Enantiomer separation of chiral pharmaceuticals by capillary electrochromatography

Enantiomer separation of chiral pharmaceuticals by capillary electrochromatography (CEC) is achieved with open-tubular capillaries (o-CEC), with packed capillaries (p-CEC) or with monolithic capillaries. In o-CEC, capillaries are coated with a thin film containing cyclodextrin derivatives, cellulose, proteins, poly-terguride or molecularly imprinted polymers as chiral selectors. In p-CEC, typical chiral HPLC stationary phases such as silica-bonded cyclodextrin or cellulose derivatives, proteins, glycoproteins, macrocyclic antibiotics, quinine-derived and 'Pirkle' selectors, polyacrylamides and molecularly imprinted polymers are used as chiral selectors. Chiral monolithic stationary phases prepared by in situ polymerization into the capillary were also developed for electrochromatographic enantiomer separation.     

Recent progress in enantiomer separation by capillary electrochromatography

Enantiomer separation by electrochromatography (CEC) can be performed in three modes: (i) open-tubular capillary electrochromatography (o-CEC), in which the chiral selector is physically adsorbed coated, and thermally immobilized or covalently attached to the internal capillary wall; (ii) packed capillary electrochromatography (p-CEC), in which the capillary is either filled with chiral modified silica particles or with an achiral packing material, and a chiral selector is added to the mobile phase; and (iii) monolithic (rod)-capillary electrochromatography (rod-CEC) in which the chiral stationary phase (CSP) consists of a single piece of porous solid. We present an overview on methods and new trends in the field of electrochromatographic enantiomer separation such as CEC with either nonaqueous mobile phases or stationary phases with incorporated permanent charges, or with packing beds consisting of nonporous silica particles or particles with very small internal diameters.     

Chromatographic evaluation and comparison of three beta-cyclodextrin-based stationary phases by capillary liquid chromatography and pressure-assisted capillary electrochromatography

Enantiomer separations were performed on three beta-cyclodextrin-based chiral stationary phases (CSP) containing the pernaphthylcarbamoylated beta-cyclodextrin (CSP 1), peracetylated beta-cyclodextrin (CSP 2) and permethylated beta-cyclodextrin (CSP 3) as chiral selectors by capillary liquid chromatography and pressure-assisted capillary electrochromatography in this study. Triethylammonium acetate/MeOH or phosphate buffer/MeOH was used as the mobile phase. The experimental factors affecting chiral separations have been examined for each CSP, including pH of the buffers, methanol content and applied voltage. Under optimal separation conditions, a number of racemic compounds were resolved into their enantiomers on three cyclodextrin-based CSP. A comparative study on the performance of three CSP revealed the presence of carbonyl functional groups as well as aromatic rings in the cyclodextrin derivatives, enhanced the interaction between the analytes and CSP, and thus improved enantioselectivity of the CSP.     

A mesoporous silica nanoparticles immobilized open-tubular capillary column with a coating of cellulose tris(3,5-dimethylphenyl-carbamate) for enantioseparation in CEC

An approach of immobilizing mobile crystalline material (MCM)-41 mesoporous silica nanoparticles on the inner wall of an open-tubular (OT) capillary as the support for coating chiral selector of cellulose tris(3,5-dimethylphenyl-carbamate) (CDMPC) was carried out. By taking advantage of the improved phase ratio of OT capillary with the immobilization of MCM-41 mesoporous material, the cellulose derivative of CDMPC as the chiral selector was simply coated on the MCM-41 nanoparticle layer via the hydrogen-bonding interaction, and the enantioseparation was successively carried out. Eight pairs of acidic, neutral and basic enantiomers were resolved in capillary electrochromatography or capillary liquid chromatography mode. The concentration of CDMPC for coating was systematically investigated to obtain the optimized chromatographic properties on enantioseparation by controlling the supposed film thickness of CDMPC on MCM-41 nanoparticle layer. Comparing with a bare fused silica capillary column coated with CDMPC under the same coating procedure as MCM-41-modified capillary did, the MCM-41-modified capillary column offered much higher enantioselectivity. This result indicated the significance of using the mesoporous nanoparticles as the electrochromatographic support to enhance the phase ratio of OT capillary column in capillary electrochromatography and capillary liquid chromatography. For investigating the effect of experimental conditions on the enantioseparation with this prepared OT capillary column, the content of organic modifier acetonitrile in the mobile phase was thus extensively evaluated to achieve a better chiral separation.     

