UNIVERSITY OF CINCINNATI

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1 UNIVERSITY OF CINCINNATI, 20 I,, hereby submit this as part of the requirements for the degree of: in: It is entitled: Approved by:

2 Chiral Separation Using Capillary Electrophoresis (CE) and Continuous Free Flow Electrophoresis (CFFE) A thesis submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in the Department of Chemistry of the College of Arts and Sciences 2003 by Yufu Liang Committee Chair: Dr. Apryll M. Stalcup i

3 ii

4 Abstract In this report, chiral separations of hydrobenzoin (HB), piperoxan (PI) and methoxyphenamine (MPA) using capillary electrophoresis (CE) are discussed. In the experimental part, separation conditions such as buffer ph, buffer concentration, CE instrumental polarity, capillary length, capillary temperature and sample matrix, etc., were considered and their impact on the chiral separations compared. All selected analytes - hydrobenzoin, piperoxan and methoxyphenamine could be well separated by CE using sulfated β-cyclodextrin (SCD) as chiral additive (CA). Preparative scale separation of MPA and PI was carried out by a continuous free flow electrophoresis (CFFE) instrument using SCD with two different degrees of substitution as CA. Rapid chiral CE methods were developed for the fractionated CFFE samples. Buffer concentration, SCD concentration, applied voltage, sample feed rate and position, and electrode wash conditions were investigated. Experiments with dyes as surrogates were also completed on the CFFE and UV-Vis instruments. The CFFE instrument PI sample capacity was investigated using optimized conditions. iii

5 ACKNOWLEDGMENTS I would like to take this opportunity to thank my research advisor, Professor Apryll M. Stalcup. To study and do research work under her guidance is a wonderful experience. Her teaching, discussion, advising and patience all have been invaluable to me. Working with the other members in her group has been a great pleasure. It is a great pleasure to thank Professors Thomas H. Ridgway and Carl J. Seliskar being my advisory committee members. The time and support given by them are greatly appreciated. Here, I also want to thank University of Cincinnati for the opportunity to study here. Finally, I would like to thank my wife May and new baby Olivia for their patience, sacrifice, encouragement and especially their unconditional love. iv

6 Table of Contents Abstract Acknowledgments iii iv Title 1 List of Figures 2 List of Tables 5 List of abbreviations 6 Chapter 1. Introduction 8 Chapter 2. Experimental Section 39 Chapter 3. Results and Discussion 45 Chapter 4. Conclusion 87 Chapter 5. Future Work 89 References 91 v

7 Chiral Separation Using Capillary Electrophoresis (CE) and Continuous Free Flow Electrophoresis (CFFE) 1

8 List of Figures Page Figure 1.1 Structure of the various natural and derivatized CDs 12 Figure 1.2 Some physicochemical properties of cyclodextrins 13 Figure 1.3 Schematics of basic CE instrumentation 18 Figure 1.4 Differential solute migration superimposed on EOF in CZE 21 Figure 1.5 Representation of the electrical double layer verses distance from the capillary wall 22 Figure 1.6 Schematic illustrating the effect of the CD charge status on the chiral separation behavior of cationic chiral compounds 25 Figure 1.7 Flow profile and corresponding solute zone 27 Figure 1.8 Schematic of CFFE instrument 34 Figure 1.9 Schematic of CFFE electrophoresis chamber 35 Figure 2.1 Structures of the compounds used in this study 40 Figure 3.1 Hydrobenzoin CE electropherogram for repeated run 46 Figure 3.2 Methoxyphenamine experiment electropherogram for sample matrix comparison 50 Figure 3.3 Methoxyphenamine CE ionic strength experiment Electropherograms 52 2

9 Figure 3.4 Influence of ionic strength on CE chiral separation 53 Figure 3.5 Hydrobenzoin CE electropherogram for fast analysis 55 Figure 3.6 Hydrobenzoin CE electropherogram for ph and resolution in reverse polarity mode 56 Figure 3.7 Methoxyphenamine CE electropherograms for effect of buffer SCD concentration on migration time and resolution 58 Figure 3.8 Effect of buffer SCD concentration on migration time and Resolution 59 Figure 3.9 Methoxyphenamine CE electropherograms for effect of electric field strength on migration time and resolution 61 Figure 3.10 Methoxyphenamine CE result for effect of electric field strength on migration time 62 Figure 3.11 Ohm s Law curve 63 Figure 3.12 Piperoxan CE separation electropherogram 65 Figure 3.13 Methoxyphenamine CFFE separation histogram 69 Figure 3.14 ph distribution of collected samples after CFFE separation (a is the corresponding histogram) 72 Figure 3.15 Dye structures used in the experiments 74 Figure 3.16 UV-Vis scan spectrum of dye experiments 75 Figure 3.17 CFFE dye experiment using 14 degree substituted SCD 77 3

10 Figure 3.18 CFFE dye experiment using 5-7 degree substituted SCD 78 Figure 3.19 Piperoxan CFFE separation using 14 degree substituted SCD as CA 81 Figure 3.20 Piperoxan CFFE separation using 5-7 degree substituted SCD 83,84 Figure 3.21 Piperoxan CFFE separation capacity investigations using 14 degree substituted SCD 86 4

11 List of Tables page Table 1.1 Some physicochemical properties of cyclodextrins 14 Table 2.1 CFFE instrument operation sequence 44 Table 3.1 CE data for methoxyphenamine 47 Table 3.2 CE data for hydrobenzoin 48 Table 3.3 Best CE separation results for the investigated analytes 67 5

12 List of Abbreviations A Absorbance α Selectivity (µ e2 /µ e1 or k 2 /k 1 ) BGE C CD CE CFFE CSP CV CZE D E ε EOF Background Electrolyte Concentration Cyclodextrin Capillary Electrophoresis Continuous Free Flow Electrophoresis Chiral Stationary Phase Crystal Violet Capillary Zone Electrophoresis Diffusion Coefficient Electric Field Dielectric Constant (C 2 /Jm) Electro-osmotic Flow η Viscosity (Ns/m 2 ) HB HPCE HPLC i.d. K Hydrobenzoin High Performance Capillary Electrophoresis High Performance Liquid Chromatography Internal Diameter Partition Coefficient k Capacity Factor 6

13 L l MPA N o.d. PB pka Q R RSD SCD SP T t UV-Vis V v W 1/2 W b ζ Total Capillary Length Effective Capillary Length Methoxyphenamine Number of Theoretical Plates Outer Diameter Patent Blue Acid Dissociation Constant Charge Resolution Relative Standard Deviation β-sulfated Cyclodextrin Stationary Phase Temperature Migration Time Ultra Violet-visible Voltage Velocity Peak width at half-height (s or min) Peak Width at Baseline Zeta Potential 7

14 Chapter 1. Introduction 1.1 Chirality and chiral separation Chirality has the characteristic of handedness, which is having the potential to exist as two nonsuperimposable structures that are mirror images. Enantiomers are two stereoisomers that exhibit non-superimposable mirror images of one another. They have the same molecular and structural formulas but different spatial arrangement of their atoms. No amount of rotation can convert one of these structures into the other. Usually, the chiral center is a carbon atom and it is linked to four different groups. This fascinating stereoisomerism is shown by many organic compounds, especially carbohydrates and many other natural substances. Some enantiomers even have more than one chiral center, so they will have more than two spatial arrangements and isomers. 1,2,3,4,5 In an achiral enviroment, enantiomers have identical physical and chemical properties, so their separation is more difficult than other chemical separations. Chemical separations play a significant role in chemical studies. In the past few decades, a significant effort has been made to develop methods for the separation of enantiomers or chiral separations. Especially during the past two decades, there has been intense interest in the development and application of chiral chromatographic, electrophoresis and capillary electrophoresis (CE) methods, particularly in the pharmaceutical industries and various research groups. Many groups are active in doing research on this topic. 6,7,8,9,10 Vigh et al did lots of theoretical model work and CE chiral 8

