Influence of structure and chirality of the selector on the chiral recognition of amino acids using ligand-exchange capillary electrophoresis

The dependence of the chiral recognition ability and enantiomer migration order on the structure, substitution pattern and chirality of chiral selectors used in ligand-exchange capillary electrophoresis is investigated. As chiral selectors different N-alkyl derivatives of proline and hydroxyproline as their copper(II) complexes are used. The influence of the position and conformation of the hydroxy group in the hydroxyproline derivatives and of the structure and chirality of the side chain on enantioselectivity is investigated. Furthermore, the effect of surfactants such as sodium dodecyl sulfate and cetyl trimethyl ammonium bromide on resolution and enantiomer migration order is studied. The investigations were carried out with three aromatic amino acids as model compounds.     

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 advances in chiral separation principles in capillary electrophoresis and capillary electrochromatography

This review summarizes recent developments in chiral separation in capillary zone electrophoresis (CZE), electrokinetic chromatography (EKC), and capillary electrochromatography (CEC) covering literature published since the year 2000. New chiral selectors and innovative approaches for CE and CEC are introduced. Recent progress in column technology for CEC is highlighted and the development of new chiral stationary phases is discussed. This review is not dedicated to list applications but will focus on new developments.     

Recent progress in chiral separation principles in capillary electrophoresis

This review summarizes recent developments in the field of chiral separations by electromigration techniques including capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), isotachophoresis (ITP), electrokinetic chromatography (EKC), and capillary electrochromatography (CEC). This overview focuses on the development of new chiral selectors and the introduction of new techniques rather than applications of already established selectors and methods. The mechanisms of the different chiral separation principles are discussed.     

Separation of basic drug enantiomers by capillary electrophoresis using ovoglycoprotein as a chiral selector: comparison of chiral resolution ability of ovoglycoprotein and completely deglycosylated ovoglycoprotein

Separations of basic drug enantiomers by capillary electrophoresis (CE) using ovoglycoprotein (OGCHI) as a chiral selector are described. The effects of running buffer pH and 2-propanol content on the migration times and resolution of basic drug enantiomers were examined using a linear polyacrylamide-coated capillary. High resolution of basic drug enantiomers was attained using a mixture of 50 mM sodium phosphate buffer (pH 4.5-6.0) and 2-propanol (5-30%) including 50 microM OGCHI. It was found that ionic and hydrophobic interactions could work for the recognition of basic drug enantiomers. Further, we compared the chiral resolution ability of OGCHI with that of completely deglycosylated OGCHI (cd-OGCHI) using them as chiral selectors in CE. OGCHI showed higher resolution for basic drug enantiomers tested than cd-OGCHI. The results suggest that the chiral recognition site(s) for OGCHI exists on the protein domain of OGCHI.     

Comparative enantioseparations with native beta-cyclodextrin and heptakis-(2-O-methyl- 3,6-di-O-sulfo)-beta-cyclodextrin in capillary electrophoresis

Twenty-three cationic chiral analytes were resolved in capillary electrophoresis using native beta-cyclodextrin and single isomer heptakis-(2-O-methyl-3,6-di-O-sulfo)-beta-cyclodextrin as chiral selectors. For 12 of 16 chiral analytes resolved with both chiral selectors the enantiomer migration order was opposite. In selected cases the structure of cyclodextrin-analyte complexes in aqueous solution was investigated using one-dimensional transverse rotating frame nuclear Overhauser and exchange spectroscopy. It was found that in contrast to mainly inclusion-type complexes between chiral analytes and beta-cyclodextrin, external complexes are formed between the chiral analytes and structurally crowded, highly charged heptakis-(2-O-methyl-3,6-di-O-sulfo)-beta-cyclodextrin.     

Chiral separation by capillary electromigration techniques

This review gives an overview of chiral separation principles and their applications in capillary electromigration techniques. The basic chiral separation principles are explained and the mechanisms discussed. Recent developments and new techniques in CZE and capillary electrochromatography (CEC) are highlighted. New chiral selectors among cyclodextrins, crown ethers, carbohydrates, macrocyclic antibiotics, proteins, chiral ion-pairing reagents, chiral surfactants and chiral metal ion complexes and their chiral recognition ability are discussed. Recent advances in chip technology for chiral separation and new approaches regarding improvement of detection sensitivity are presented. Due to the tremendous number of publications dealing with applications, in this review only recent applications are summarized.     