Separation of enantiomers by capillary electrochromatography

The current popularity of capillary electrochromatography (CEC) has led to an increasing number of studies on the development and evaluation of enantioselective CEC systems. These studies clearly demonstrate that the most prominent advantage of electrically driven separation methods, the vastly increased column efficiency as compared to pressure-driven chromatography, can also be experimentally achieved for the separations of enantiomers. In analogy to high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), several approaches have been used. The addition of a chiral selector to the mobile phase is the simplest method. Less erroneous and more elegant approaches are those that use open-tubular, conventional packed, and monolithic columns containing chiral stationary phases that stereoselectively interact with enantiomers. This review evaluates the new techniques and compares them to enantioselective HPLC and CE. Further, it describes the various concepts of enantioselective CEC and focuses on the current ‘state-of-the-art' column technology.     

Enantiomer separation by capillary electrochromatography on a cyclodextrin-modified monolith

A chiral monolithic stationary phase was prepared by packing a capillary with bare porous silica and sintering the silica bed at high temperature. The resulting silica monolith was polymer-coated with Chirasil-Dex, a permethylated beta-cyclodextrin covalently linked via an octamethylene spacer to dimethylpolysiloxane. Subsequently, Chirasil-Dex was thermally immobilized on the silica support and a chiral monolith of very high stability (30 kV, more than 400 bar pressure) was obtained. The enantiomer separation of various chiral compounds by monolithic (rod) capillary electrochromatography (rod-CEC) was feasible. This method was compared with capillary liquid chromatography (LC) in a single-column mode using unified equipment. About two to three times higher efficiency was found in the rod-CEC mode as compared to rod-LC. The influence of pressure-driven flow support on efficiency, resolution, elution time and baseline stability was investigated. The amount and nature of organic modifier strongly influences efficiency and resolution.     

Comparative study on the enantiomer separation of 1,1'-binaphthyl-2,2'diyl hydrogenphosphate and 1,1'-bi-2-naphthol by liquid chromatography and capillary electrophoresis using single and combined chiral selector systems

The chiral recognition ability of single and dual selectors, that were used as additives, have been investigated by HPLC and CE. Native beta- and gamma-cyclodextrins, permethylated beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, cholic acid and taurodeoxycholic acid sodium salts were applied as chiral selectors, whereas the atropisomers of 1,1'-binaphthyl-2,2'-diyl hydrogenphosphate, and 1,1'-bi-2-naphthol served as model compounds. It was found that all investigated selectors, except for gamma-cyclodextrin, display the same affinity pattern for binaphthyl enantiomers, i.e., binding the S more strongly than the R enantiomer. However, the differences in the phase distribution of chiral selectors led to the opposit elution order of enantiomers: with cyclodextrins, the first eluted is S enantiomer, while R is the first eluted for bile salts. Under the conditions studied, cyclodextrins (except gamma-cyclodextrin), as well as cholic acid sodium salts acting singly, enable the separation of 1,1'-binaphthyl-2,2'-diyl hydrogenphosphate enantiomers both by HPLC and CE methods, while 1,1'-bi-2-naphthol enantiomers were resolved only under CE conditions with permethylated cyclodextrin or bile salts. In both techniques the application of dual systems could improve resolution or make it worse (oreven cancel), depending on the sign of enantioselectivity of particular selectors, their concentrations and localization: mobile or stationary phase. It has been found that the mechanism of separation as well as interactions occurring between two selectors may be followed by using combined HPLC and CE methods. The obtained results proved that, as well as beta-CD, TM-beta-D and gamma-CD also form inclusion complexes with cholic acid sodium salts. The reversal of elution order may be realized by two procedures: changing a single selector, i.e., cyclodextrin on cholic acid sodium salt or vice versa, and by changing the proportion of selectors in the combined bile salt-cyclodextrin system.     