15 separations using CDs or their derivatives as chiral additives (CA). 11,12,13,14,15,16,17 Stalcup et al have been involved in chiral separations in various research efforts. 18,19,20,21,22,23,24,25,26,27,28 The motive for this is the desire to develop and exploit good science and the increasing pressure by the regulatory authorities over the past ten years against the marketing of racemic mixtures. 29 The U.S. Food and Drug Administration (FDA) requires biotoxicity and bioefficacy reports on not only each enantiomers but also the racemate for marketing the drug. 30 Many other countries also require consideration of enantiomers as different entities and to test their pharmacological, pharmacokinetic and toxicological properties separately. 31 Two enantiomers of chiral drugs may have totally different effects in the human body. One enantiomeric form may have medicinal benefit while the other form may be cause significant undesirable effects. 32 It was reported recently that about 88% of all synthetic chiral drugs are currently made as the racemic mixture and in year 2000, chiral drug sales accounted for one third of all pharmaceutical products sold world-wide. 33 Chiral separation strategies are also employed in food, beverage and agrochemical industries. It is a great challenge for the industries to seek techniques that can be easily, efficiently and commercially applied to their manufacture of enantiomeric products. The agrochemical, food and beverage industries are also concerned about potential new related regulatory pressures. 34,35,36 In the 1960 s, chemists began to use chiral stationary phases (CSP) in both liquid chromatography (LC) and gas chromatography (GC) to separate enantiomers. Gil-Av 9

16 and co-workers at the Weizmann Institute of Science, Israel first reported a CSP for GC. 37 Many CSP are developed and commercially available now, so many enantiomeric separations have become a somewhat routine process. Most of the CSP columns commercially available are very expensive. Some of the separation methods are not very efficient, or easy and in preparative scale, all strategies have their advantages and drawbacks. Analytical scale separations only supply a method to detect the existence of optical isomers and evidence of separation. Some of the preparativescale chiral separation methods suffer apparent drawbacks. For instance, preparative LC has a high cost, unavailability of chiral stationary phases, consumption of large amounts of predominantly organic mobile phase, and solubility problems associated with the sample. 9,38 Further effort must be devoted to this area. 10

17 1.2 Cyclodextrin (CD) Among the most useful chiral additives, 39 cyclodextrins (CDs) are a family of several minor oligosaccharides and three major, well-known cyclic oligosaccharides formed from 6 (α-cd), 7 (β-cd), and 8 (γ-cd) glucose residues bonded via a 1,4-linkage. They are widely used CA for chiral separations by CE, HPLC and etc., either in their native or in their derivatized forms. 6-8,10-25,27,28 They can be schematically viewed as a truncated cylinder. The schematic picture of these molecules and the size of the internal hydrophobic cavity can be found in Figure 1.1, 40 Figure1.2 and Table The wider mouth of the cyclodextrin cavity is lined with secondary hydroxyls (large rim), while the narrower mouth is lined with primary hydroxyls and glycosidic oxygens (small rim). The mouths have some level of Lewis base character due to the nonbonding electron pairs of the glycosidic oxygens direct towards the cavity. 9 Sterically, secondary hydroxyls are able to form internal H-bonds; the most favorable H-bond is between OH group on C2 carbon of one glucose unit and the OH group on C3 carbon of an adjacent unit. β-cd forms a complete H-bond belt around the mouth and has a quite rigid structure. Thus, it has low solubility in water (less than 1.85g/100ml at room temperature). Since one glucose unit is distorted from horizontal configuration in α- CD and no H-bond belt formed, it is more easily dissolved in water (14.5g/100ml). The 8-glucose residue γ-cd is quite flexible in structure, and formation of internal H-bonds is not as favorable as it is in β-cd. It is also more soluble in water (23.2g/100ml) than β-cd

18 Figure 1.1 Structure of the various natural and derivatized CDs 40 12

19 b a c Figure 1.2 Some physicochemical properties of cyclodextrins (data in table 1.1) 9 13

20 Table 1.1 Some physicochemical properties of cyclodextrins 9 CD Dimensions Cavity Molecular Specific Solubility Volume mass optical in water A o A o3 rotation at 25 C a b c [α] D 25 (g 100ml -1 ) α β γ

21 The cavity of cyclodextrin is hydrophobic, so hydrophobic interactions with hydrophobic groups of analyte can occur. One of the most remarkable properties of CDs is their ability to bind chiral organic molecules stereoselectively. In general, size, shape, and a charge of the hydrophobic molecules are the most critical for the strength of the inclusion. This characteristic of CDs has led to wide applications in analytical sciences, especially for enantioseparations in GC, HPLC and CE. One of the inherent problems with native CDs, however, is their low solubility. It is for this and other reasons that a number of derivatized CDs have been produced. As well as increasing their solubility, extended applicability has been found for these functionalized CD. For instance, SCD has been successfully employed in HPLC and CE for chiral separations of many pharmaceutical compounds. 6,42 When CDs or derivatized CDs are used as CA or CSP, a variety of interactions (e.g. H- bonds, hydrophobic and electrostatic interactions) with an analyte will be involved, although inclusion complexation was reported as the main interaction mechanism due to their special structures. 2,6,24,39 15

22 1.3 HPCE (CE) Historical Background and Development Electrophoresis is the primary method for the identification and quantitation of biological molecules such as DNA and protein analysis. In early research work, slabgel, cellulose or paper was used as the necessary supporting material to provide electrophoresis physical support and mechanical stability. Modern CE is the marriage of the powerful separation mechanisms of electrophoresis with the instrumentation and automation concepts of chromatography, especially GC and HPLC. 1,2,3 Since the first modern capillary zone electrophoresis was introduced by Hjerten in 1967, CE technology has been developed rapidly. 1 Using capillary walls to replace the stab-gel provides the possibility of easily fulfilling the task of mechanical support, background electrolyte requirement and instrumentation automation. 1,2,3 Capillary zone electrophoresis (CZE) is the simplest mode of CE. Some buffer or background solutions must be used to maintain sufficient conductivity and ph value. Homogeneous electrolyte medium is used in the buffer. When ionic compounds pass through the capillary driven by applied high voltage, they are separated into discrete bands if an individual solute s mobility is sufficiently different from all others in the sample. First CE enantioseparation was reported by Gassmann et al. in Electrophoretic separations of enantiomer pairs have grown constantly using CE since then and especially the early 1990s. 44 CE chiral separation by using chiral additives in the buffer 16

23 is an efficient and sensitive technique; many enantiomers can be separated analytically. 19,20 CE can provide the separation conditions for analytical separations, but it cannot be applied directly for preparative scale separation. However, CE chiral separation does not need the expensive chiral stationary phase and large volume of mobile phase that is required in HPLC chiral separation. It also requires a smaller amount of sample. All these advantages make CE an attractive choice for analytical chiral separation CE Instrumentation Figure 1.3 shows the basic CE instrumentation. 1 Separation by electrophoresis is obtained by differential migration of solutes in an electric field. In CE, electrophoresis is performed in a narrow-bore capillaries, typically 25 to 75 µm i.d., which are usually filled only with buffer solution. The buffer reservoirs contain the buffer solution with the capillary ends and the electrodes dipping in. After loading the sample into the capillary by either electrical or hydrodynamic injection, electrophoresis separation is performed in the capillary between the two buffer reservoirs and electrodes by applying an electric field. On-line detection (usually UV detection) is the most common method of detection. The advantages of using a capillary are: a high voltage power supply can provide a high electrical field ( V/cm) through the capillary; the large surface area-to-internal volume ratio enables efficient joule-heat disspation. 1 17

24 detector Capillary electrode HV electrode power supply buffer + sample buffer - Figure 1.3 Schematic of basic CE instrumentation 18