毛细管电泳新型手性分离体系研究进展

在现代分离科学中,手性化合物的分离分析一直是研究的重点和难点。相比于高效液相色谱（HPLC）、气相色谱（GC）等传统色谱分析方法,毛细管电泳（CE）技术凭借其高效率、低消耗、分离模式多样化等诸多优势,已经发展成为手性分离研究领域最有应用前景的分析方法之一。近年来,研究人员在CE手性分析方法的构建过程中,基于毛细管电动色谱（EKC）、配体交换毛细管电泳（LECE）、毛细管电色谱（CEC）等各种基础电泳模式,不断地对传统手性分离体系进行优化和改造,构建出了许多高性能的新型手性CE分离体系。如利用各类功能化离子液体以"手性离子液体协同拆分""手性离子液体配体交换""离子液体手性选择剂"等模式设计出多种基于离子液体的CE手性分离体系;利用纳米材料独特的尺寸效应、多样性、可设计性等特点,直接或与传统手性选择剂有机结合构建CE手性分离体系。此外,金属有机骨架材料修饰、低共熔溶剂修饰、非连续分段式部分填充等各式新颖的CE手性分离体系也都被研究人员成功开发,并表现出较大的发展潜力。该综述将对近年来（尤其是2015~2019年）此类新型CE手性分离体系的发展状况进行梳理,并结合相应的手性识别机理研究和手性CE方法实际应用情况,对该领域存在的问题及发展前景进行分析和展望。     

分子印迹聚合物在毛细管电色谱拆分手性药物中的研究进展

手性药物通过与生物体内生物大分子之间的手性匹配与分子识别来发挥药理作用。两个对映体与体内手性环境相互作用的不同导致每个对映体表现出不同的药理活性、代谢过程、代谢速率及毒性等药代动力学特征。因此发展手性药物的拆分方法,对于手性药物的开发和生产过程的质量监控具有重要意义。分子印迹聚合物（MIPs）是以目标分子作为模板而制备的高分子聚合物,它具有特定的空间分子结构和官能团,对目标分子具有高度的特异性识别能力。基于该特点,MIPs非常适合于手性药物的拆分和纯化。毛细管电色谱（CEC）可同时基于毛细管电泳和液相色谱的分离机理对目标物进行分离,因此具有高分离效率和高选择性的特点。将MIPs材料作为CEC的固定相,可将这两种技术的优势结合,从而实现对手性药物的高效拆分。MIPs材料在1994年首次应用于CEC手性拆分,此后该研究领域开始获得关注和发展。MIPs材料主要通过4种模式在CEC中实现手性拆分,分别是作为开管柱、填充柱和整体柱的固定相以及分离介质中的准固定相。该综述以这4种模式作为分类基准,根据MIPs制备所需的材料和分离对象对其在CEC手性拆分中的应用进行了总结,揭示了MIPs在CEC手性拆分中的潜力,同时评述了这4种模式各自的优势与不足,并对将来MIPs在CEC手性拆分中的发展进行了展望。     

Separation of ofloxacin enantiomers by capillary electrophoresis with fluorescence detection using deoxyribonucleic acid oligonucleotides as chiral selectors

This study used capillary electrophoresis with fluorescence detection- and a partial-filling mode-based method for chiral separation of ofloxacin. The deoxyribonucleic acid oligonucleotides with different base sequences were studied as potential chiral selectors including deoxyribonucleic acid tetrahedron, G-quadruplex, and G-riched double-strand deoxyribonucleic acid. Under the optimized conditions, all the deoxyribonucleic acid chiral selectors exhibited excellent chiral separation capabilities with a resolution higher than 1.5. The electrophoretic behavior of the ofloxacin enantiomer might result from the intermediate conjugate with different stabilities between chiral selectors and analytes by a combination of the hydrogen bond and spatial recognition structure. Moreover, satisfactory repeatability regarding run-to-run and interday repeatability was obtained, and all the relative standard deviation values of migration times and resolutions were below 4% (n = 6). Conclusively, both spatial structure and arrangement of the G bases potentiated the chiral separation capability of deoxyribonucleic acid for ofloxacin enantiomer. This work offered a stepping stone for enantioseparation using deoxyribonucleic acid as chiral selectors.     

