Improved preparative-scale. continuous, free-flow electrophoretic separation of the enantiomers of terbutaline utilizing equal-but-opposite enantiomer mobilities.

The factors that influence yield and product purity in the continuous, preparative-scale electrophoretic separation of the enantiomers of terbutaline when using the principle of equal-but-opposite effective mobilities were studied. The sodium salt of heptakis-6-sulfato-beta-cyclodextrin was used as the resolving agent, in acidic, isopropanol-containing background electrolytes, in the continuous, free-flow, preparative electrophoretic instrument, the Octopus. By matching the linear velocity of the feed solution to that of the background electrolyte, lateral hydrodynamic dispersion was minimized resulting in a nonelectrophoresed feed band that was only three fractions (about 3 mm) wide as it exited the 0.5 m long separation channel. The multiple of residence time and applied potential was also optimized, constrained by migration of the front of heptakis-6-sulfato-beta-cyclodextrin out of the separation zone, leading to the recovery of 95% of both enantiomers in better than 99.99% purity, at a production rate of 0.1 mg/h.     

Use of full-column imaging capillary isoelectric focusing for the rapid determination of the operating conditions in the preparative-scale continuous free-flow isoelectric focusing separation of enantiomers

A rapid, simple method is proposed here for the identification of the experimental conditions that lead to satisfactory preparative-scale isoelectric focusing enantiomer separations in continuous free-flow electrophoretic units. The method first calls for the use of a commercially available, full-column imaging capillary electrophoretic system to find the background electrolyte composition that generates the largest pI difference between the bands of the enantiomers. The method then calls for the finding of the minimum residence time that permits full development of the pH gradient across the separation chamber of the continuous free-flow electrophoretic unit by measuring the pH in the sample-free carrier electrolyte fractions collected during these runs. Finally, the quality of the predicted preparative-scale separation is verified by analyzing the enantiomer-containing collected fractions by capillary electrophoresis using a 14-sulfated, single-isomer cyclodextrin as resolving agent. The pI difference values and production rate values observed in this work agree well with the literature values that were obtained by much more time-consuming methods.     

Experimental verification of a predicted, previously unseen separation selectivity pattern in the capillary electrophoretic separation of noncharged enantiomers by octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin

The capillary electrophoretic separation of noncharged enantiomers with single-isomer anionic resolving agents is reexamined here with the help of the charged resolving agent migration model. Two general model parameters have been identified that influence the effective mobility, separation selectivity and mobility difference curves of the enantiomers: parameter b, called binding selectivity (K(RCD)/K(SCD)), and parameter s, called size selectivity (mu(o)RCD/mu(o)SCD). Analysis of the model in terms of these parameters indicates that in addition to the known, previously observed separation selectivity vs. resolving agent concentration patterns, a new pattern, increasing separation selectivity with increasing resolving agent concentration, is also possible provided that (i) K(RCD)/K(SCD)<1 and mu(o)RCD/mu(o)SCD>1 and (K(RCD)mu(o)RCD)/(K(SCD)mu(o)SCD)>1, or (ii) K(RCD)/ K(SCD)>1 and mu(o)SCD/mu(o)SCD<1 and (K(RCD)mu(o)RCD)/(K(SCD)mu(o)SCD)<1. This hitherto unseen separation selectivity pattern was experimentally verified during the capillary electrophoretic separation of the enantiomers of O-isopropyl p-nitrophenyl methylphosphonate with the single-isomer octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin as resolving agent.     

Experimental verification of a predicted, hitherto unseen separation selectivity pattern in the nonaqueous capillary electrophoretic separation of weak base enantiomers by octakis (2,3-diacetyl-6-sulfato)-gamma-cyclodextrin

The capillary electrophoretic separation of cationic enantiomers with single-isomer multivalent anionic resolving agents was reexamined with the help of the charged resolving agent migration model. Three general model parameters were identified that influence the shape of the separation selectivity and enantiomer mobility difference curves: parameter b, the binding selectivity (K(RCD)/K(SCD)), parameter s, the size selectivity (mu0(RCD)/mu0(SCD)), and parameter a, the complexation-induced alteration of the analyte's mobility (mu0(RCD)/mu0). In addition to the previously observed discontinuity in separation selectivity that occurs as mu(eff) of the less mobile enantiomer changes from cationic to anionic, a new feature, a separation selectivity maximum was predicted to occur in the resolving agent concentration range where both enantiomers migrate cationically provided that (i) K(RCD)/K(SCD) <1 and mu0(RCD)/mu0(SCD) >1 and (K(RCD)mu0(RCD))/(K(SCD)mu0(SCD)) > 1, or (ii) K(RCD)/K(SCD) >1 and mu0(RCD)/mu0(SCD) <1 and (K(RCD)mu0(RCD))/(K(SCD)mu0(SCD)) <1. This hitherto unseen separation selectivity pattern was experimentally verified during the nonaqueous capillary electrophoretic separation of the enantiomers of four weak base analytes in acidic methanol background electrolytes with octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin (ODAS-gammaCD) as resolving agent.     