25 1.3.3 CE theory Electrophoresis mobility Separation by electrophoresis is based on differences in solute velocity in an electric field. The velocity v of an ion can be given by v = µ e E (1) where µ e is the electrophoretic mobility, E is the applied electric field. The velocity is controlled by two competing forces applied electric force (F E ) and the frictional force (F F ) from the medium. 1 For spherical particle, these forces are given by F E = q E (2) F F = -6πηrv (3) where q is the ion charge; η is the solution viscosity; r is the ion radius. When electrophoresis reaches a steady state, the two forces are equal but opposite, 1 so the mobility in terms of physical parameters is: µ e = q 6πηr (4) Small and highly charged particles have high mobilities whereas large and minimally charged particles have low mobilities Electroosmotic flow (EOF) The electro-osmotic flow of the bulk solution causes movement of nearly all species in the same direction, regardless of their charge. Under normal conditions (i.e., negatively charged capillary surface), the flow is from the anode to cathode. Anions usually will 19

26 be flushed towards the cathode since the magnitude of the flow can be more than an order of magnitude greater than their electrophoretic mobilities. Thus cations, neutrals, and anions can be electrophoresed in a single run since they all migrate in the same direction. This description is shown in Figure It is the pumping mechanisms of CE. This occurs because of the dissociation of the capillary surface silanol groups (SiOH SiO - + H + ), creating an electrical double layer shown in Figure Smoluchowski defined the EOF in 1903 [1] as v eof = (εξ/η) E (5) or µ eof = εξ/η (6) where v eof is the EOF velocity; µ eof is the EOF mobility ; ε is the dielectric constant and ξ is the zeta potential which is essentially determined by the surface charge on the capillary wall (strongly ph dependent, higher ph, higher zeta potential). Considering all the parameters of the pumping mechanisms in CE, the apparent solute mobility can be defined as: µ app = µ e + µ eof (7) And in terms of experimentally measurable quantities, the apparent mobility is given by l ll µ app = = (8) te tv 20

27 EOF Figure 1.4 Differential solute migration superimposed on EOF in CZE 21

28 Capillary wall N N N N N N N N N Stern Compact layer Diffuse layer layer Absorbed layer Interface Potential d 0 Compact layer Diffuse layer Distance from the capillary wall Figure 1.5 Representation of the electrical double layer verses distance from the capillary wall 45 22

29 where µ app is the apparent solute mobility; V is the applied voltage; l is the effective capillary length (to the detector); L is the total capillary length; t is the migration time, which is the time needed for particular solute travel the effective capillary length, and E is the electric field strength (applied voltage divided by the total capillary length) Chiral separation mechanism of CE with CD additive There are many reported results using functionalized classes of electrophoretic chiral selectors based on cyclodextrins. 46 Cyclodextrin-based CE chiral separation behavior can be strongly influenced by ph, not only because the ph can change the EOF, but also change the ionization state of some of functionalized CDs. With a chiral additive in the system, the electrophoretic mobility of a solute in CZE was given by Wren and Rowe as: 47 µ f + K[ CA] µ c µ = (9) 1+ K[ CA] Where µ f is the mobility in free state; µ c is the mobility in the complex state and K is the association constant between an analyte and CA. For enantiomer separations, in the case where the molecular weight of the CA is much greater than that of the analyte, an approximation can be made, that is µ c = µ CA. And for enantiomers µ 1,f = µ 2,f. Thus, the mobilities of two enantiomers can be compared as below: 47 23

30 [ µ 1, c µ 1, f ][ K1 K 2 ][ CA] µ 1 µ 2 = (10) [1 + K [ CA]][1 + K [ CA]] 1 From equation 10, it is apparent that if the following conditions are met there will be no separation: if the mobilities of the free state are equal to the complexed state; if the association constants of the enantiomers are equal; and if the CA concentration is zero or too high. 2 As an example, when CD is used as the chiral additive, the CD concentration plays a very important role for chiral separation by CE, and thus this parameter should be carefully controlled, for optimized separation conditions. The CD can function as a moving stationary phase. This can benefit the separation of uncharged chiral compounds when CD is charged. On the other hand, native CD is always neutral so it can only move with the bulk solution dragged by EOF. The small difference between the two complex constants of the enantiomers with CD finally causes the different mobility that enables the analytes to be separated. The above description is illustrated in Figure 1.6 (cationic chiral compound as example). 40 Wren and Rowe found that the optimum CD concentration is dependent upon the formation constants of the diastereomers formed during the electrophoresis [ CD ] = opt [ K (11) 1/ 2 1 K 2 ] Where [CD] opt is the optimum CD concentration that should be used; K 1, K 2 are the diastereomers formation constants of the two enantiomers separately. 24

31 Detection R-Analyte strong binding CD µ (high) R Cation enantiomer CD Negatively charged CD µ (low) S S-Analyte weak binding CD Cation enantiomer R-Analyte strong binding µ (low) R Cation enantiomer S-Analyte weak binding Neutral CD Cation enantiomer µ (high) S Figure 1.6 Schematic illustrating the effect of the CD charge status on the chiral separation behavior of cationic chiral compounds 40 25

32 CE efficiency CE has higher efficiency than HPLC. This is the result of two main factors. 1 First, as mentioned before, there is no stationary phase in CE system, so all the disadvantages related to stationary phase, such as mass transfer resistance (between the stationary phase and the mobile phase) and other dispersion mechanisms (eddy diffusion and stagnant mobile phase) have been avoided. Secondly, in a pressure-driven flow system such as HPLC, laminar or parabolic flows exist. This is caused by the frictional forces at the liquid-solid boundaries, so there is a radial velocity gradient throughout the tube. The fluid flow velocity is greatest in the middle of the tube and almost zero near the tube wall. Thus, the analytical peak is broadened (Figure 1.7). 1 However, in electrically driven systems, EOF is generated homogeneously along the capillary; there is no gradient. An uniform flow velocity vector exist across the tube. Only in the very near capillary wall region (the double layer region) the flow rate approaches zero. So the peak shape is much better than hydrodynamic driven flow (Figure 1.7). 1 Dispersion, or spreading of the solute zone, results from differences in solute velocity within that zone. Under ideal conditions, the sole contribution to solute-zone broadening in CE can be considered to be longitudinal diffusion (along the capillary). Radial diffusion can be ignored due to the plug flow profile. The Einstein equation can describe the dispersion caused by diffusion in liquid. 1 26

33 HYDRODYNAMIC FLOW + - ELECTROOSMOTIC FLOW + - Figure 1.7 Flow profile and corresponding solute zone 27

34 σ 2 DL = 2 Dt = (12) V 2 2 µ app Where D is the diffusion coefficient of an individual solute. The number of theoretical plates, N, is given by N = L 2 / σ 2 (13) Substituting equation (12) into equation (13) yields a fundamental electrophoretic expression for plate number. N µ appv = (14) 2D The equations show that the parameters such as higher voltage, high mobility and low diffusion coefficient solutes, which can reduce the diffusion effect, leads to higher theoretical plate numbers and better efficiency. The theoretical plate number can be determined directly from the electropherogram by using: 2 t N = 5.54 (15) W1/ 2 In addition to longitudinal diffusion, there are some other factors including Joule heating, injection length, sample adsorption, mismatch conductivities of sample and buffer, unlevel buffer reservoirs and detector cell size that can also affect the CE efficiency. 1 28

35 CE resolution In separation science, resolution of the sample components is the ultimate goal. Thus, resolution is the main factor to evaluate a separation method. Resolution is most simply defined as: 1 R s 2( t2 t1) t2 t1 = = (16) W + W 4σ 1 2 Where t is the migration time; W is the baseline peak width (in time); σ is the temporal standard deviation. However, in CE, the separation depends on efficiency, as well as the selectivity. Since the solute zones are very sharp, small differences in solute mobility (<0.05% in some cases) 1 are often sufficient for complete resolution. Thus, the resolution of two sample components can be expressed in efficiency and selectivity as: 1 R s = 4 µ N µ (17) Where µ = µ 2 - µ 1 ; µ = µ 2 + µ 1 2 Substituting equation (14) into equation (17) yields an expression for resolution in terms of EOF, diffusion, selectivity and electrical field strength. R s 1 = 4 2 ( µ ) D V ( µ + µ ) eof 1/ 2 V = ( µ ) (18) D ( µ + µ ) eof 29