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.     

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.     

Designed combination of chiral selectors for adjustment of enantioseparation selectivity in capillary electrophoresis.

In this study an attempt has been made to explain the reasons for changing the enantioseparation selectivity in some dual cyclodextrin (CD) systems compared to the use of single chiral selectors in capillary electrophoresis (CE). An explanation for selectivity changes is proposed based on the effect of the chiral selector on the mobility of the analyte. In order to support the proposed mechanism, several dual systems were designed on the basis of the known recognition pattern of enantiomers for individual CDs. In most cases the separation selectivity could be adjusted in a designed way. There was no experimental evidence for simultaneous binding of a given chiral analyte with both chiral selectors or of chiral recognition of an analyte complex with one CD by another CD.     

Evaluation of new adsorbed coatings in chiral capillary electrophoresis and the partial filling technique

When using chiral selectors and the partial filling technique in capillary electrophoresis, a suitable and reproducible suppression of the electroosmotic flow is still a challenging issue, and there are a number of reasons to find alternatives to the use of covalently coated capillaries for such a particular application. In this paper, new achiral, neutral, and water-soluble polymers are evaluated as adsorbed polymers for the suppression of electroosmotic flow (EOF) when employing chiral capillary electrophoresis and the partial filling technique. Four chiral selectors, namely a cationic cyclopeptide, vancomycin, human serum albumin and riboflavin binding protein have been chosen for this study and some analytes such as derivatized amino acids, promethazine and prilocaine have been used as test compounds. Reproducibility of migration times, resolution, and selectivity as well as efficiency are reported to critically evaluate the performance of the adsorbed coatings. Results are compared to parallel data obtained with fused-silica and polyvinyl alcohol-coated capillaries.     

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.     

Chiral separations in capillary high-performance liquid chromatography and nonaqueous capillary electrochromatography using helically chiral poly(diphenyl-2-pyridylmethyl methacrylate) as chiral stationary phase.

Enantioseparations in nonaqueous capillary electrochromatography (CEC) are reported in this study for the first time, using wide-pore aminopropyl silica gel coated with helically chiral poly(diphenyl-2-pyridylmethyl methacrylate) (PDPM) as chiral stationary phase (CSP). The anodic electroosmotic flow (EOF) in a methanolic solution of ammonium acetate was used for the migration of neutral analytes through the packed bed in the capillaries. Four different techniques, high-performance liquid chromatography (HPLC) in common-size columns, capillary HPLC, pressure-assisted CEC and CEC were compared from the viewpoint of separation parameters. The latter three were performed with the same experimental setup, varying the relative contribution of the pressure-driven and the electrically driven flow to the overall mobility of the analyte. Capillary HPLC offers clear advantages compared to enantioseparations in common-size columns. However, for a given particle size of the packing material, CEC was not obviously advantageous compared to pressure-driven separations.     