Capillary electrophoretic separation of the enantiomers of organophosphates with a phosphorus stereogenic center using the sodium salt of octakis(2,3-diacetyl-6-sulfo)-gamma-cyclodextrin as resolving agent

The sodium salt of the single-isomer, chiral resolving agent, octakis(2,3-diacetyl-6-sulfo)-gamma-cyclodextrin (ODAS-gammaCD) has been used for the capillary electrophoretic separation of the enantiomers of alkylarylphosphates which carry a phosphorus-based stereogenic center. The effective mobilities and separation selectivities were measured at different ODAS-gammaCD and methanol concentrations to find the conditions under which the minor enantiomers could be adequately quantitated in samples obtained by chemical resolution of the racemic mixtures. This work extends the utility of ODAS-gammaCD to a hitherto unexplored field, the capillary electrophoretic separation of the enantiomers of organophosphorus compounds.     

Preparative-scale isoelectric trapping enantiomer separations

The new Gradiflow BF200 IET unit, developed for isoelectric trapping protein separations has been modified and used to carry out preparative-scale enantiomer separations. Hydroxypropyl beta-cyclodextrin was used as the chiral resolving agent to induce an isoelectric point difference between the enantiomers. Three isoelectric membranes with isoelectric points below, in between and above the isoelectric points of the complexed enantiomers were used to trap the separated enantiomers in the anodic and cathodic separation compartments of the Gradiflow BF200 IET apparatus, respectively. The production rates were about 15 times higher than those previously obtained with another isoelectric trapping device and about 30% higher than those obtained in a continuous free-flow electrophoretic device operated in the isoelectric focusing mode. The remarkable separation speed observed in the modified Gradiflow BF200 IET unit is attributed to the favorable interplay of the short electrophoretic transfer distance, the high electric field strength and the large effective surface areas of the isoelectric membranes.     

Separation selectivity patterns in the capillary electrophoretic separation of anionic enantiomers by octakis-6-sulfato-gamma-cyclodextrin

The capillary electrophoretic separation of anionic enantiomers with multiply-charged, single-isomer, anionic resolving agents was reexamined with the help of the charged resolving agent migration model. Three general model parameters were identified that influence the shape of the separation selectivity and enantiomer mobility difference curves: parameter b, the binding selectivity (K(RCD)/K(SCD)), parameter s, the size selectivity (micro0RCD/micro0SCD), and parameter a, the complexation-induced alteration of the analyte's mobility (micro0SCD/micro0). Function analysis of the model indicates that there are six unique separation selectivity vs. resolving agent concentration patterns: in two of the patterns, separation selectivity asymptotically increases to the limiting value set by parameter b; in two other patterns, separation selectivity passes a local maximum and asymptotically decreases to the limiting value set by parameter b; and in the last two patterns, separation selectivity passes a local maximum, decreases to unity, then, after reversal of the intrinsic migration order, asymptotically increases to the limiting value set by parameter b. Though the patterns with asymptotically increasing selectivities were observed in earlier work, this paper reports the first experimental verification of the existence of the local selectivity maximum during the capillary electrophoretic separation of the enantiomers of several weak acids in high pH background electrolytes with octakis-6-sulfato-gamma-cyclodextrin as the resolving agent.     

Stability constants of amino acids, peptides, proteins, and other biomolecules determined by CE and related methods: recapitulation of published data

The stability (affinity, association, binding, complexation, formation) constant characterizes binding interaction between the analyte and the complexing agent. Knowledge of the stability constant makes possible the prediction and estimation of the binding behavior of constituents (amino acids, peptides, proteins, drugs, antibiotics, enzymes, enantiomers) to their partners, and the finding of a suitable partner for the given analyte to form a stable complex. The present paper summarizes the stability constant determination methods and the approaches used to evaluate the experimental data. Further, the paper recapitulates the published stability constant values determined, mainly, by capillary electrophoretic methods, taken from the Web of Science database covering the last decade. Details of the experimental conditions employed for the determination of the stability constants are also given. The review attempts to give a critical evaluation of the problems that accompany the determination of stability constant and discusses their solution.     

