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CYCLODEXTRINS AND MICELLES IN SEPARATIONS by TIMOTHY JOSEPH WARD, B.S. A DISSERTATION IN CHEMISTRY (ANALYTICAL) Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Approved 1 /1 May, 1987 h'C^ ACKN0WLEDGMENT5 I give my deepest appreciation to Dr. Armstrong for his guidance and constant support. His encouragement helped me to grow as an independent researcher. I also thank the members of my committee for their constructive criticism. I would like to express my love and gratitude to Karen, for her companionship and help made the entire process easier and more enjoyable. I give thanks and praise to our Lord Jesus Christ through whom all things are possible. Finally, I want to dedicate this dissertation to my mother and father, who placed such a high value on education. Their love and encouragement sustained me through the years. 11 TABLE OF C0NTENT5 ACKN0WLEDGMENT5 i i LI5T0FTABLE5 vi LI5T0FFIGURES viii PREFACE Xi CHAPTER PART ONE CYCL0DEXTRIN5 IN SEPARATI0N5 \. INTRODUCTION 2 Traditíonal Methods for Resolving Enantiomers 2 Separation of Enantiomers by HPLC 4 Cyclodextrins 8 Structure and Physical Properties 11 Preparation of Bonded Phases 20 11. MATERIALS AND METHODS 29 Instrumentation 29 111 Chemicals 30 Methods 31 III. RESULTS AND DI5CU55I0N 34 Enantiomeric Separations 34 Dansy] Amino Acids 35 Crown Ethers 43 Drug Stereoisomers 54 Routine Separations 59 Mechanism of Separation 67 Parameters Which Affect Separation 80 IV. CONCLUSIONS 92 REFERENCES 94 APPENDIX 99 PART TWO MICELLESINSEPARATIONS V. MICELLES 101 Introduction 101 Micelle Structure and Properties 101 Micelles in Chromatography 102 iv VI. MATERIALS AND METHODS 106 VII. RESULT5ANDDISCU55I0N 108 Theory 111 Chromatographic Considerations 116 VIII. CONCLUSIONS 123 REFERENCES 124 LISTOFTABLES PART ONE CYCLODEXTRINS IN SEPARATIONS 1. Physical Properties of Cyclodextrins 19 2. Optical Resolution of the Enantiomers of 36 Dansylamino Acids 3. Retention, Separation, and Resolution Parameters 41 for the Separation of Other Enantiomers on the p-CD Stationary Phase 4. Separation Data for Several Racemic 46 2,2*-Binaphthol Derivatives on p-CD Stationary Phase 5. Binding Constants of Four 2,2'-Binaphthol 53 Derivatives withp-CD 6. Separation of Enantiomeric Drugs 55 7. Separation of Diastereomeric Drugs 57 8. Retention, Separation, and Resolution Parameters 62 for the LC Separation of Routine Substances on the p-CD Column 9. Structures and Chromatographic Data for a Series 65 of Nicotine Alkaloids VI 10. Comparison of Enantiomeric Separations Using 88 Different Mobile-Phase Components with the p-CD Column 11. Effect of Flow Rate on the Chromatographic 89 Separation Factor and Resolution of the (+) and (-) Enantiomers of DDDD PART TWO MICELLESINSEPARATIONS 12. Variations in Solute Diffusion Coefficients in the 109 Absence and Presence of SDS Micelles 13. Variations in Solute Diffusion Coefficients 112 in the Presence of Sodium Methylsulfate 14. Calculated Values of the Observed Solute Diffusion 115 Coefficients for Compounds Which Bind toSDSMicelles 15. Variations in the Number of Theoretical Plates (n) for 117 Various Solutes as a Function of SDS Micelle Concentration in the Mobile Phase Vll LISTOFFIGURES PART ONE CYCLODEXTRINS IN SEPARATIONS 1. A Schematic Showing Different Chiral Stationary 5 Phases Developed by Pirkle and Coworkers (A-C) and Related Chiral Stationary Phases Developed by Oi and Coworkers (D-F) 2. A Schematic Showing the Structure of Three 9 Different Charge Transfer Adducts That Have been Bonded to or Adsorbed on Silica Gel and Cram's Crown Ether Stationary Phase 3. Structure of p-Cyclodextrin (A) and Two of the 12 Glucopyranose Units that lllustrate Details of the a-( 1,4) Glycosidic Linkage (B), C1 (D) Chair Conformation, and the Numbering System Employed to Describe the Ring System 4 lllustration of the Size and Shape of a 15 p-Cyclodextrin Molecule 5. A Schematic Showing the Relative Size of a, p, and 17 y CyclodextrinMolecules 6. A Schematic of the Reactions Used to Produce the 22 Nitrogen Linked Bonded Phase 7. Diagram of the Non-Nitrogen Containing Bonded Phase 26 Vlll 8. Chromatographic Separation of the Enantiomers of 58 a Mixture of Racemic (i)-[DDDD], Dansyl-D,L- Threonine [Dns Thr], and Dansyl-D,L-Phenylalanine [DnsPhe]onthep-CDC5P 9. Structures of 2,2'-Binaphthy1diyl Crown Ethers and 44 Analogues 10. LC separation of Enantiomers of Crown Ether 48 Compounds 8, X 2, i, and 15 11. CD Spectra of (-)-(5)-4 and (+)-(R)-4 51 12. Chromatogram Showing the Near Baseline Separation 60 of a Series of Heterocyclic Aromatic Compounds 13. Schematic of a Cyclodextrin Molecule, Represented 68 as a Truncated Cone, Which Forms a Reversible Inclusion Complex with Mephobarbital 14. Structures of Different Drugs Separated