36 From the equation, resolution increases by square root of applied voltage. When µ and µ eof are nearly same but with different signs, highest resolution will be obtained. 30

37 1.4 CFFE Historical Background and development There are two approaches for obtaining preparative or semi-preparative quantities of pure R,S; D,L or +,- separated enantiomers. One is chiral synthesis; the other is chiral separation of the racemic mixtures. Chiral synthesis often needs very strict conditions and may not apply to all chiral compounds, so it may be difficult to realize. Thus, in some cases, chiral separation may be the more practical option. CE and HPLC are good chiral separation methods, but they are primarily analytical scale chiral separation methods. Convenient and practical preparative scale chiral separation methods need to be developed. 26 Some preparative chiral chromatography research has been carried out. 48 Several groups have actively been attempting to conduct electrophoretic enantiomer separations at the preparative scale. Recently, methods have been investigated as approaches to preparative or semi-preparative chiral separations. Complete resolution of piperoxan enantiomers 21 and partial resolution of mg quantities of terbutaline enantiomers 22 by using classical gel electrophoresis was reported by Stalcup et al. 25 However, batch processes or non-continuous introduction of sample in those experiments limits the sample throughput. Preparative continuous free flow electrophoresis was first reported in Pioneering works on continuous free flow electrophoresis (CFFE) have been conducted by several groups (e.g., H. Wagner et al 49,50,51,52,53 and K. Hannig et al 54 ; P. R. Brown et 31

38 al 55,56 and L. S. Rodkey 57 ; R. Kessler 58 ) during late 1980s and early 1990s. Recently, Stalcup et al 18,27,28 reported that preparative scale chiral separations can be performed by CFFE. An average processing rate of 0.5 mg/h for Ritalin 27 and 0.45 mg/h for racemic piperoxan 28 were reached by using SCD as the CA. Other CFFE results were also reported recently by C. F. Ivory et al 59, G. Vigh et al 60,61,62 and G. Weber et al 499,63,64. Truly preparative chiral separation could be fulfilled by Continuous free flow electrophoresis (CFFE), although it is still far from being a routine chiral separation method. The advantage of the CFFE method is both the sample and background electrolyte (BGE) are continuously introduced into the electrophoresis chamber through a single inlet or a series of inlet ports. Thus, it combines the tremendous resolving power of electrophoresis with a controlled continuous feed process CFFE instrumentation The principles for CFFE instrumentations are similar to CE instrumentation. However, the sample and BGE are continuously introduced. The process is totally a free solution electrophoresis process. All the solutions, buffer, anode and cathode electrode wash, anode and cathode sheath wash and the sample are introduced by peristaltic pumps. In the system used in this study, circulating coolant was introduced by PTFE capillaries counter-current to the buffer and sample flows. The coolant absorbs Joule heating created by the electrophoresis system. The separation is performed in the interstitial spaces between the capillaries in the chamber. The rectangular dimension of the 32

39 separation chamber produces a buffer curtain when buffer is introduced at the top end of the chamber; the sample is introduced from the same end. An electric field is applied perpendicular to the sample and electrolyte flow, so as to induce lateral displacement of the sample components towards the respective counter electrode according to their electrophoretic mobilities. The separated sample components and the buffer solution travel through an array of outlet tubes and are collected by a fraction collector at the electrophoresis chamber outlet end. To realize chiral separation, as other chiral separation methods, a chiral selector is needed in the buffer curtain. Detailed schematics can be seen in Figure and Figure

40 Sample Fractions collector Buffer + - Figure 1.8 Schematic of CFFE instrument 18 34

41 + Sheet Sample inlets Sheet Buffer Buffer Membrane Membrane + electrode wash electrode wash Coolant Figure 1.9 Schematic of CFFE electrophoresis chamber 35

42 1.4.3 CFFE theory When charged analytes interact with the electric field in CFFE, they are deflected laterally toward their respective counter-electrode. The angle of deflection can be obtained by using equation (19). 56 µ V i tan Θ = (19) ν l Where: Θ -- deflection angle; µ i apparent or intrinsic mobility of the solute; ν -- the linear velocity of the electrolyte; V/l the field strength across the electrophoretic chamber. For the separation of enantiomers in the presence of a chiral selector such as CD, the parameter of interest is the difference of the deflection angle between the individual enantiomers. The free enantiomers have the same mobility and assuming, to a first approximation, the complexes formed by each of enantiomers with cyclodextrin also have the same another mobility. Thus, the separation equation can be obtained as equation (20) and (21). 18 This relationship is analogous to the cyclodextrin-mediated CE separation of enantiomers equation derived by Wren and Rowe (refer to CE section equation (10)). ( µ ) Θ a (20) 1 Θ 2 1 µ 2 Where: a = V/νl; Θ 1 Θ 2 a ( µ f µ c )( K1 K 2 )[ CD] ( [ ])( [ ]) 1+ K1 CD 1+ K 2 CD (21) 36

43 µ f -- mobility of the free solute; µ c mobility of the complex; K 1, K 2 enantiomer- cyclodextrin binding constants; [CD] concentration of CD. Equation (21) predicts that, as in the case of CE, both the differences of the free and complexed analyte mobilities and the differences in binding constants will influence the separation results. Also, there will be an optimum CD concentration as in CE for best separation. The equation also tells that higher electric field strength and lower buffer flow rate will improve the separation. 18 It should be noted that this model makes several assumptions. First, the separation buffer and the CA are uniformly distributed in the chamber. Second, the electric field across the separation chamber is also uniform. Third, the flows in the electrophoresis chamber are uniform. However, results from dye studies 28 show that this model is oversimplified. 37

44 1.5 Outline of experiments The project involved investigating chiral recognition for chiral additive and analyte pairs, developing an analytical method for rapid analysis of samples from CFFE instrument, and developing a method for preparative chiral separation. Three compounds methoxyphenamine (MPA), hydrobenzoin (HB) and piperoxan (PI) were investigated. The structure and some physical properties can be seen in the experimental section. MPA and PI are basic solutes; both have high affinity for SCD as is indicated by early migration in carrier mode CE using SCD as chiral additive. HB is a neutral analyte and has less affinity for SCD. Chiral separation of these three compounds by CE can help screen separation and operational parameters for CFFE. Capillary electrophoresis (CE) and CFFE were used as analytical scale and preparative scale chiral separation methods. CE parameters, such as ph, buffer concentration (ionic strength), sample matrix, applied voltage, CE operating mode and SCD concentration influence on the chiral separation was investigated. The influence of CFFE parameters, such as buffer concentration, SCD concentration, electrode washing conditions, electrode sheath washing conditions, ph of the solutions, applied voltage and SCD degree of substitution influence on the CFFE chiral separation were also investigated. Additional studies under selected conditions were applied to CFFE for piperoxan preparative scale separation. The sample capacity was investigated. Two dyes, patent blue (PB) and crystal violet (CV) were used as surrogates to investigate the CFFE performance. 38