Enhancement of selectivity and resolution in the enantioseparation of uncharged compounds using mixtures of oppositely charged cyclodextrins in capillary electrophoresis

The enantiomeric separation of some nonsteroidal anti-inflammatory drugs (NSAIDs) was investigated in capillary electrophoresis (CE) using dual systems with mixtures of charged cyclodextrin (CD) derivatives. A significant enhancement of selectivity and resolution could be achieved in the enantioseparation of these analytes in their uncharged form by the simultaneous addition of two oppositely charged CD derivatives to the background electrolyte. The combination of the single-isomer cationic CD, permethyl-6-monoamino-6-monodeoxy-beta-CD (PMMAbetaCD) and the single-isomer polyanionic CD, heptakis-6-sulfato-beta-cyclodextrin (HSbetaCD) in a pH 2.5 phosphoric acid-triethanolamine buffer, was designed and employed for the enantioseparation of profens. The improvement in selectivity and resolution can be attributed to the fact that the two CDs, which lead to independent and enantioselective complexation with the analyte enantiomers, have not only opposite effects on the electrophoretic mobility of these compounds but also opposite affinity patterns towards the enantiomers of these compounds. Binding constants for these enantiomers with each CD were determined using linear regression approach, in order to be able to predict the effect of the concentrations of the two CDs on enantiomeric selectivity and resolution in such dual systems.     

Separation of enantiomers of ephedrine by capillary electrophoresis using cyclodextrins as chiral selectors: comparative CE, NMR and high resolution MS studies

The enantiomer migration order (EMO) of ephedrine was investigated in the presence of various CDs in CE. The molecular mechanisms of chiral recognition were followed for the ephedrine complexes with native α- and β-CD and heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-CD (HDAS-β-CD) by CE, NMR spectroscopy and high-resolution MS. Minor structural differences were observed between the complexes of ephedrine with α- and β-CD although the migration order of enantiomers was opposite when these two CDs were applied as chiral selectors in CE. The EMO was also opposite between β-CD and HDAS-β-CD. Significant structural differences were observed between ephedrine complexes with the native CDs and HDAS-β-CD. The latter CD was advantageous as chiral CE selector not only due to its opposite electrophoretic mobility compared with that of the cationic chiral analyte, but also primarily due to its enhanced chiral recognition ability towards the enantiomers of ephedrine.     

Synthesis of a single-isomer sulfated beta-cyclodextrin carrying nonidentical substituents at all of the C2, C3, and C6 positions and its use for the electrophoretic separation of enantiomers in acidic aqueous and methanolic background electrolytes. Part 2: Heptakis(2-O-methyl-6-O-sulfo) cyclomaltoheptaose

The sodium salt of heptakis(2-O-methyl-6-O-sulfo)cyclomaltoheptaose (HMS), the second single-isomer, sulfated beta-CD carrying nonidentical substituents at all of the C2, C3, and C6 positions, has been synthesized, analytically characterized, and used for the capillary electrophoretic separation of the enantiomers of a group of 23 weak base analytes in acidic aqueous and methanolic BGEs. HMS interacted strongly with only about half of the analytes studied. The good separation selectivities and favorable normalized EOF mobilities allowed for rapid, efficient separation of the enantiomers of 19 of the 23 weak base analytes in the aqueous BGEs, often with separation selectivity values complimentary to those obtained with other single-isomer sulfated CDs. HMS did not prove to be as good a resolving agent in acidic methanolic BGEs as its counterpart, heptakis(2-O-methyl-3-O-acetyl-6-O-sulfo)cyclomaltoheptaose.     

Synthesis of mono-6-deoxy-6-N,N,N',N',N'-pentamethylethylenediammonio-cyclomaltoheptaose, a single-isomer, monosubstituted, permanently dicationic beta-CD and its use for enantiomer separations by CE

The dichloride salt of mono-6-deoxy-6-N,N,N',N',N'-pentamethylethylenediammonio-cyclomaltoheptaose (PEMEDA-BCD), the first single-isomer, monosubstituted, permanently dicationic beta-CD has been synthesized, analytically characterized, and used for the capillary electrophoretic separation of the enantiomers of a group of analytes in acidic and basic BGEs. When the concentration of PEMEDA-BCD was changed in the BGEs, the resulting effective mobilities of the analytes and the respective separation selectivities followed the predictions of the ionic strength-corrected charged resolving agent migration model. Good separation selectivities and favorable normalized EOF mobilities allowed for the rapid, efficient separation of the enantiomers of anionic, weak acid and nonionic analytes in the low- and/or high-pH BGEs.     

