Recent innovations in enantiomer separation by electrochromatography utilizing modified cyclodextrins as stationary phases.

Enantiomer separation by electrochromatography employing modified cyclodextrins as stationary phases is performed in two ways. (i) Polysiloxane-linked permethylated beta-cyclodextrin (Chirasil-Dex 1) or related selectors are coated and immobilized onto the inner surface of a capillary column. Enantiomer separation is performed in the open tube and the method is referred to as open-tubular capillary electrochromatography (o-CEC). (ii) Silica-linked native beta-cyclodextrin, permethylated beta-cyclodextrin (Chira-Dex 2) or hydroxypropyl-beta-cyclodextrin are filled into a capillary column and the bed is secured by two frits. Enantiomer separation is performed in a packed column and the method is referred to as packed capillary electrochromatography (p-CEC). In a unified instrumental approach, method (i) as well as method (ii) can be operated both in the electro- and pressure-driven modes (o-CEC vs. open-tubular liquid chromatography (o-LC) and p-CEC vs. p-LC). It is demonstrated that the electro-driven variant affords higher efficiencies at comparable elution times. Employing a single open-tubular column coated with Chirasil-Dex 1, a unified enantioselective approach can be realized in which the same selectand is separated using all existing chromatographic modes for enantiomers, i.e., gas chromatography (GC), super-critical fluid chromatography (SFC), o-LC and o-CEC. As the chiral selector is utilized as a stationary phase, an additional chiral selector may be added to the mobile phase. In the resulting dual chiral recognition systems, enhancement of enantioselectivity (matched case) or compensation of enantioselectivity (mismatched case) may be observed. The overall enantioselectivity is dependent on the sense of enantioselectivity of the selectors chosen and their influence on the electrophoretic and electroosmotic migration of the enantiomers of a selectand.     

A validated gas chromatographic method for the evaluation of enzymatic enantioselectivity in kinetic resolution applications

An enantioselective gas chromatography (GC) method has been developed and validated for determination of the enantiomers of citronellol in kinetic resolution experiments. S-(-)-beta-Citronellol is a precursor of rose oxide. After solid-phase extraction (SPE) with ethyl acetate, the enantiomers of R-(+)-beta-citronellol and S-(-)-beta-citronellol and their corresponding acetate- and butyrate esters were separated through enantioselective GC respectively. The method was validated and found to be reproducible, specific, accurate, and precise. Analyte recoveries and detection limits were also determined. The applicability of this method was shown in a kinetic resolution experiment using lipase A of Bacillus subtilis.     

Fundamental aspects of chiral separations by capillary electrophoresis

A review is presented that surveys the basic theory of direct separation of enantiomers by capillary electrophoretic (CE) techniques. These separations are based on the formation of diastereomeric complexes between the enantiomeric analytes and a chiral selector added to the electrolyte solution. The review covers a comprehensive treatment of the equations needed for optimization of selectivity coefficients, resolution and analysis time in the zone electrophoretic mode. In this context, it takes into account combined equilibria of complexation and protonation/deprotonation as well as complexation and paritition into micelles. On the basis of these equations, the benefits of charged selectors and the optimization potential inherent to pH tuning can be documented. In addition, the review deals with some basic aspects of chiral isoelectric focusing and briefly discusses indirect enantioseparation. In a subsequent section a survey is given on particularfeatures of the various types of chiral selectors. Finally, the recent developments in preparative enantioseparation in continuous free-flow system and by use of isoelectric membranes are discussed.     

pKa shift-associated effects in enantioseparations by cyclodextrin-mediated capillary zone electrophoresis.

Enantioselective migration of dansylated (Dns) amino acids in the presence of hydroxypropylated-beta-cyclodextrin under acidic conditions near the pI value of the analytes was investigated by means of capillary zone electrophoresis. Based on the migration data, the pH dependence of the complexation constants was evaluated, as well as the variation of the complex mobilities with pH. As a result of these data, the migration behavior in the pH region near the pI could be understood, which, in some instances, includes the reversal of migration order upon variation of selector concentration. The enantioselective pKa shifts upon complexation could be quantitated for the carboxylic and the amino group separately. pKa shifts were found in the order of 0.8 pI units, the differences between the enantiomers being up to 0.25 pH units. These data were in agreement with the pI shifts reported from isoelectric focusing experiments. The accurate determination of the pI values of the Dns amino acids makes it possible to calibrate the pI scale in isoelectric focusing in the presence of chiral selectors.     

