivatives 124 6.3.1 Mobile Phase Effects on Retention and Stereoselectivity 127 6.3.2 Effect of the Solute Structure on Stereoselectivity 139 6.3.3 Influence of Temperature 147 6.3.4 Quantitative Analysis 150 6.3.5 Preparative Chromatographie Applications 155 6.3.6 Micro-LC (ji-LC) Applications 160 6.4 Benzoyl Cellulose Beads in the Pure Polymerie Form 163 6.5 Microcrystalline Tribenzoylcellulose 174 6.6 Conclusions 178

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7 Separation of Enantiomers by Protein-Based Chiral Phases

Stig Alienmark

7.1 Introduction 183 7.1.1 Early Observations of Chiral Discrimination by Proteins 183

X Contents

7.1.2 Affinity Chromatography and the Reciprocity Principle 185 7.1.3 Immobilized Proteins for Chiral Separations 185 7.2 Various Principles Used for Immobilization of Proteins

to Liquid Chromatography Supports 186 7.2.1 Non-crosslinked Phases 186 7.2.1.1 Physical Immobilization by Adsorption 186 7.2.1.2 Heterobifunctional Reagents for the Functionalization

of Silica Matrices 186 7.2.1.3 Covalent Binding of Proteins via Homobifunctional Reagents . . . . 187 7.2.2 Crosslinked Phases 187 7.2.2.1 Physical Immobilization via Homobifunctional Reagents 187 7.2.2.2 Immobilization by Covalent Binding and Crosslinking 187 7.3 General Techniques for Regulation of Retention and Resolution.

Mobile Phase Optimization 188 7.4 Different Protein Phases for Chiral Reversed Phase Liquid

Chromatography and their Use 190 7.4.1 Serum Albumin (BSA and HSA) 191 7.4.2 Orosomucoid or or1-Acid Glycoprotein (AGP) 193 7.4.3 Ovomucoid (OVM) 194 7.4.4 Cellobiohydrolase-I (CBH-I) 196 7.4.5 Avidin 198 7.4.6 Chymotrypsin (CT) 199 7.4.7 Ovotransferrin or Conalbumin 200 7.5 Selected Examples of Useful Applications 201 7.5.1 Determination of Reaction Stereochemistry 201 7.5.1.1 Stereoselective Monitoring of Pharmacokinetics 201 7.5.1.2 Stereoselectivity in Enzyme-Catalyzed Reactions 205 7.5.2 Determination of Enantiomeric Purity 207 7.5.3 Preparative-Scale Isolation of Pure Enantiomers 209 7.5.4 Studies of Drug-Albumin Binding 209 7.6 Some Experimental Observations Related to the Mechanism

of Retention and Chiral Recognition 210 7.7 Special Microanalytical Techniques 211 7.7.1 Miniaturization of the Liquid Chromatographie System 211 7.7.2 Capillary Electrophoresis 212 7.8 Some Concluding Remarks 213

8 Optically Active Polyacrylamide/Silica Composites and Related Packings and their Application in Chiral Separations

Joachim N. Kinkel

8.1 Introduction 217 8.2 The Chromatographer's View:

How to Define the "Ideal Support?" 219

Contents XI

8.3 The Synthesis of Polymer/Silica Composites 223 8.3.1 The Deposition of Preformed Polymers on the Surface

of Inorganic Oxides 224 8.3.2 Preparation of Preformed Polymers by Copolymerization

of Optically Active Monomers and Comonomers Bearing Silane Functional Groups and Subsequent Reaction with the Silica Surface 232

8.3.3 Copolymerization of Optically Active Monomers with Vinyl- or (Meth)acryloxypropyl Modified Silica 233

8.3.4 Graft Polymerization of Optically Active Vinyl Compounds to Silica and Polar Modified Silica 237

8.3.5 Graft Polymerization of Optically Active Vinyl Compounds by UV-Radiation of Photoinitiator-Modified Silica 244

8.3.6 Graft Polymerization of Optically Active Vinyl Compounds Initiated by Ceric Nitrate/Diol Silica Redox Systems in Diluted Nitric Acid Solutions 246

8.4 Chromatographie Characterization of Optically Active Polyacrylamide/Silica Composites 247

8.4.1 Comparison of 'Brush-type' Monomeric Packings and Polymer Composites 247

8.4.2 Influence of the Vinyl Group Substituent of Optically Active Polyacrylamides on the Chromatographie Resolution of Enantiomers 250

8.4.3 Influence of Solvent on Retention, Enantioselectivity and Resolution 255

8.4.4 Structure/Enantioselectivity Relationship of Composites, Derived from the Ephedrin Skeleton 261

8.5 Chromatographie Applications 269

9 Ion-Pair Chromatography in Enantiomer Separations

Curt Pettersson and Eve Heldin

9.1 Introduction 279 9.2 Ion-Pair Chromatography 279 9.2.1 Batch Extraction 279 9.2.2 Liquid-Liquid Chromatography 281 9.2.3 Liquid-Solid Chromatography 283 9.1 Separation of Enantiomers Using a Chiral Counter Ion 285 9.3.1 Normal Phase Chromatography 287 9.3.1.1 Structure and Enantioselectivity 287 9.3.1.2 Mobile Phase Composition and Separation 295 9.3.1.3 Solid Phase and Separation 297 9.3.2 Reversed Phase Chromatography : 301 9.4 Tartaric Acid Derivatives - Uncharged Chiral Selectors 302