Continuous fractionation of enantiomer pairs in free solution using an electrophoretic analog of simulated moving bed chromatography

Continuous fractionation of the left and right enantiomers of Piperoxan was performed in free solution in a vortex-stabilized electrophoresis apparatus. Sulfated beta-cyclodextrin was used as the chiral selector. A capillary electrophoresis (CE) study of the separation of Piperoxan enantiomers was carried out in order to find the buffer conditions that produce the maximum peak separation time between the two enantiomers and the optimal chiral selector concentration. These peak separation times were then used to calculate the electrophoretic mobilities of the enantiomer-ligand complexes. The difference in electrophoretic mobilities, when used in a preliminary model of the enantiomer separation, indicated that, by imposing a fluid flow opposite the direction of electromigration, it would be possible to force the fast and slow enantiomers to move in opposite directions within the vortex-stabilized apparatus. Using the predictions of the preliminary separation model, the vortex stabilized electrophoresis apparatus was configured with a feed port at the center of the chamber axis and offtake ports near the cathode and anode. This allowed for continuous operation of the apparatus. Continuous fractionations were completed at throughputs of 1.5 and 4.0 mg/h with both offtakes showing greater than 99% enantiomeric purity at 4.0 mg/h using CE. Fractionation was achieved at a throughput of 10 mg/h, but while the slow enantiomer was recovered with greater than 99% purity, only 96% enantiomeric purity of the fast stereoisomer was achieved. The loss of resolution at higher volumetric throughputs supports our hypothesis that a mobility-dependent "window" of operation exists in which two solutes can be completely separated.     

Efficient resolution of 2,2'-dihydroxy-1,1'-binaphthyl by inclusion complexation with chiral N-(3-chloro-2-hydroxypropyl)-N,N,N-trimethylammonium chloride

The complete resolution of 2,2[prime or minute]-dihydroxy-1,1[prime or minute]-binaphthyl into its enantiomers by inclusion complexation with a commercially available derivative of choline, is reported. The two enantiomers are recovered in >99% ee from the inclusion complexes by simple dissolution in a diethyl ether-water medium and the resolving agent can be recycled.     

Resolution of the enantiomers of oxamniquine by capillary electrophoresis and high-performance liquid chromatography with cyclodextrins and heparin as chiral selectors

The methods of separation of the enantiomers of the chiral drug oxamniquine are compared, between HPLC with either cyclodextrins and their related derivatives as chiral selectors in the mobile phase or immobilisedin a chiral stationary phase (as Cyclobond I and II) and between capillary zone electrophoresis (CZE) where the cyclodextrins are added to the buffer solution. The HPLC experiments, which included structured method optimisation were largely unsuccessful in resolving the enantiomers, with the exception of when a Chiral-AGP protein stationary phase was introduced into the programme. However although this chiral stationary phase provided baseline resolution of the enantiomers the stability of the method was suspect to small changes in the pH (0.2 units). In contrast the CZE method developed for both cyclodextrins and their derivatives gave good resolution of the enantiomers and method stability (R.S.D. <1%, N = 10 on precision). The basis of the interaction mechanism between selector and selectand was shown as a 1:2 relationship of cyclodextrin to analyte by NMR. In addition the polysaccharide, heparin was investigated as a chiral additive and excellent resolution of the oxaminiquine was achieved with 3 mM heparin in 50 mM sodium dihydrogenphosphate (pH 3.0) as buffer in CZE, which also gave a stable procedure. This method allowed the detection of each of the enantiomers in the presence of the other down to 0.23% (m/m). The overall composition of the heparin material from different sources can however be slightly variable and this can result in small differences in resolution capability.     

Synthesis of heptakis(2-O-methyl-3-O-acetyl-6-O-sulfo)-cyclomaltoheptaose, a single-isomer, sulfated beta-cyclodextrin carrying nonidentical substituents at all the C2, C3, and C6 positions and its use for the capillary electrophoretic separation of enantiomers in acidic aqueous and methanolic background electrolytes

The sodium salt of heptakis(2-O-methyl-3-O-acetyl-6-O-sulfo)cyclomaltoheptaose (HMAS), the first single-isomer, sulfated beta-cyclodextrin carrying nonidentical substituents at all of the C2, C3, and C6 positions, has been synthesized, analytically characterized, and used for the capillary electrophoretic separation of the enantiomers of a group of 24 weak base pharmaceuticals in acidic aqueous and acidic methanolic background electrolytes. HMAS interacted more strongly with most of the analytes studied than heptakis(2,3-di-O-methyl-6-O-sulfo)cyclomaltoheptaose, but less strongly than heptakis(2,3-di-O-acetyl-6-O-sulfo)cyclomaltoheptaose, the respective analogs with identical substituents at the C2 and C3 positions. The good separation selectivities and favorable normalized electroosmotic flow mobilities allowed for rapid, efficient separation of the enantiomers of 19 of the 24 weak base analytes in the aqueous and methanolic background electrolytes. The trends in the effective mobilities and separation selectivities as a function of the HMAS concentration closely followed the predictions of the ionic strength-corrected charged resolving agent migration model.     