on 71 p-Cyclodextrin Bonded Phase 15. Chromatogram Showing the LC Separation of 73 d- and 1-Propranolol on Two 25-cm p-Cyclodextrin Columns 16. Computer Projections of Inclusion Complexes of 76 (A) d-Propranolol and (B) 1-Proprano1o1 in p-Cyclodextrin, From X-Ray Crystallographic Data IX 17. Graph Showing the Inf luence of Percentage 81 Methanol in the Aqueous Mobile Phase on the Capacity Factor, k', for the (+) and (-) Enantiomers of DDDD on a 25-cm p-Cyc1odextrin Column 18. Plot Showing the Influence of the Percent Methanol 84 on the Resolution of the Enantiomers of DDDD on a 25-cm p-Cyclodextrin Column 19. Enantiomeric Separation and Resolution of 86 Hexobarbital on the p-Cyclodextrin Chiral Stationary Phase as a Function of the Percentage of Methanol in theMobile Phase PART TWO MICELLES INSEPARATIONS 20. Typical Cross-Sectional Schematic Representing 103 the Classical View of an Aqueous Micelle 21. SimplifiedModel Showing a "Membrane" or 121 "Bilayer-Like" Area When Surfactant Adsorbs on LC Bonded Media X PREFACE The resolution of enantiomers traditionally has been considered one of the more difficu1t problems in separation science. This arises from the fact that enantiomers have identical physical and chemical properties with the exception that they rotate plane polarized light in opposite directions. The separation of enantiomers using cyclodextrins is described in Part One of this dissertation. Cyclodextrins are nonionic, cyclic, chiral carbohydrates composed of glucopyranose units bound through a-( 1,4) linkages. By covalently bonding cyclodextrins to silica gel particles, an effective chiral stationary phase for high performance liquid chromatography (HPLC) has been produced. The packing is hydrolytically stable under standard HPLC conditions. The ability of cyclodextrins to form inclusion complexes with a wide variety of water insoluble, sparingly soluble, and soluble compounds make it particularly useful for enantiomeric separations. The formation of inclusion complexes is affected by a number of factors, such as hydrophobic effects, hydrogen bonding at the mouth of the cyclodextrin cavity, dipole-dipole interactions or the release of high energy water or modifer from the cyclodextrin cavity during inclusion complex formation. The effects of temperature, mobile phase composition, pH, flow rate and solute structure on resolution and separation efficiency are examined as well as possible modes of mechanism by which XI separation occurs. The results provide important insight into the inf luence of experimental parameters upon these separations. Part Two of this dissertation examines the use of micelles in chromatography. Micelles are dynamic species consisting of aggregated surfactants in an aqueous continuum. While micellar mobile phases have been known to produce unusual selectivities for various compounds in HPLC, they have offered little or nothing for improved chromatographic efficiency. A study was performed to determine the loss of efficiency in micellar mediated separations. Results indicate that poor mass transfer in the stationary phase is predominately responsible for the low efficiencies observed in HPLC separations. Xll PART ONE CYCLODEXTRINS IN 5EPARATI0N5 CHAPTERI INTRODUCTION Traditional Methods for Resolving Enantiomers Enantiomers are compounds which cannot be superimposed on their mirror images. The resolution of enantiomers has historically been one of the more difficult separation problems. This is attributable to the fact that enantiomers have identical physical and chemical properties with the exception that they rotate plane polarized light in opposite directions. When a compound is capable of rotating plane polarized light it is said to be optically active. In 1848 Pasteur reported the first successful separation of enantiomers from a racemic mixture (1). While examining the crystal structure of the sodium ammonium salt of racemic tartaric acid, he noticed that two types of crystals were present. One was identical to the sodium ammonium salt of (+)-tartaric acid (which had been discovered previously and shown to be dextrorotatory) while the other crystals were nonsuperimposable mirror reflections of (+)-tartaric acid. Pasteur separated the two types of crystals under a microscope with tweezers. Upon dissolving each crystal separately in water and placing the solution in a polarimeter, Pasteur found that they rotated plane polarized light in opposite directions by an equal amount. Since few organic compounds crystallize into distinct crystals (containing separate enantiomers) that are amenable to mechanical separation, this method was not generally applicable. i 3 Enantiomeric separations have traditionally been performed by conversion

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