Thermodynamic study of N-trifluoroacetyl-O-alkyl nipecotic acid ester enantiomers on diluted permethylated beta-cyclodextrin stationary phase

Thermodynamic studies were performed on 12 pairs of N-trifluoroacetyl-O-alkyl nipecotic acid ester enantiomers on diluted permethylated beta-cyclodextrin stationary phase (CP Chirasil-Dex CB). The influence of ester alkyl group structure on interaction with permethylated beta-cyclodextrin (Me-CD) and enantioselectivity was studied. The types of alkyl groups studied included n-alkyl (C1-C5) and groups containing branching at differing locations relative to the chiral center of the molecule. The results show that for a given molecular weight, the n-alkyl esters have stronger interactions with Me-CD than esters containing branched alkyl groups. However, although having weaker interactions with Me-CD, esters containing alpha-branched alkyl groups exhibit higher enantioselectivity than the corresponding n-alkyl or beta-branched isobutyl esters. From the retention data, thermodynamic parameters were estimated using the retention increment method and enthalpy-entropy compensation plots (ln R' versus deltaH) were constructed. The results suggest that ester enantiomers with branching at the alpha-carbon of the ester alkyl group have additional and/or different types of enantioselective interactions with Me-CD than the C1-C5 n-alkyl esters or beta-branched isobutyl ester. In order to obtain a qualitative sense of the interaction with Me-CD, structures of the diastereomeric complexes formed between Me-CD and some of the ester enantiomers were modeled using simulated annealing molecular dynamics.     

Enantiomeric separation of chiral peptide nucleic acid monomers by capillary electrophoresis with charged cyclodextrins

Direct chiral separation of chiral peptide nucleic acid (PNA) monomers has been achieved for the first time by capillary electrophoresis (CE) with charged cyclodextrins as chiral selectors added to the electrophoretic buffer. Selectively modified 6-deoxy-6-N-histamino-beta-cyclodextrin and sulfobutyl ether-beta-CD were successfully used as chiral selectors for the enantiomeric separation of chiral monomers based on different aminoethylamino acids bearing thymine or adenine as nucleobases. Chiral separations were obtained at low selector concentrations (1-3 mM) with good enantioselectivity and resolution factors. Separations were optimized as a function of pH in order to exploit the effect of the electrostatic interactions between the oppositely charged selector and selectand. The method has been applied to the analysis of the enantiomeric excess of chiral monomers used for the solid phase synthesis of chiral PNA oligomers. CE chiral analysis showed that a very high enantiomeric purity was generally achieved in the synthesis of all monomers, except for histidine and aspartic acid based monomers in which ca. 10% of the "wrong" enantiomer was always present.     

Separation of brompheniramine enantiomers by capillary electrophoresis and study of chiral recognition mechanisms of cyclodextrins using NMR-spectroscopy, UV spectrometry, electrospray ionization mass spectrometry and X-ray crystallography

Opposite migration order was observed for the enantiomers of brompheniramine [N-[3-(4-bromphenyl)-3-(2-pyridyl)propyl]-N,N-dimethylamine] (BrPh) in capillary electrophoresis (CE) when native beta-cyclodextrin (beta-CD) and heptakis(2,3,6-tri-O-methyl)-beta-CD (TM-beta-CD) were used as chiral selectors. NMR spectrometry was applied in order to obtain information about the stoichiometry, binding constants and structure of the selector-selectand complexes in solution. The data were further confirmed by UV spectrometry and electrospray ionization mass spectrometry. The structure of the complexes in the solid state was determined using X-ray crystallography performed on the co-crystals precipitated from the 1:1 aqueous solution of selector and selectand. This multiple approach allowed an elucidation of the most likely structural reason for a different affinity (binding strength) of BrPh enantiomers towards beta-CD and TM-beta-CD. However, the question about a force responsible for the opposite affinity pattern of BrPh enantiomers towards these CDs could not be answered definitely.     

Enantiomer separation by pressure-supported electrochromatography using capillaries packed with Chirasil-Dex polymer-coated silica.

Pressure-supported electrochromatography using capillaries packed with permethyl-cyclodextrin covalently linked via an octamethylene spacer to dimethylpolysiloxane and immobilized on silica (Chirasil-Dex silica) has been employed as an efficient and rapid method for the enantiomer separation of various racemic compounds. By comparing this method with micropacked liquid chromatography (LC), employing the same column in a unified instrumental setup, micropacked capillary electrochromatography (CEC) shows higher column efficiencies and hence better resolution factors. The influence of type and concentration of buffer, amount and nature of organic modifier, and pressure support is investigated.     

