Separation of binaphthyl enantiomers by capillary zone electrophoresis and electrokinetic chromatography

The capillary electrophoretic (CE) separation of the enantiomers of three binaphthyl compounds is investigated. Several CE modes such as cyclodextrin (CD) modified capillary zone electrophoresis (CZE) (CD-CZE), micellar electrokinetic chromatography (MEKC), cyclodextrin electrokinetic chromatography (CD-EKC), etc. are employed for the simultaneous enantiomer separation of the three solutes. The successful separation was achieved by combining two modes, in other words by using more than two chiral selectors. A development of the CE enantiomer separation is demonstrated for the binaphthyl compounds. The enantioselectivity of binaphthyl compounds is alo briefly discussed.     

Characterization of sulfated beta-cyclodextrins and determination of enantiomeric purity of (1 R,2S)-ephedrine by capillary zone electrophoresis

Matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize two different sulfated beta-cyclodextrins (HS-beta-CD). It was found that the HS-beta-CDs have broad heterogeneity in terms of sulfation degree. The average sulfate contents were in the range of 6 to 8 per CD molecule. Furthermore the sugar moieties of both HS-beta-CDs were sulfated either at position 6 or at positions 2 and 6, but not at position 3. Enantiomeric separation by capillary zone electrophoresis (CZE) using the HS-beta-CDs as chiral selectors showed that these CDs exhibited similar chiral selectivity and resolution of the ephedrine enantiomers. One of the CDs was employed for the enantiomeric purity evaluation of (1R,2S)-ephedrine (or (-)-ephedrine) by capillary zone electrophoresis. Quantification was done by comparison between the corrected peak areas of the minor enantiomer and (-)-ephedrine.     

Separation of neutral and basic enantiomers by cyclodextrin electrokinetic chromatography using anionic cyclodextrin derivatives as chiral pseudo-stationary phases

Separations of neutral and basic racemates were performed using five different anionic cyclodextrin (CD) derivatives as chiral selectors, viz. carboxymethylated β-CD, β-CD phosphate sodium salt, sulfobutyl ether β-CD sodium salt, carboxymethylated γ-CD, and γ-CD phosphate sodium salt. For the separation of neutral racemates, an untreated fused silica capillary was employed and various neutral racemates were successfully separated. Since the pH of the buffer affected the electroosmotic flow (EOF), the resolution was improved by changing the buffer pH. A polyacrylamide coated capillary was employed for the separation of basic racemates to suppress EOF and to prevent adsorption of cationic analyte on the capillary surface. By choosing an appropriate type and concentration of anionic CD, about 40 basic racemates were successfully separated. Some rough binding constants of basic analytes with an anionic β-CD were measured to discuss the optimum concentration of the CD. The migration direction was dependent on the binding constants and the concentration of the CD. The analyte strongly bound to the anionic CD migrated towards the anode but the weakly bound one moved towards the cathode. Anionic γ-CDs were also very useful for the separation of basic enantiomers. Five neutral CDs were employed as chiral selectors to compare selectivity between charged and neutral CDs, and eleven racemates could only be resolved using anionic CDs. The separation of some basic racemates in human plasma was also described. The direct injection of plasma samples was possible for some enantiomers that did not interact strongly with plasma proteins.     

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.     

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.     

Enantioseparation of new nucleoside analogs, related to d4T and acyclovir, by chiral capillary electrophoresis using highly sulfated beta-cyclodextrins

Baseline separation of some new acyclic nucleosides which are potential antiviral agents was achieved using cyclodextrin capillary zone electrophoresis (CD-CZE). A method for the enantiomeric resolution of these compounds and determination of their enantiomeric purity was developed using anionic CDs (highly sulfated-CD or highly S-CD) as chiral selectors and capillaries, which were dynamically coated with polyethylene oxide (PEO). Operational parameters including (i) the nature and concentration of the chiral selectors, (ii) organic modifiers, (iii) temperature, and (iv) applied voltage were investigated. The use of charged CDs provides (i) a supplementary driving force for the compounds in a running buffer and (ii) enantiomeric resolution by inclusion of compounds in the CD cavity. The highly S-CD was found to be the most effective complexing agent and allowed good enantiomeric resolution. The complete resolution of five nucleoside analogs was obtained using 25 mM phosphate buffer, pH 2.5, containing either highly S-alpha-CD, S-beta-CD or S-gamma-CD at 30 degrees C with an applied field of 0.30 kV/cm. The apparent association constants of the inclusion complexes were calculated. The enantiomer migration order for the molecules investigated was determined and the detection limit of enantiomeric impurities was found to vary between 0.34 to 3.56 ng.mL(-1) for the first enantiomer.     

Separation of enantiomers in capillary electrophoresis with contactless conductivity detection

Contactless conductivity detection is successfully demonstrated for the enantiomeric separation of basic drugs and amino acids in capillary electrophoresis (CE). Derivatization of the compounds or the addition of a visualization agent as for indirect optical detection schemes were not needed. Non-charged chiral selectors were employed, hydroxypropylated cyclodextrin (CD) for the more lipophilic basic drugs and 18-crown-6-tetracarboxylic acid (18C6H4) for the amino acids. Acidic buffer solutions based on lactic or citric acid were used. The detection limits were determined as 0.3 microM for pseudoephedrine as an example of a basic drug and were in the range from 2.5 to 20 microM for the amino acids.     