Enantiomeric separations of ruthenium(II) polypyridyl complexes using high-performance liquid chromatography (HPLC) with cyclodextrin chiral stationary phases (CSPs)

Rapid, highly efficient, analytical resolution of the enantiomers of eight different monomeric ruthenium(II) polypyridyl complexes has been achieved using HPLC with cyclodextrin chiral stationary phases. This technique also proved capable of separating both of the diastereomers and the enantiomers of one dinuclear complex in a single run, whereas similar efforts with another dinuclear complex gave only one stereoisomer cleanly. Factors such as the stereochemistry of the chiral selectors, solvent polarity, and salt effects can be altered to provide precise control of the enantioselective interactions. The ability to quickly and quantitatively determine the enantiopurity of a given ruthenium complex allowed facile reexamination and optimization of the commonly used bulk resolution procedures based on diastereomeric coprecipitation with sodium arsenyl (+)-tartrate or sodium arsenyl (-)-tartrate salts.     

Enantioselective analysis of ofloxacin enantiomers by partial-filling capillary electrophoresis with bacteria as chiral selectors

The enantiomeric separation of ofloxacin enantiomers (OFLX) was achieved by using capillary electrophoresis partial-filled with Escherichia coli, Pseudomonas aeruginosa (Gram-negative), and Staphylococcus aureus (Gram-positive) as chiral selectors. Experimental parameters, including the concentration of background electrolyte, applied voltage, length of the filled bacteria plug, and pH of the buffer, were intensively investigated. Baseline separation of OFLX could be achieved within 7 min by using E. coli and P. aeruginosa as chiral selectors under the following conditions: electrophoretic buffer composed of 10 mM phosphate buffer at pH 7.4, applied voltage at 15 kV, and the bacteria (6.0 × 10(8) cells/mL) were injected into the capillary by gravity with injection height of 17.5 cm for 180 s (E. coli), 300 s (P. aeruginosa), and 300 s (S. aureus), respectively. E. coli and P. aeruginosa had better chiral selectivity for OFLX than S. aureus, which was in good agreement with OFLX having better antimicrobial activity on Gram-negative rather than Gram-positive bacteria. A novel method was developed for the enantioselective separation of enantiomers using bacteria as chiral selectors, which provides a new approach for antimicrobials enantioselective analysis, chiral pharmacodynamics, and chiral pharmacokinetics studies.     

Modulating the Enantiodiscrimination Features of Inherently Chiral Selectors by Molecular Design: A HPLC and Voltammetry Study Case with Atropisomeric 2,2’-Biindole-Based Monomers and Oligomer Films

A family of inherently chiral electroactive selectors based on the 2,2’-biindole atropisomeric scaffold, of easy synthesis and modulable functional properties, is studied in cascade in two enantioselection contexts. They are at first investigated as probes in enantioselective HPLC, studying molecular structure and temperature effects, and achieving very efficient semipreparative enantioseparation. The enantiomers thus obtained, of remarkable chiroptical features (optical rotation as well as circular dichroism), are successfully applied as selectors in chiral voltammetry in different media for discrimination of the enantiomers of chiral electroactive probes, either by conversion into enantiopure electroactive electrode surfaces by electrooligomerization on glassy carbon substrate (the two monomers with shorter alkyl chains), or as chiral additive in achiral ionic liquid (the monomer with longest alkyl chains). Discrimination is conveniently and reproducibly achieved in terms of significant potential differences for the two enantiomers, specularly inverting either probe or selector configuration. In one case successful discrimination is also observed with the two probe enantiomers concurrently present, either as racemate or with enantiomeric excesses, neatly accounted for by the peak current ratios.     