Enantioseparation of chiral sulfoxides and sulfinate esters by capillary electrophoresis

Forty-one chiral sulfoxides and sulfinate esters were separated using sulfated beta-cyclodextrin and carboxymethyl beta-cyclodextrin as chiral selectors. Binding constants of some analytes to both chiral selectors were measured in order to examine and help explain the observed migration behavior and enantioselectivity trends. Overall, sulfated beta-cyclodextrin separated a greater number of compounds, and had better separating capabilities than did carboxymethyl beta-cyclodextrin for these analytes. This was true even though all of the analytes showed much stronger binding to carboxymethyl beta-cyclodextrin than to sulfated beta-cyclodextrin. General procedures to optimize the separation, by varying pH, selector concentration, and organic modifier concentration were examined and discussed. Chiral selector concentration had the greatest effect on enantioseparation, with higher concentrations of selector giving better peak-to-peak separations. Organic modifier had an adverse affect on resolution, with increasing amounts giving lower mobility differences. Lastly, pH had only a minimal effect on separation.     

Open-tubular capillary electrochromatography using a polymeric surfactant coating

A stable polyelectrolyte multilayer (PEM) coating was investigated for use in open-tubular capillary electrochromatography (o-CEC). In this approach, the PEM consisted of the cationic polymer of a quaternary ammonium salt, poly(diallyldimethylammonium chloride) and the anionic polymeric surfactant, poly(sodium undecylenic sulfate). Both the cationic and anionic polymers were physically adsorbed to the surface of a fused-silica capillary by use of a simple coating procedure. This procedure involved an alternate rinse of the positively and negatively charged polymers. The performance of the PEM coating as a dynamic stationary phase was evaluated by use of electrochromatographic experiments and showed good selectivity for both phenols and benzodiazepines. Reproducibility of the PEM coating was also evaluated by calculating the relative standard deviations (RSDs) of the electroosomotic flow (EOF). The run-to-run and capillary-to-capillary RSD values of the EOF were less than 1.5%. The endurance of the coating was more than 100 runs. The importance of the PEM coating was illustrated by comparing separations on a bare uncoated capillary with the coated capillary. In addition, the chromatographic performance using o-CEC and micellar electrokinetic chromatography (MEKC) was compared for the separation of benzodiazepines.     

Cyclocholates as Chiral Selectors for Capillary Gas Chromatography – Effect of Temperature Conditioning on the Chromatographic Behavior of the Stationary Phase

The suitability of cyclocholates as chiral selectors in gas chromatography has been evaluated. We present the synthesis and characterization of two cyclocholates, viz. 3α,7α-diacetoxycyclo[3]cholate and 3α,7α-diacetoxycylo[4]cholate. Mixtures of these new selectors with polysiloxanes were tested as chiral stationary phases in capillary gas chromatography. Several enantiomer separations of common racemates were achieved with the 3α,7α-diacetoxycyclo[3]cholate at 10% in OV-1701 (w/w). It was shown that column efficiency was strongly dependent on temperature and that enantioselectivity was very sensitive to column conditioning. This chromatographic behavior suggested that cyclocholates were only dispersed in polysiloxane. Thus, it was assumed that chiral discrimination occurred via enantioselective adsorption interactions of enantiomers at the surface of the solid chiral selector dispersed in the polysiloxane matrix OV-1701.     

吸附固定相电色谱和动态改性电色谱的手性分离

对动态改性电色谱手性分离进行了研究。电色谱柱填充强阴离子交换固定相（SAX0,添加在流动相中的磺化β－环糊精（S－CD）动态地吸陵于SAX填料表面,形成一层准手性固定相。色氨酸、阿托品和异博定对映体在本体系获得了很好的分离,它们的分离分别为2．06,10．1和1．96,对映体峰的柱效价于85,000塔板数／米和412,000塔板数／米之间。连续运行17次,死时间和色氨酸对映体的电色谱保留因子的相对标准偏差分别为0．53％,0．62％和0．69％。此外,以吸附于SAX填料的牛血清白蛋白和S－CD为手性固定相进行了电色谱手性分离的研究。在这两种体系下分离色氨酸对映体的分离度分别为3．86和2．97。吸附S－CD柱电色谱和动态改性电谱的重现性进行子比较,发现动态改性电色谱有更好的重现性。     

Chemically modified chiral monolithic silica column prepared by a sol-gel process for enantiomeric separation by micro high-performance liquid chromatography

In this work a new type of chiral monolith silica column was developed for the chiral separation by micro high-performance liquid chromatography (micro-HPLC). The chiral monolith column with a continuous skeleton and a large through-pore structure was prepared inside a capillary of 100 microm I.D. by a sol-gel process, and chemically modified with chiral selectors, such as L-phenylalaninamide, L-alaninamide and L-prolinamide, on the surface of the monolithic silica column. Based on the principle of ligand exchange, these chiral monolithic columns were successfully used for the separation of dansyl amino acid enantiomers, as well as hydroxy acid enantiomers by micro-HPLC. The chromatographic conditions, the enantioselectivity and the performance of columns are discussed.     