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.     

Peak splitting in the CE separation of enantiomers caused by organic solvents in the sample

Two robust chiral standard separation systems were developed for the analysis of the chiral purity of chemically different model compounds applied in homogeneous asymmetric hydrogenation catalysis. Sulfated CDs were used as chiral selectors as they allow the analysis of neutral, acidic as well as basic compounds in the same electrophoretic system. Poorly water-soluble amines were dissolved in different organic solvent/buffer mixtures. Reproducibly, depending on the amount of organic solvent in the sample solution, peak splitting occurred and/or more peaks than expected were observed, implying impure model compounds. The dependence of the "chiral purity" on experimental parameters, e.g., kind and amount of sample solvent, length of injection plug, inner surface modification of the capillary, kind of sulfated CD, hydrophobicity, and basicity of the analytes, etc. was investigated. It is gathered that different equilibrium constants of the strong binding basic analytes and highly sulfated CD complex in the organic phase of the injection plug and the aqueous electrolyte phase are resulting in two different mobility zones for each enantiomer. It follows that each enantiomer is showing two peaks instead of one. Experimental strategies are shown to avoid these peak splitting/artificial impurity effects and obtain the "real" chiral purity picture of the samples.     

毛细管电泳手性分离进展*

评述了近年来毛细管电泳手性分离的进展。以各种手性选择剂的发展为线索介绍了毛细管电泳手性分离理论、方法及应用。简要说明了分离中应注意的一些关键问题。     

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.     

Fast enantiomeric separation of basis drugs by electrokinetic chromatography. Application to the quantitation of terbutaline in a pharmaceutical preparation

Electrokinetic chromatography (EKC) using micelles of bile salts alone or mixed with sodium dodecyl sulfate (SDS) and neutral, anionic, or cationic cyclodextrins (CDs) in the separation buffer has been employed in order to achieve fast enantiomeric separation of basic drugs. A study of the enantiomeric separation ability of these chiral selectors concerning four basic drugs (epinephrine, terbutaline, clenbuterol, and salbutamol) has been carried out under different experimental conditions. The best chiral selectors to perform the enantiomeric separation of these drugs were neutral beta-CD derivatives, specifically permethylated beta-CD PM-beta-CD. The effect of the PM-beta-CD concentration, temperature, and applied voltage on the enantiomeric resolution of the basic drugs was investigated. The use of a 25 mM ammonium acetate buffer (pH 5.0), 30 mM in PM-beta-CD together with an applied voltage of 20 kV and a temperature of 15 degrees C enabled the individual and fast enantiomeric separation of epinephrine, norepinephrine, terbutaline, clenbuterol, and salbutamol each one into its two enantiomers in less than 3 min. The EKC method was validated (precision and accuracy) to quantitate terbutaline in a pharmaceutical preparation, obtaining a limit of detection of 4 microg/mL.     

Evaluation of the enantioseparation capability of the novel chiral selector clindamycin phosphate towards basic drugs by micellar electrokinetic chromatography

To date, a series of chiral selectors have been utilized successfully in capillary electrophoresis (CE). Among these various chiral selectors, macrocyclic antibiotics have been demonstrated to represent powerful enantioselectivity towards many chiral compounds. Differing from macrocyclic antibiotics, the use of lincosamide antibiotics as chiral selectors has not been reported previously. In our recent work, clindamycin phosphate belonging to the group of lincosamides has been first used as a chiral selector in capillary zone electrophoresis (CZE). In this paper, a micellar electrokinetic chromatography (MEKC) method has been developed for the evaluation of enantioseparation capability of this novel chiral selector towards several racemic basic drugs. As observed during the course of this work, clindamycin phosphate allowed excellent separation of the enantiomers of nefopam, citalopram, tryptophan, chlorphenamine, propranolol and metoprolol, as well as partial enantioresolution of tryptophan methyl ester and cetirizine. In this MEKC chiral separation system, different types of anionic surfactants, organic additives and background electrolytes were tested, and satisfactory enantioseparations of basic drugs above-mentioned were achieved using sodium dodecyl sulfate (SDS) as the surfactant, isopropanol as the organic additive, and phosphate as the background electrolyte. Furthermore, both migration times and enantioseparation of the analytes were influenced by several experimental parameters such as pH of the BGE, clindamycin phosphate and SDS concentrations, phosphate and isopropanol concentrations, and applied voltage. Consequently, the effects of these factors on enantioseparations of the studied basic drugs were systematically investigated in order to evaluate the stereoselectivity of clindamycin phosphate in MEKC.     