45 Chapter 2. Experimental Section 2.1 Materials The phosphate buffer (100 mm, ph 2.5) was obtained from Bio-Rad laboratories (Hercules, CA, USA) and used for the CE experiments. Phosphate buffer for CFFE samples CE analysis were prepared from buffers obtained from Sigma Diagnostics (St. Louis, MO, USA). The sulfated β-cyclodextrins (SCD) (nominal degree of substitution 14; lot # 1210s and sulfated groups 5-7 SCD, lot # 1123) were obtained from Michigan Diagnostic Corp. (Troy, Michigan, USA). All the CE experiments were done with the same lot of SCD. Doubly de-ionized water (18.2 MΩ cm) used for CE solutions was prepared by passing doubly distilled water through a NanoPure treatment system (Barnstead, Boston, MA, USA). The (±) hydrobenzoin, methoxyphenamine hydrochloride and piperoxan were obtained from Sigma Chemical Company (St. Louis, MO, USA) (Figure 2.1 shows the structure and some properties). Crystal violet and patent blue VF were obtained from Aldrich Chemical Company (Milwaukee, WI, USA). Other materials such as citric acid, sodium hydroxide, phosphoric acid, hydroxide ammonium acetate and etc. were analytical grade and obtained from Fisher Chemical/Fisher Scientific (Fair Lawn, NJ, USA). Filters (0.2 µm) were obtained from Nalge Nunc International Corporation (Rochester, NY, USA). The fused-silica capillaries were obtained form Bio-Rad Laboratories, Inc. (Hercules, CA, USA). 39

46 OCH 3 CH 3 CH 2 CHNHCH 3 Methoxyphenamine, Hydrochloride 65 C 11 H 17 NO HCl, FW pka = OH C OH C H H Hydrobenzoin 65 C 14 H 14 O 2, FW O O CH2 N Piperoxan, d,l form hydrochloride 65 C 14 H 20 ClNO 2, FW pka = 8.57, UV max = 275 nm Figure 2.1 Structures of the compounds used in this study 40

47 2.2 Apparatus BioFocus 2000 and 3000 CE instruments (Bio-Rad Laboratories; Richmond, CA, USA) were used. A Dell P-III Dimension PC and Gateway 2000 PC were connected to the CE instruments for instrument control and data handling. A Beckman Coulter P/ACE TM MDQ Capillary Electrophoresis System interfaced to an HP P-II PC was also used (Beckman Coulter, Inc., Fullerton, CA, USA). The CE column (Biocap TM Bare Silica Capillary, 50µm 375µm 10m) was obtained from Bio-Rad Co. A Perkin Elmer Lambda 20 UV-Vis absorption spectrometer (Shelton, CT, USA) was used for the dye experiments. The syringe filter (0.2 µm style) was obtained from Fisher Scientific (Pittsburgh, PA, USA). The CFFE prototype instrument was on loan from Varian, Inc. (Wakefield, RI, USA). 41

48 2.3 Methods and procedure CE methods All the CE solutions were prepared with18.2 MΩ water, filtered and degassed by using the 0.2 µm syringe filter before use. For all buffers, ph adjustments were done with phosphoric acid or sodium hydroxide solution after the addition of the SCD. Phosphate buffer (50 mm and 10 mm) with varied ph values were used for CE experiments. MPA and HB CE samples were previously dissolved in water, added 1:3 (V/V) volume of the CE running buffer to dilute before use. Sonication was used when preparing hydrobenzoin sample solutions. Piperoxan was dissolved in the CE buffer. The final analyte concentration was about 0.1 mg/ml. Three lengths of capillary columns (23 cm, 25 cm and 35 cm respectively) were used in the CE experiments. The capillary was thermostatted at 20 C or 25 C. The applied high voltage was varied from 4 KV to 12 KV. UV on-line detection was carried out at the capillary anode end (reverse polarity) or the capillary cathode end (normal polarity) at 200 or 214 nm wavelengths. Between runs, the capillary was rinsed by 0.5 M NaOH and D.I. water for 60 seconds each. Various SCD concentrations (0.75% to 2.5%) and ph values were used during the experiments. The sample matrix influence on the separation also was investigated. Carrier mode (reversed polarity mode) and normal polarity mode CE methods each was investigated for two of the compounds (MPA and HB). 42

49 2.3.2 CFFE methods Citric acid buffers with various concentrations of SCD were used for the CFFE experiments. Preliminary experiments on the CFFE instrument were run using crystal violet and patent blue dye. Some parameters of the instrument were investigated. All the CFFE solutions were filtered through a 0.45 µm nylon filter prior to use. Ice was used to make chilled water in the coolant system. MPA and piperoxan preparative scale chiral separation was investigated under different running conditions. Details of the conditions and various buffer compositions used in the CFFE experiments are presented along with the histograms in the results section. The CFFE instrument was operated in a constant voltage mode. Table 2.1 shows the detail operation sequence required. 43

50 Table 2.1 CFFE instrument operation sequence Time/min Operation Pre-run Pump water, check to make sure flow in all 48 collection tubes and adjust the collector end flow rate; apply coolant 0-10 Begin pumping buffer; no applied voltage Begin pumping sample; no applied voltage Apply initial voltage (typically 60 V) Increase voltage stepwise to final operating voltage 50 Begin fraction collection Post-run 0 Shut down voltage 0-60 Rinse the chamber and electrode compartments with 0.1 M NaOH Rinse the chamber and electrode compartments with water 44

51 Chapter 3. Results and Discussion 3.1 CE results and discussion General observations Data from electropherograms, and experimental conditions for MPA and HB CE experiments are shown in Table 3.1 and Table 3.2. CE experiment results are also shown in the electropherogram figures. The influence of SCD concentration, ph value of the buffer, buffer concentration (ion strength), applied voltage (electric field strength), capillary column length (also related to the electric field strength) and the sample matrix on the separation of the two enantiomer compounds were examined and detailed discussion about all these influences is addressed in following sections. Because CE is a very sensitive analysis method, many factors influence the CE reproducibility 67. For quantitative and qualitative analysis, conditions must be well controlled so as to obtain good reproducibility. Our goal was to investigate the influence of CE operation factors on chiral separation and develop fast CE analytical methods for the collected CFFE samples. Figure 3.1 shows typical hydrobenzoin CE results. The second electropherogram was from a repeated run with the same vials of buffer. The slight shift in migration time for the second enantiomer may be the result of buffer depletion. Thus, for long CE experiments, the buffer should be refreshed after each run. 45

52 Absorbance (AU) First run Second run Time (min) Sample: HB in buffer, ph 3.24; B capillary; buffer: ph 3.0, 50 mm phosphate, 2% SCD; HV: 8 KV; Reverse polarity Figure 3.1 Hydrobenzoin CE electropherogram for repeated run 46

53 Table 3.1 CE data for Methoxyphenamine Figure # Sample matrix (Buffer + SCD) Column Buffer ph 3.3 a 25 mm phos % 3.3 b 2.5 mm phos % 3.3 c 2.5 mm phos % Buffer component (phos, SCD) A mm, 1.5% A mm, 1.5% A mm, 1.5% Field strength (V/cm) Migration time (min) Analyte mobility (10-3 cm 2 /v min) Rs CE mode / / Normal / / Normal / / Normal 3.7 a 25mM % C mM,0.5% / / Reverse 3.7 b 25mM + 1.0% C mM,2.0% / / Reverse 3.9 a 25mM + 1.0% B mM,2.0% / / Reverse 3.9 b 25mM + 1.0% B mM,2.0% / / Reverse 3.9 c 25mM + 1.0% A mM,2.0% / / Reverse 3.9 d 25mM + 1.0% A mM,2.0% / / Reverse Column dimensions: A (50 µm 375 µm 23 cm), B (50 µm 375 µm 25 cm), C (50 µm 375 µm 35 cm). 47

54 Table 3.2 CE data for hydrobenzoin Figure # Sample matrix Column Buffer ph 3.1 HB in buffer ph HB in buffer ph HB in buff, ph 9.24,0.75%SCD 3.6 HB in buff, ph 3.24, 1.5%SCD No Fig HB in buff, ph 9.24,0.75%SCD Buffer component (phos, SCD) B mm 2.0% Field strength (V/cm) Migration time (min) Analyte mobility (10-3 cm 2 /v min) Rs CE mode / / Reverse B mm / / Reverse 2.0% A mM, 0.75% / / Normal B mM,1.0% / / Reverse A mM, 1.0% / / Normal HB: hydrobenzoin. Column dimensions: A (50 µm 375 µm 23 cm), B (50 µm 375 µm 25 cm), C (50 µm 375 µm 35 cm). 48