XII Contents

9.4.1 Liquid Tartaric Acid Ester as Mobile or Liquid Stationary Phases 302 9.4.2 Tartaric Acid Esters as Dynamically Modified Stationary Phases . . 304 9.5 Miscellaneous 307 9.5.1 Proteins as Chiral Mobile Phase Additives 307 9.5.2 Crown Ethers as Dynamically Modified Stationary Phases 308

10 Separations of Enantiomeric Compounds by Chiral Selectors in the Mobile or Solvent Phase

Brian J. Clark

10.1 Introduction 311 10.2 Nuclear Magnetic Resonance (NMR) 312 10.2.1 Chiral Shift Reagent Additives 312 10.2.2 Chiral Solvating Agents 312 10.2.3 Cyclodextrins and their Derivatives 313 10.3 Thin Layer Chromatography (TLC) 313 10.3.1 Chiral Stationary Phase TLC Plates 315 10.3.2 Enantioselectors Added to the Developing Solvent 316 10.4 Chiral Separations by Capillary Electrophoresis 316 10.4.1 Chiral Ligand Exchange in Capillary Electrophoresis 317 10.4.2 Chiral Micellar Separations by Capillary Electrophoresis 317 10.4.3 Chiral Separations with Cyclodextrins in Capillary Electrophoresis 319 10.5 Separations by Mobile Phase Additives in HPLC 322 10.5.1 Mobile Phase Additives in Ligand Exchange HPLC 322 10.5.2 Proteins as Chiral Selectors in HPLC 323 10.5.3 Inclusion Complexation in Chiral Separations by HPLC 324

11 Chiral HPLC in the Pharmaceutical Industry

Ian M. Mutton

11.1 Introduction 329 11.1.1 Chiral Drugs: Single Enantiomers or Racemates? 329 11.1.1.1 Background 329 11.1.1.2 Biological Relevance of Stereospecifity 329 11.1.1.3 Regulatory Issues 330 11.1.1.4 The Pharmaceutical Industry : 330 11.1.2 The Analyst's Role 331 11.1.3 Techniques for Chiral Analysis 332 11.1.3.1 Spectroscopy 332 11.1.3.2 Chiral Chromatography 332 11.2 Chiral Discrimination Using HPLC 333 11.2.1 General 333 11.2.2 Resolution Using Diastereomer Formation 333

Contents XIII

11.2.3 Resolution Using Chiral Stationary Phases (CSPs) 335 11.2.3.1 Introduction 335 11.2.3.2 Type I CSPs 335 11.2.3.3 Type II CSPs 337 11.2.3.4 Type III CSPs 340 11.2.3.5 Type IV CSPs 342 11.2.3.6 Type V CSPs 343 11.2.3.7 Customized CSPs 345 11.2.4 Resolution Using Chiral Mobile Phase Additives (CMPAs) 346 11.2.4.1 Introduction 346 11.2.4.2 Cyclodextrins as CMPAs 346 11.2.4.3 Ligand Exchangers as CMPAs 347 11.2.4.4 Proteins as CMPAs 347 11.2.4.5 Chiral Ion-Pair Chromatography 347 11.2.5 Chiro-Optical Detection 348 11.2.6 Preparative Chiral Separatibns 348 11.3 Current and Future Trends 349 11.4 Conclusion 351

12 Bioanalytical Applications of Enantioselective High-Performance Liquid Chromatography

Terence A. G. Noctor

12.1 Introduction 357 12.1.1 Enantioselective Chromatographie Bioanalysis 358 12.2 Enantioselective Bioanalysis Using Chiral Derivatization 358 12.2.1 Indirect Chiral Resolution of Enantiomeric Amines

in Biological Fluids 359 12.2.2 Indirect Chiral Resolution of Enantiomeric Alcohols

in Biological Fluids • 361 12.2.3 Indirect Chiral Resolution of Enantiomeric Acids

in Biological Fluids 361 12.3 Direct High-Performance Liquid Chromatographie Separation

of Enantiomers Using Chiral Stationary Phases 363 12.3.1 Protein-Based CSPs in the Analysis of Drug Enantiomers

in Biological Fluids 364 12.3.1.1 a rAcid Glycoprotein 366 12.3.1.2 Ovomucoid 370 12.3.1.3 Serum Albumins 371 12.3.2 Cyclodextrin-Based CSPs 373 12.3.3 Brush Type CSPs 375 12.3.1 Polysaccharide-Based CSPs 377 12.3.4.1 Bioanalytical Applications of Derivatized Cellulose CSPs 377 12.3.4.2 Bioanalytical Applications of Derivatized Amylose CSPs 380

XIV Contents

12.3.5 Biomedical Separations Using Other Types of CSP 380 12.3.5.1 CSPs Based on Synthetic Polymers 381 12.3.5.2 CSPs Based on Ternary Complexation

(Ligand Exchange Chromatography) 381 12.4 Multidimensional Approaches to Chiral Analysis by HPLC 381 12.4.1 Sequential Achiral-Chiral Chromatography in Bioanalysis 382 12.4.2 Determination of Enantiomers in Biological Matrices

Using Serially Coupled Achiral-Chiral Chromatography 383 12.4.3 Determination of Enantiomers in Biological Matrices

Using Achiral-Chiral Chromatographie Systems, Coupled by Means of a Switching Valve 385

12.5 Direct Chromatographie Bioanalysis of Enantiomers Using Chiral Mobile Phase Additive 389

12.5.1 Chiral Ligand Exchange Additives to the Mobile Phase 389 12.5.2 Chiral Mobile Phase Additives Forming Diastereomeric lon-Pairs . 389 12.5.3 Cyclodextrins as Chiral Mobile Phase Additives 390 12.6 Conclusion 391

Index 397