Enantioseparation of derivatized amino acids by capillary isoelectric focusing using cyclodextrin complexation

Enantioseparation of dansylated as well as 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC)-derivatized amino acids by means of capillary isoelectric focusing using various cyclodextrin derivatives is demonstrated. Separation is based on the enantioselective shift of the isoelectric points upon complexation with the chiral selectors. The zwitterionic, diastereomeric analyte-cyclodextrin complexes exhibited differences in the pI values up to more than 0.25 pI units. Enantioresolution was achieved for a number of derivatized amino acids and various selectors added to the carrier ampholyte solution. The hydroxypropyl-beta-cyclodextrin proved to be the best selector for this purpose. Enantioseparation as dependent on the selector concentration was evaluated in a range between 5 and 30 mM. Separation could be attained down to selector concentrations corresponding to a degree of complexation as low as 30%. The peaks appear according to the degree of complexation between the positions adopted without and with full complexation. The kinetics of complex formation and dissociation was fast enough in most instances to produce single peaks, even with complexation degrees near 0.5 and significant pI shifts. Peak widths were slightly enlarged in these instances. The method offers excellent perspectives for preparative applications.     

α－溴丙酸甲酯与β－环糊精衍生物手性识别过程的模 拟研究

应用分子动力学方法,模拟研究了α－溴丙酸甲酯分别与全甲基－β－环糊精(PMBCD)、七(2,6-二-O-丁基-3-O-丁酰基)-β－环糊精(DBBBCD)的手性识别过程。结果表明,α－溴丙酸甲酯对映体与PMBCD,DBBBCD的优先结合点位于环糊精空腔的内部,其对映体与所述环糊精的手性识别机理和形成腔内结合物有关;对映体在环糊精空腔内的结合并非传统意义上的紧密包合,对映体在腔内可以上下运动和转动;从对平衡构象的结构分析发现,所述对映体与环糊精衍生物的手性识别与葡萄糖单元的C(2),C(3)所提供的手性环境密切相关。而且,分子模拟方法得到的对映体保留顺序与气相色谱实验结果是一致的。     

Enantioselective Voltammetric Sensors: New Solutions

A review of new approaches and solutions in the development and application of enantioselective voltammetric sensors for the recognition of optical isomers of biologically active compounds and medicines is presented. The main methods of electrode modification by enantioselective selectors are discussed, i.e., the application of inclusion complexes, molecularly imprinted polymers, elements of living systems and their analogs, inorganic and organic materials with the effect of chirality, and also supramolecular structures. The main analytical characteristics of some sensors and sensor systems of the electronic tongue type for the recognition and determination of enantiomers in various samples are presented. Methods of processing of voltammetric data for the elimination of the effect the memory of measurements and the cleanup of analytical signals at low concentrations of enantiomers are considered.     

Enantioselective determination of the organochlorine pesticide bromocyclen in spiked fish tissue using solid-phase microextraction coupled to gas chromatography with ECD and ICP-MS detection

A method for enantioselective determination of bromocyclen enantiomers in fish tissue has been developed. The enantiomers were resolved by capillary gas chromatography (GC) using a commercial chiral column (CP-Chirasil-Dex CB) and a temperature program from 50 degrees C (held for 1 min), raised to 140 degrees C at 40 degrees C min(-1) and then raised at 0.2 degrees C min(-1) to 155 degrees C. This enantioselective gas chromatographic separation was combined with a clean-up/enrichment procedure based on solid-phase microextraction (SPME). Under SPME optimized conditions, precision, linearity range and detection limits of the developed SPME-enantioselective GC procedure were evaluated and compared using two different detection systems: a classical electron-capture detection (ECD) and an element specific detection using inductively coupled plasma mass spectrometry (ICP-MS). The SPME-GC-ECD method exhibited an excellent sensitivity, with detection limits of 0.2 ng L(-1) for each enantiomer of bromocyclen. Although ICP-MS offered poorer detection limits (7 ng L(-1) as Br, equivalent to 36 ng L(-1) of each enantiomer) than conventional ECD detector, it proved to be clearly superior in terms of selectivity. The relative potential and performance of the two compared methods for real-life analysis has been illustrated by the determination of enantiomers of bromocyclen in spiked tissue extracts of trout.     