Enantioselective strong cation-exchange molecular recognition materials: design of novel chiral stationary phases and their application for enantioseparation of chiral bases by nonaqueous capillary electrochromatography

New chiral stationary phases derived from enantiomerically pure derivatives of cysteine carrying sulfonic acid groups are synthesized and evaluated for enantiomer separation of chiral bases by non aqueous capillary electrochromatography after bonding to a linker and grafting upon thiol-modified silica particles. Structural modifications of these low molecular weight chiral selectors are investigated and discussed in terms of apparent enantioselectivities and resolution factors based on the enantiomeric separations of a set of chiral bases including beta-blockers, beta-sympathomimetics and other basic drugs. The influence of the mobile phase constitution and its flow velocity on the enantioseparation by nonaqueous capillary electrochromatography is also briefly evaluated and discussed for the chiral substances investigated.     

胶束电动毛细管色谱法分析手性化合物

对近年来胶束电动毛细管色谱法（ＭＥＣＣ）分析手性化合物方面的工作进行了评述,简述了ＭＥＣＣ分离手性化合物的原理,并探讨了几种ＭＥＣＣ手性分离体系的分离机理。     

Enantiomer separation by capillary electrochromatography using fritless packed columns.

This review summarizes recent developments in the field of enantiomer separation by capillary electrochromatography using fritless packed columns. Various enantiomers have been separated by employing fritless packed columns prepared in a fused silica capillary either by the immobilization of chiral packing materials by sintering or sol-gel technology or by in situ polymerization of a mixture containing chiral selectors. The details of the column preparation procedures and the attainable column performance are described.     

Study of physically absorbed stationary phases for open tubullar capillary electrochromatography.

A novel method based on the adsorption of positively charged compounds on the wall of a fused-silica capillary was applied to prepare stationary phases for open tubular capillary electrochromatography (OTCEC). The positively charged substances including cationic surfactant such as cetyltrimethylammonium bromide (CTAB) and basic chiral selectors such as protein, peptide and amino acid were physically adsorbed onto the capillary wall under specially selected conditions. The adsorbed stationary phase of CTAB was used to separate neutral compounds, while the others were used for chiral separations. The run-to-run reproducibility of retention time was rather good with relative standard deviation (RSD) values of less than 2.3%. The separation efficiency was excellent with the highest theoretical plate number of up to 590000/m and the average one above 250000/m. Stored at 2-8 degrees C in the refrigerator, the adsorbed stationary phase can last at least one month. It was observed that the UV spectra for the enantiomers are significantly different due to the diastereomeric interactions of enantiomers with the chiral stationary phase in the detection window. With the use of the same capillary, the same instrument, and the same mobile phase, the superiority of OTCEC over open tubular liquid chromatography (OTLC) and capillary zone electrophoresis (CZE) was illustrated.     

Comparing cyclodextrin derivatives as chiral selectors for enantiomeric separation in capillary electrophoresis

A total of 26 different cyclodextrin (CD) derivatives with different functional groups and degrees of substitution were tested against 35 basic pharmaceutical compounds in an effort to investigate their effectiveness as chiral selectors for enantiomeric separation in capillary electrophoresis (CE). Testing was performed under the same conditions using a low pH buffer (25 mM phosphate buffer at pH approximately 2.5). Five CD derivatives, namely, highly sulfated-beta-CD, highly sulfated-beta-CD, hydroxypropyl-beta-CD (degree of substitution approximately 1), heptakis-(2,6-O-dimethyl)-beta-CD, and heptakis(2,3,6-O-trimethyl)-beta-CD were identified to be most effective for enantiomeric separations and have a wide range of enantiomeric selectivity towards the model compounds. Over 90% of the model compounds were enantiomerically resolved with the five identified CD derivatives, at a minimum resolution of 0.5. An additional 20 compounds were also tested to demonstrate the validity of the identified CD derivatives. The five CD derivatives were recommended as the starting chiral selectors in developing enantiomeric separation methods by CE.     

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.     

Development of stereoselective nonaqueous capillary electrophoresis system for the resolution of cationic and amphoteric analytes

A stereoselective ion-pair nonaqueous capillary electrophoresis (NACE) method employing the partial filling technique with N-derivatized amino acids, e.g., (R)- and (S)-3,5-dinitrobenzoyl-leucine (DNB-Leu), as chiral selector for the separation of "pseudoenantiomeric" cinchona alkaloid derivatives and other structurally related basic compounds like the enantiomers of mefloquine is presented. Originating from NACE with cinchona alkaloid derivatives as chiral counterions, this method was developed by application of the reciprocity principle of chiral recognition, which was proven to be valid for stereoselective ion-pair capillary electrophoresis (CE). A variety of basic and amphoteric selectands (SAs) could be well resolved. Thereby, the separation was primarily based on stereoselective ion-pair formation of corresponding SA stereoisomers and mobility differences of free and complexed (ion-paired) SAs. Additionally, in the case of diastereomeric SAs, naturally existing mobility differences between the diastereomers played also a role, but was shown by control experiments with racemic DNB-Leu and without selector (SO) to be of minor contribution to overall separation selectivity. Due to its simplicity, speed, and good reproducibility, the established method can be utilized for fast screening of cationic as well as amphoteric chiral compounds, and therefore is a valuable tool in the development of new chiral selectors and chiral stationary phases. Small sample amounts of the SO (4-5 mg) and only analytical amounts of SAs are needed, and about 20-50 compounds per day can be tested.