A Practical Approach to Chiral Separations by Liquid Chromatography

A Practical Approach to
Chiral Separations by
Liquid Chromatography

Edited by
G. Subramanian




            Weinheim • New York
VCH         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




#
V III     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
*

    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
        C hromatography 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
          t o 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
X II       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   X III


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 r Acid 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
X IV    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


                                                                          397
Index