Physically adsorbed chiral stationary phase of avidin on monolithic silica column for capillary electrochromatography and capillary liquid chromatography

The physical adsorption method proposed previously has been successfully applied to a monolithic silica column. By virtue of the physical adsorption, a chiral stationary phase of avidin was prepared onto the silica monolith. The phase ratio of resulting stationary phase was evaluated with frontal analysis. The method proved to be comparable in phase ratio to the chemical bonding methods used in high-performance liquid chromatography (HPLC). Enantiomer separations were carried out in capillary electrochromatography (CEC) and capillary liquid chromatography (CLC) modes. Due to its larger phase ratio, the resulting column showed more powerful separation capability as compared to open-tubular CEC (OTCEC). Twelve chiral compounds were baseline-resolved. The resulting column showed high separation efficiency, with average theoretical plate numbers of 66 000/m for CLC and 122 000/m for CEC. Good reproducibility was observed, with RSD value less than 1.3% for retention time, retention factor and separation factor, and less than 6.6% for plate counts and resolution (n = 40). Fast separations were achieved with a short column. The test enantiomers were baseline-resolved within 4 min under CLC and CEC modes. In addition, field-enhanced sample injection (FESI) was coupled to CLC as well as CEC to improve the detection sensitivity.     

New amphiphilic aminosaccharide derivatives as chiral selectors in capillary electrophoresis

Two amphiphilic aminosaccharide derivatives were investigated as chiral selector additives in capillary electrophoresis. Each substance has a glucosamine backbone carrying three hydrocarbon chains as the hydrophobic region and three carboxylic groups as the hydrophilic region, which is an artificial biologically active compound. Using each compound as a chiral selector, the optical resolution of dansylated amino acids or new quinolone antibacterial agents (NQs) was observed. Increasing the concentration of the chiral selector or the ionic strength of running solution led to successful optical resolution. In consideration of the chemical structure of each selector and the migration behavior of the enantiomers, the resolution seemed to be based on micellar electrokinetic chromatography mode. Both selectors differed in their enantioselectivity for dansylated amino acids or NQs although the chemical structures were similar.     

聚丙烯酸酯类毛细管整体柱的制备及其在加压毛细管电色谱中的应用

毛细管电色谱(CEC)是毛细管电泳(CE)和微径液相色谱(micro—HPLC)技术的结合,是集CE的电子迁移机制和micro-HPLC的分配分离机理发展起来的一种高效微分离技术．CEC以塞子流型的电渗流代替抛物线流型的压力流,具有CE的高效性,能够分离电中性化合物而具有HPLC的高选择性．     

Enantiomeric separation of hydroxy acids and carboxylic acids by diamino-beta-cyclodextrins (AB,AC,AD) in capillary electrophoresis

Selectively modified 6,6'-dideoxy-6,6'-L-diamino-beta-cyclodextrins (AB, AC, AD) were successfully used as chiral selectors for the enantiomeric separation of hydroxy acids and carboxylic acids (in particular, phenoxyalkanoic acid herbicides) in capillary electrophoresis (CE). Chiral separations were obtained at a low selector concentration (1 mM) with good enantioselectivity and resolution factors. Separations were optimized as a function of pH. The different position of the charged groups on the upper rim greatly influenced the separation, accounting for electrostatic interactions between the protonated amino groups of the cyclodextrins (CDs) and the carboxylate of the selectands. The best enantiomeric separation of hydroxy acids was obtained with the AC regioisomer, whereas carboxylic acids were well resolved only by the AB regioisomer. A recognition model is proposed, based on two-dimensional nuclear magnetic resonance (2-D NMR) experiments, in which the orientation of the guest in the inclusion complex is determined by the electrostatic interactions between the selectand and the CD upper rim.