Chiral capillary electrophoresis-mass spectrometry: modes and applications

A review is presented to highlight several approaches for coupling capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS) for analysis of chiral compounds. A short discussion of commercially available CE-MS instruments and interface design is followed by a detail review on various modes of chiral CE-MS. In general, for each CE-MS mode, the capabilities, applications and limitations for chiral analysis have been pointed out. The first mode, chiral capillary zone electrophoresis-mass spectrometry (CZE-MS) in which neutral derivatized cyclodextrins (CDs) are used is possible using either column coupling with voltage switching or a partial-filling technique (PFT). However, some applications of direct coupling of CZE-MS mode are also reported. The second mode is a chiral electrokinetic chromatography-mass spectrometry (EKC-MS) in which a charged chiral selector such as a sulfated beta-CD or a vancomycin could be conveniently employed. This is because these chiral selectors have a significantly higher countercurrent electrophoretic mobility which prevents the entrance of these selectors into the mass spectrometer. The combination of counter-migration and PFT demonstrates that this synergism could be successfully applied to chiral analysis of a broader range of compounds. It is well-known that the on-line coupling of micellar electrokinetic chromatography to mass spectrometry (MEKC-MS) is problematic because the high surface activity and nonvolatile nature of conventional surfactant molecules lower the electrospray ionization efficiency. However, a recent report demonstrates that this hyphenation is now possible with the use of molecular micelles. Various MEKC-ESI-MS parameters that can be used to optimize both chiral resolution and ESI response are discussed. Finally, two recent examples that demonstrate the feasibility of using either open-tubular or packed chiral CEC with MS are reviewed. This survey will attempt to cover the state-of-the-art on various modes of CE-MS from 1998 up to 2002.     

Novel enantioselective strong cation exchangers based on sulfodipeptide selectors: evaluation for enantiomer separation of chiral bases by nonaqueous capillary electrochromatography

Strong cation exchange (SCX)-type chiral stationary phases (CSPs) based on beta-amino sulfonic acid-terminated dipeptide derivatives as chiral selectors, immobilized on thiol-modified silica particles (3.5 microm), were synthesized and applied to enantiomer separations of chiral bases by nonaqueous capillary electrochromatography (CEC). The effect of structural variations of the sulfodipeptide selectors on the separation factors alpha was investigated. These studies included variation of the acid-terminal amino sulfonic acid residue, variation of the configurations, i.e., comparison of the diastereomeric (S,S)- and (R,S)-configurations of the sulfodipeptides, and finally comparison of sulfodipeptide selectors with corresponding beta-amino sulfonic acid analogs. In general, the capillary columns (100 microm ID) packed with the new SCX-type CSPs showed enantioselectivity for an elaborated set of chiral basic drugs in CEC acting by an enantioselective cation-exchange retention mechanism. N-[N-(4-Allyloxy-3,5-dichlorobenzoyl)-leucyl]-2-amino-3,3-dimethylbutane sulfonic acid, in particular with (R,S)-configuration, turned out to be a more effective SCX-type selector than a more rigid analog based on N-[N-(4-Allyloxy-3,5-dichlorobenzoyl)-leucyl]-2-pyrrolidinemethane sulfonic acid. Both of the former diastereomers were capable to baseline-resolve the enantiomers of ca. 40% of the tested basic chiral solutes including sympathomimetics and beta-blockers, while for the latter SCX-type CSPs only 10-20% of the selected solutes afforded resolutions > 1.5.     

Chiral separations by capillary electromigration techniques in nonaqueous media. II. Enantioselective nonaqueous capillary electrochromatography

A review on the advantages, peculiarities, and the potential of enantioselective capillary electrochromatography (CEC) in nonaqueous media is presented. Some fundamentals on CEC with particular focus on enantioselective CEC are discussed. The strategies, concepts, preferentially utilized chiral selectors and column technologies that have been utilized to succeed in highly efficient enantiomer separations by nonaqueous CEC are described thoroughly.     

Enantiomer separations by capillary electrochromatography using chiral stationary phases

The applicability of capillary electrochromatography (CEC) using packed capillary column to enantiomer separations was investigated. As chiral stationary phases, OD type packing materials of 5 and 3 microm particle diameters, originally designed for conventional high-performance liquid chromatography (HPLC) were employed. The chiral packing materials were packed by a pressurized method into a 100 microm I.D. fused-silica capillary. Several racemic enantiomers, such as acidic, neutral and basic drug components, were successfully resolved, typically by using acidic or basic solutions containing acetonitrile as mobile phases. The separation efficiencies for some enantiomers in the chiral CEC system using the 5 microm OD type packing were superior to those obtained in HPLC using chiral packings. The plate heights obtained for several enantiomers were 8-13 microm or the reduced plate height of 1.6-2.6, which indicates the high efficiency of this chiral CEC system.