55 3.1.2 Influence of sample matrix In Figure 3.2 (a), methoxyphenamine was dissolved in water, filtered, degassed, and the sample was directly injected into the capillary. When only the MPA water solution was injected into the column, the peak shape from the UV on-line detector was very poor, with no apparent separation. The BGE used was 50 mm phosphate with ph 3.0. Compared with the BGE zone, the sample zone has lower ionic strength, so the sample zone has a greater EOF power than the BGE zone. Thus, an electro-osmotic pressure developed at the boundary between the sample and BGE zones. This pressure difference causes a pressure driven hydrodynamic flow. As described previously, laminar flow will result in zone broadening. This degraded the resolution. Figure 3.2 (b) showed the effect of diluting the water sample with running buffer (3:1 V/V). As can be seen in the figure a better separation (with better peak shape and better resolution) was obtained. 49

56 Absorbance (AU) a Migration time (min) Absorbance (AU) b Migration time (min) Sample: a-mpa in water; b-mpa water solution and buffer (3:1 volume); A capillary; buffer: ph 3.0, 50 mm Phosphate, 0.5% SCD; HV: 12 KV; Reverse polarity Figure 3.2 Methoxyphenamine experiment electropherogram for sample matrix comparison 50

57 3.1.3 Influence of ionic strength Buffer ionic strength influence on CE chiral separation was investigated. When 50 mm phosphate buffer were used, MPA enantiomers had lower mobilities than when 10 mm phosphate buffer was used in normal mode (Table 3.1), and the peak resolution was also affected (Figure 3.3, a). The recorded current during the separation running was high, so more Joule heat was created. If excessive Joule heat cannot be efficiently dissipated, both peak efficiency and resolution will decrease. When 10 mm phosphate and lower concentration of SCD buffer was used, a good separation was obtained (Figure 3.3, b and c). The recorded current was greatly decreased; under these conditions a less Joule heat was generated so the CE coolant system dissipated it efficiently and higher voltage can be applied. For normal polarity mode, the detector is on the cathode side and analytes move with EOF (protonated MPA migrates ahead of EOF) to the detector while the complex migrate to the anode side, so the lower ion strength and lower SCD concentration, the less migration time is needed (See Table 3.1 and Figures 3.3 and 3.4). 51

58 0.023 Absorbance Time (min) a Sample: MPA + 25 mm phosphate % SCD; A capillary; buffer: ph 8.76, 50 mm phosphate, 1.5% SCD; HV: 8 KV; Normal polarity; I ave = 116 µa Absorbance (AU) Time (min) Sample: MPA+2.5 mm phosphate % SCD; A capillary; buffer: ph 8.76, 10 mm phosphate, 1.5% SCD; HV: 10 KV; Normal polarity; I ave = 42 µa b 0.08 Absorbance (AU) c Time (min) Sample: MPA mm phosphate % SCD; A capillary; buffer: ph 8.76, 10 mm phosphate, 1.5% SCD; HV: 8 KV; Normal polarity; I ave = 34 µa Figure 3.3 Methoxyphenamine CE ionic strength experiment electropherograms 52

59 Migration time (min) Faster isomer Slower isomer Phosphate concentration (mm) Sample: MPA + 25 mm phosphate % SCD; A capillary; buffer: ph 8.76, 50 mm phosphate, 1.5% SCD; HV: 8 KV; Normal polarity; I ave = 116 µa Sample: MPA mm phosphate % SCD; A capillary; buffer: ph 8.76, 10 mm phosphate, 1.5% SCD; HV: 8 KV; Normal polarity; I ave = 34 µa Figure 3.4 Influence of ionic strength on CE chiral separation 53

60 3.1.4 Influence of buffer ph As theoretically predicted, the ph value of the buffer system will affect the EOF so as to influence the separation. During the experiments, ph values of 3.0 for low ph value and about 9.0 for high ph were used. The obtained results are reported in Table 3.1, 3.2 and in the electropherograms. When normal polarity mode was used, high ph buffers were chosen, since the high EOF could reduce the migration time. In this mode, fast CE analysis can be obtained (Figure 3.3 b for MPA and Figure 3.5 for HB). Resolutions were lower in this mode compared with reverse polarity mode (see Table 3.1 and Table 3.2). When carrier mode (reverse polarity mode) was used, low ph buffers were used which resulted in low EOF. Thus, the SCD could form complex with the analytes and carry the analytes to the anode side detector (see Table 3.1 and Table 3.2). But in reverse polarity mode, when an intermediate ph value was used, the two opposite movements EOF and SCD analyte complexes resulted in better resolution (Table 3.2, Figure 3.6). 54

61 Absorbance (AU) Time (min) Sample: HB in buffer, ph 9.24, 0.75% SCD; A capillary; buffer: ph 8.44, 50 mm phosphate, 0.75% SCD; HV: 8 KV; Normal polarity Figure 3.5 Hydrobenzoin CE electropherogram for fast analysis 55

62 Absorbance (AU) Migration Time (min) Sample: HB in buffer, ph 3.24; B capillary; buffer: ph 6.0, 50 mm phosphate, 1.0% SCD; HV: 12.0 KV; Reverse polarity Figure 3.6 Hydrobenzoin CE electropherogram for ph and resolution in reverse polarity mode 56

63 3.1.5 Effect of SCD concentration In the normal polarity mode, concentrations from 0.75%-1.5% SCD and high ph ( ) were used (Tables 3.1 and 3.2). If high concentration SCD was used, the analyte migration time was long and sometimes the analyte could not be detected at the cathode end. With increasing SCD concentration, the migration times increase, because in the normal polarity mode, the free analytes migrate toward the cathode with the EOF while the SCD drags the analytes to the opposite end. In contrast, when reversed polarity mode was used, the higher SCD concentration caused higher analyte mobility, thus the migration time was reduced. In this mode, SCD carries the analytes to anode end. Methoxyphenamine has a very high affinity to SCD, so the SCD concentration can be low and separation can still be obtained. If SCD concentration was too high, it tended to increase the ionic strength and decrease the mobility. Figure 3.7 and 3.8 showed the effect of SCD concentration on the CE chiral separation in reverse polarity mode. 57

64 Absorbance (AU) a b Time (min) Sample: a-mpa + 25mM phosphate % SCD, b-mpa + 25mM phosphate + 1.0% SCD; C capillary; buffer: ph 3.0, a-50mm phosphate + 0.5% SCD, b-50mm phosphate + 2.0% SCD; HV: 12 KV; Reverse polarity Figure 3.7 MPA CE electropherograms for effect of buffer SCD concentration on migration time and resolution 58

65 migration time (min) Slow isomer Fast isomer SCD concentration (%) SCD concentration vs. migration time Resolution SCD concentration (%) SCD concentration Vs. resolution Sample: a-mpa + 25mM phosphate % SCD, b-mpa + 25mM phosphate + 1.0% SCD; C capillary; buffer: ph 3.0, 50mM phosphate + SCD; HV: 12 KV; Reverse polarity Figure 3.8 Effect of buffer SCD concentration on migration time and resolution 59