Enantiomer separations by nonaqueous capillary electrophoresis using octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin

The newest member of the single-isomer isomer sulfated cyclodextrin family, octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin (ODAS-gamma-CD) was used for the first time as a resolving agent for the nonaqueous capillary electrophoretic separation of the enantiomers of 26 weak base pharmaceuticals in an acidic methanol background electrolyte. The solubility limit of ODAS-gamma-CD at room temperature proved to be 55 mM in this background electrolyte, which afforded good, fast enantiomer separations for most of the basic drugs tested. For all the bases studied, the effective mobilities and separation selectivities were found to follow the predictions of the charged resolving agent migration model of electrophoretic enantiomer separations. The effective mobilities of the weakly binding weak bases remained cationic throughout the entire 0 to 45 mM ODAS-gamma-CD concentration range; separation selectivities increased as the ODAS-gamma-CD concentration was increased. The effective mobilities of the moderately binding weak bases became anionic in the 2.5 to 45 mM ODAS-gamma-CD concentration range; separation selectivities first increased as the effective mobilities approached zero, then decreased again as the ODAS-gamma-CD concentration was increased further. The effective mobilities of the strongly binding weak bases became anionic in the 0 to 2.5 mM ODAS-gamma-CD concentration range; separation selectivities decreased as the ODAS-gamma-CD concentration was increased above 2.5 mM.     

Nonaqueous capillary electrophoretic separation of basic enantiomers using octakis(2,3-O-dimethyl-6-O-sulfo)-gamma-cyclodextrin, a new, single-isomer chiral resolving agent

The enantiomers of 34 pharmaceutical weak-base analytes were separated by nonaqueous capillary electrophoresis in acidic methanol background electrolytes using the sodium salt of the new, single-isomer chiral resolving agent, octakis(2,3-O-dimethyl-6-O-sulfo)-gamma-cyclodextrin (ODMS). The effective mobilities, separation selectivities and peak resolution values of the weak-base analytes were determined as a function of the ODMS concentration in the 0-40 mM range and were found to follow the theoretical predictions of the charged resolving agent migration model (CHARM model) modified for ionic strength effects. Fast, efficient separations were achieved for both comparatively small and large enantiomers.     

The role of achiral sorbent matrix in chiral recognition of amino acid enantiomers in ligand-exchange chromatography

Summary As an alternative to the known three-point interaction model describing recognition of optical isomers by a chiral resolving agent, a new concept has been developed stating that two interaction points between the resolving agent and the enantiomers are also sufficient for achieving chiral recognition of the latter, provided that the diastereomeric adducts formed by the resolving agent with the enantiomers additionally interact with a non-chiral chromatographic sorbent. This concept is based on the results of ligand-exchange chromatography of -amino acid enantiomers with copper(II) complexes of chiral bifunctional ligands as the resolving agents in chromatographic systems.     

Single-isomer sulfated alpha-cyclodextrins for capillary electrophoresis. Part 2. Hexakis(6-O-sulfo)-alpha-cyclodextrin: synthesis, analytical characterization, and initial screening tests

The second member of the family of single-isomer sulfated alpha-cyclodextrins, the sodium salt of hexakis(6-O-sulfo)-alpha-cyclodextrin (HxS), has been synthesized, analytically characterized, and used as the resolving agent for the capillary electrophoretic separation of the enantiomers of nonionic, weak-acid and weak-base analytes present in our initial screening kit. HxS interacted less strongly with many of the analytes tested than the larger-ring analogs, heptakis(6-O-sulfo)-beta-cyclodextrin (HS) and octakis(6-O-sulfo)-gamma-cyclodextrin (OS). For some of the analytes, the separation selectivities obtained with HxS were complementary to those observed with hexakis(2,3-di-O-acetyl-6-O-sulfo)-alpha-cyclodextrin (HxDAS), HS, and OS. For all analytes, the effective mobilities and separation selectivities as a function of the background electrolyte concentration of HxS followed the trends that were found for HxDAS, HS, and OS.