66 3.1.6 Influence of the electric field strength Applied electric field strength is another vital parameter influencing the migration times and peak efficiencies in chiral CE. Capillary column length and the applied high voltage between the two electrodes were changed to examine the effect of the electric field strength in our investigation. Migration time for the analytes arriving the detector changed with the electric field strength (Figure 3.9, Figure 10 and see tabulated results in Table 3.1 and Table 3.2). The higher applied voltage led to faster migration, narrower peaks and higher resolution. But if the applied high voltage was too high, the current in the capillary would increase, thus electrical noise increased. This was observed during the experiments. Figure 3.11 showed that when 50 mm phosphate was used as buffer and applied voltage was above 12 KV, the curve deviated from Ohm s Law for this CE system. Joule heating became a problem when applied voltage was set above 12 KV and caused the resistance decreased. Increasing ionic strength, increasing SCD concentration and increasing applied voltage all can cause current increasing. 60

67 Absorbance (AU) a b Time (min) Sample: MPA + 25 mm phosphate + 1.0% SCD; B capillary; buffer: ph 3.0, 50 mm phosphate, 2.0% SCD; HV: a-10 KV, b-8 KV; Reverse polarity 0.02 Absorbance (AU) d c Time (min) Sample: MPA + 25 mm phosphate + 1.0% SCD; A capillary; buffer: ph 3.0, 50 mm phosphate, 2.0% SCD; HV: c-10 KV, d-12 KV; Reverse polarity Figure 3.9 Methoxyphenamine CE electropherograms for effect of electric field strength on migration time and resolution 61

68 25 Fast isomer Slow isomer 20 Migration time (min) Electric field strength (V/cm) Sample: MPA + 25 mm phosphate + 1.0% SCD; A capillary; buffer: ph 3.0, 50 mm phosphate, 2.0% SCD; Reverse polarity Figure 3.10 Methoxyphenamine CE result for effect of electric field strength on migration time 62

69 Current (micro A) Applied voltage (KV) Sample: HB in buffer; Column: 50 µm 375 µm 25 cm; Buffer: 50 mm Phosphate, 1.0% SCD, ph 3.24; Instrument: Bio-Rad 2000; Reverse polarity Figure 3.11 Ohm s Law Curve 63

70 3.1.7 Piperoxan CE chiral separation Figure 3.12 shows the electropherogram and the separation conditions for fast piperoxan CE analysis. Because piperoxan has the similar ionic property as methoxyphenamine, it should perform similar in CE system. Thus, similar CE conditions from methoxyphenamine experiment were applied to piperoxan CE experiments. This method was applied to the CFFE sample fractions analysis. Baseline of the electropherogram is very clear. The two peaks are well separated (Rs = 1.5). CE analysis can be completed within 3 minutes (migration time: 2.50/2.83 minutes). This allows fast analysis of the collected CFFE piperoxan samples. 64

71 Sample: Piperoxan in buffer; 21.0cm/31.2cm total, 75 µm i.d. capillary; buffer: ph3.0, 10 mm phosphate, 2.0% SCD; HV: 12KV; Normal polarity; UV 214 nm; 2.0 psi, 5.0 seconds injection; Beckman Coulter CE system. Figure 3.12 Piperoxan CE separation electropherogram 65

72 3.1.8 Best results for CE experiments Table 3.3 showed the best results for shortest analysis time and highest resolution. The shortest analysis times for all the analytes were obtained in normal polarity mode with high ph buffer (8.40 for HB and 8.76 for MPA), in which the analytes moved in the bulk solution driven by the EOF; low ionic strength was chosen to enhance the EOF. The best resolution was obtained in reverse polarity mode with low ph buffer (ph 3.0). In this mode, analytes were dragged back by SCD while the EOF pushed the free analytes toward opposite direction. For the CFFE samples analysis, conditions were chosen which provided the shortest analysis time. 66

73 Table 3.3 Best CE separation results for the analytes Analyte Methoxyphenamine Hydrobenzoin Shortest analysis time 2.22/ /3.57 (min) CE mode Normal Normal Resolution Mobility (10-3 cm 2 /v min) Conditions 19.1/ /11.8 Sample: MPA+2.5 mm phosphate % SCD; A capillary; buffer: ph 8.76, 10 mm phosphate, 1.5% SCD; HV: 10 KV; Normal polarity; I ave = 42 µa Sample: HB in buffer, ph 9.24, 0.75% SCD; A capillary; buffer: ph 8.40, 50 mm phosphate, 1.0 % SCD; HV: 10 KV; Normal polarity. Analyte Methoxyphenamine Hydrobenzoin Best resolution CE mode Reverse Reverse Analysis Time (min) 21.45/ /52.73 Mobility (10-3 cm 2 /v min) Conditions 4.1/ /1.2 Sample: MPA + 25mM phosphate + 1.0% SCD; C capillary; buffer: ph 3.0, 50mM phosphate + 2.0% SCD; HV: 12 KV; Reverse polarity Sample: HB in buffer, ph 3.24; B capillary; buffer: ph 3.0, 50 mm phosphate, 2% SCD; HV: 8 KV; Reverse polarity 67

74 3.2 CFFE results and discussion Methoxyphenamine CFFE separation Methoxyphenamine CFFE separation results According to the CE results, MPA had relatively high affinity for SCD. MPA was introduced in the center of the CFFE chamber (vial 24 position). In the absence of SCD, MPA should be deflected towards the cathode which corresponds to vial 48. However, in the presence of SCD, MPA should be deflected towards the anode which corresponds to vial 1. In addition, there are 7 ports for buffer introduction into the electrophoresis chamber. Port 1 and 7 are used for sheath washes; port 1 is on the cathodic end and port 7 is on the anodic end. There are also two introduction ports for electrode washes. Prior studies with ritalin and piperoxan showed that electrode wash conditions were critical in the CFFE preparative chiral separations. Prior studies also showed that there were SCD depletion (cathode side) and accumulation (anode side) zones in the chamber 28. Thus, the SCD concentration is not uniform during the separation. MPA CFFE preparative separation results are shown in Figure In Figures 3.13 a, b and c, all conditions were the same except that different compositions were introduced into the chamber from 5 and 6 buffer introduction position. The best resolution was obtained when an artificial depletion zone was made by introducing distilled deionized water into buffer ports 5 and 6 (Figure 3.13 c) which correspond to the SCD accumulation zones when the same buffer is introduced into all of the buffer ports (Figure 3.13 a). When using the buffer without SCD in 5 and 6 buffer inlets 68

75 Corrected peak Area MPA CFFE 400V 24inlet all same buffer sample Beckman CE histogram Vial Number a Separation sample 3.46mg/h; 24 inlet position; Applied voltage 400V; Buffer: 0.075% SCD (0.375mM), 7.5mM acetic acid, ph 4.31; Cathodic wash: 1.5% SCD, 10 mm acetic acid, ph 8.93; Cathodic sheath wash: 0.300% SCD, 20 mm Acetic acid, ph 3.76; Anodic wash: 10mM acetic acid, ph 3.1; Anodic sheath wash: 20 mm ammonium acetate, ph 6.7; All introduction positions are same buffer; I = 505mA-527 ma. Corrected Peak Area MPA CFFE 400V 24inlet 5&6 noscd sample Beckman CE histogram b Separation sample 3.46mg/h; 24 inlet position; Applied voltage 400V; Buffer: 0.075% SCD (0.375mM), 7.5mM acetic acid, ph 4.31; Cathodic wash: 1.5% SCD, 10 mm acetic acid, ph 8.93; Cathodic sheath wash: 0.300% SCD, 20 mm Acetic acid, ph Anodic wash: 10mM acetic acid, ph 3.1; Anodic sheath wash: 20 mm ammonium acetate, ph 6.7; 5 & 6 introduction positions are buffer w/o SCD; I = 524mA-514 ma Vial Number Corrected Area MPA CFFE 400V 24inlet 5&6 Water sample Beckman CE histogram c Separation sample 3.46mg/h; 24 inlet position; Applied voltage 400V; Buffer: 0.075% SCD (0.375mM), 7.5mM acetic acid, ph 4.31; Cathodic wash: 1.5% SCD, 10 mm acetic acid, ph 8.93; Cathodic sheath wash: 0.300% SCD, 20 mm Acetic acid, ph 3.76; Anodic wash: 10mM acetic acid, ph 3.1; Anodic sheath wash: 20 mm ammonium acetate, ph 6.7; 5 & 6 introduction positions are DI water; I = 523mA-553 ma Vial Number Corrected Area MPA CFFE 300V 5&6 noscd samples Beckman CE histogram d Separation sample 8.65mg/h; 24 inlet position; Applied voltage 300V; Buffer: 0.075% SCD (0.375mM), 7.5mM acetic acid, ph 4.31; Cathodic wash: 1.0% SCD, 10 mm acetic acid, ph 8.75; Cathodic sheath wash: 0.150% SCD, 20 mm Acetic acid, ph 3.84; Anodic wash: 10mM acetic acid, ph 3.1; Anodic sheath wash: 20 mm ammonium acetate, ph 6.7; 5 & 6 introduction positions are buffer w/o SCD; I = 196mA-208 ma Vial Number Corrected Area MPA CFFE 300V all buffer sample Beckman CE histogram Vial Number e Separation sample 8.65mg/h; 24 inlet position; Applied voltage 300V; Buffer: 0.075% SCD (0.375mM), 7.5mM acetic acid, ph 4.31; Cathodic wash: 1.0% SCD, 10 mm acetic acid, ph 8.75; Cathodic sheath wash: 0.150% SCD, 20 mm Acetic acid, ph 3.84; Anodic wash: 10mM acetic acid, ph 3.1; Anodic sheath wash: 20 mm ammonium acetate, ph 6.7; All introduction positions are same buffer; I = 300mA-334 ma. Figure 3.13 Methoxyphenamine CFFE separation histogram 69

76 (Figure 3.13 b) to generate an artificial depletion zone, better separations result than when using the same buffer throughout all the buffer inlets (compare a and b) was obtained. Some pure optical isomers were obtained. In Figures 3.13 d and e, 300V applied voltage was used and 1.0% and 0.15% instead of 1.5% and 3.0% SCD were used for cathodic wash and cathodic sheath wash. The sample loading to the system was very high (8.65 mg/h). The analytes almost stayed at the introducing position or even deflected to cathodic side due to the SCD depletion. There are several reasons that may have caused the loss of separation. First, the system may be overloaded; second, SCD concentration in the buffer was probably too low; third, the SCD concentrations in the cathodic sheath wash and cathodic wash may be too low; fourth, 300V may be too low for this system. When 400 V were used, the analytes migrated further in the anodic direction, so better separation was obtained. 70

77 Collected samples ph distribution after CFFE separation For CFFE, normal operation with uniform buffer introduction, the buffer was not homogeneous in the chamber even though the same buffer was used 28. Ions migrated to the corresponding electrode end and caused the ionic strength and ph value to shift in the separation chamber. Thus, SCD and ion depletion and accumulation zones were created. The ph distribution in the samples obtained from the vials in 300V, 5 and 6 positions are distilled deionized water MPA separation is shown in Figure It shows that the ph values in most of the chamber are similar (around 3-4), only the electrode ends are varied. The ions from electrode and sheath wash solutions can pass through the membrane in the electric field so as to contribute to the change in ph. 71

78 Corrected Peak Area MPA CFFE 300V 5 & 6 water samples Beckman CE histogram a Vial Number Collected sample ph distribution after CFFE separation 8 ph b Vial number Introduction conditions: separation sample 8.65mg/h; 24 inlet position; Applied voltage 300V; Buffer: 0.075% SCD (0.375mM), 7.5mM acetic acid, ph 4.31; Cathodic wash: 1.0% 14 subs SCD, 10 mm acetic acid, ph 8.75; Cathodic sheath wash: 0.15% 14 subs SCD, 20 mm Acetic acid, ph 3.84; Anodic wash: 10mM acetic acid, ph 3.1; Anodic sheath wash: 20 mm ammonium acetate, ph 6.7; 5 & 6 introduction positions are DI water, 2-4 positions are same buffer; I = 225mA-222 ma. Figure 3.14 ph distribution of collected samples after CFFE separation (a is the corresponding histogram) 72

79 3.2.2 Dye experiments Dye experiments on UV-Vis Spectrometer The crystal violet and patent blue dyes structures are shown in Figure Dyes were dissolved in the CFFE buffers with SCD (0.375 mm SCD) and without SCD. Figure 3.16 shows the UV-Vis spectrum. Maximum absorptions were in nm. For patent blue, the absorption maximum was independent of SCD type used (curves 4, 5 and 6 in Figure 3.16). Visually, patent blue did not change color when SCD added. For crystal violet VF, there are apparent absorption shifts to the longer wavelength direction when it combined with SCD. In the presence of the 5-7 degree substituted SCD, the crystal violet absorption peak shifted less than in the presence of 14 degree substituted SCD (curves 1, 2 and 3 in Figure 3.16). This is consistent with the visually observation (it tended to become much more blue when combined with 14 degree substituted SCD). These properties make it possible for us to distinguish the dyes movement in the separation chamber. In the absence of SCD, the crystal violet was deflected cathodically in the chamber. In the presence of SCD, the crystal violet sample solution was blue and was deflected anodically. This is consistent with previous observations made in this laboratory

80 H 3 CH 2 C N+ CH 2 CH 3 H 3 C N+ CH 3 Cl- C C +Na-O 3 S SO 3 - N CH 2 CH 3 H 3 C N N CH 3 CH 2 CH 3 CH 3 CH 3 Patent blue VF Crystal Violet Figure 3.15 Dye structures used in the experiments 74

81 CV crystal violet; PB patent blue; NEWCD 5-7 degree substituted SCD; OLDCD 14 degree substituted SCD;.SP file name extension; mm SCD. Figure 3.16 UV-Vis scan spectrum of dye experiments 75

82 Dye experiments on CFFE The crystal violet and patent blue dyes (structures see Figure 3.15) separation supplied a visual method to investigate the effect of various instrumental parameters on the separation. The cationic crystal violet complexes strongly with SCD while zwitterionic patent blue displays minimal or no interaction with SCD. Thus, patent blue stayed essentially at the introducing position and crystal violet moved to the anode direction if SCD was used as the chiral additive. Different substituted degree SCD with dyes behavior in CFFE experiments was compared in Figure 3.17 and Figure Figure 3.17 illustrates observations made of dye trajectories in the CFFE chamber during pre-equilibration using 14 degree substituted SCD conditions. While Figure 3.18 shows actual pictures obtained using 5-7 degree substituted SCD conditions. In particular, it should be noted that the difference in sample stream outlets are enhanced when the dye sample stream is introduced at position 32. It should also be noted that in both cases, the trajectory followed by the crystal violet does not follow a straight line. This nonlinearity is not accounted for in the model. Further more, dispersion in the sample stream does not seem to be pathlength dependent. Several conclusions can be drawn from these two Figures. First, the CFFE system needed some time to reach equilibrium; secondly, if 5-7 degree substituted SCD was used as chiral additive, the separation power will be lower than the 14 substituted SCD as chiral additive (CV migrated to vial 16 position compared to vial 3 position); 76

83 Inlet: iiiiii I Input + _ Vial #: , Exit Time: (0-5min) (5-10 min) (10-20 min) Applied voltage: 350V; I = mA; 24 sample inlet position; 14 subs SCD used Sample inlet Input + _ Vial #: , Exit Time: (0-5min) (5-10 min) (10-20 min) Applied voltage: 350V; I = mA; 32 sample inlet position; 14 subs SCD used. Figure 3.17 CFFE dye experiment using 14 degree substituted SCD 77

84 Sample inlet Input + _ Vial # Exit Time: (0-5min) (5-20min) (20-30 min) Stable Applied voltage: 350V; I = ma; 32 sample inlet position; 5-7 subs SCD used. Figure 3.18 CFFE dye experiment using 5-7 degree substituted SCD 78