Separation of enantiomers: needs, challenges, perspectives

Chiral drugs, agrochemicals, food additives and fragrances represent classes of compounds with high economic and scientific potential. First the present implications of their chiral nature and necessity of separating enantiomers are summarised in this article. In the following a brief overview of the actual approaches to perform enantioseparations at analytical and preparative scale is given. Challenging aspects of these strategies, such as problems associated with data management, choice of suitable chiral selectors for given enantioseparations and enhanced understanding of the underlying chiral recognition principles, are discussed. Alternatives capable of meeting the requirements of industrial processes, in terms of productivity, cost-effectiveness and environmental issues (e.g., enantioselective membranes) are critically reviewed. The impact of combinatorial methodologies on faster and more effective development and optimisation of novel chiral selectors is outlined. Finally, the merits and limitations of most recent trends in discrimination of enantiomers, including advances in the fields of sensors, microanalysis systems, chiroptical methods and chemical force microscopy are evaluated.     

Synthesis and Applications of Functionalized Magnetic Nanomaterials in Enantioseparation

Efficient enantioselective separation is a challenging task due to the identical physical and chemical properties of enantiomers. Functionalized magnetic nanomaterials modified with chiral ligands on their surface possess both magnetic property and chiral recognition ability, and have demonstrated great potential in chiral discrimination. This review summarizes the applications of magnetic nanomaterials modified with various chiral selectors (e.g., β-cyclodextrins, polymers, proteins, amino acids and cellulose) in enantioselective separation. After proper preparation and modification, these functionalized magnetic nanomaterials are effective for enantioseparation. Therefore, enantioseparations based on functionalized magnetic nanomaterials are convenient, economical and effective.     

The effect of conductivity tuning in chiral separations by CE; Using hydroxypropyl-β-cyclodextrin in combination with tetraalkylammonium ions

Summary Different approaches how to handle the electromigration dispersion process that occurs in separation and determination of enantiomers are presented. The use of cyclodextrins as chiral selectors in resolution enantiomers involves the possibility to tune the conductivity of the sample band in order to obtain symmetrical and efficient peaks. Determination of impurities that migrate in the rear part of an overloaded main peak can be accomplished if the conductivity of the background electrolyte (BGE) is adapted to the conductivity of the sample band. This strategy was shown in determination of the content of D-sotalol in a mixture of L and D-sotalol. The efficiency and the symmetry of the overloaded L-sotalol peak was substantially improved by substitution of tetrabutylammonium ions for tetrapentylammonium ions as co-ions in the BGE. In this system it was possible to determine 0.2% w/w of the chiral impurity D-sotalol. A resolution model is presented and used qualitatively in the study where the complexation between the tetraalkyllammonium ions and the cyclodextrins is taken into account.     

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.     

On the use of solid phase ion exchangers for isolation of amino acids from liquid samples and their enantioselective gas chromatographic analysis

This work focuses on the development of a suitable working procedure for preconcentration of amino acids enantiomers from water samples using a solid phase extraction. The three types of ion exchangers with various capabilities have been used. The effect of experimental conditions in SPE procedure employing strong anion exchange (SAX), weak (WCX) and strong cation exchange (SCX) cartridges (such as sample volume, pH, origin of elution solvent and its volume) on effective preconcentration of the model set of amino acids has been studied in detail. The enantiomers of isolated and preconcentrated amino acids have been analysed by GC on three capillary columns coated with chiral selectors. The different amino acids derivatives have been investigated in order to achieve optimal resolution of biogenic amino acids and their enantiomers. The best separation of amino acid enantiomers has been obtained on a Chirasil-L-Val column analysing their N-TFA methyl esters. It has been shown that SCX-SPE cartridge with sulfonic groups attached on silicagel support is most suitable for isolation and preconcentration of amino acids from water samples. For this sample treatment procedure, the overall recovery of extraction process has been calculated as an average value from three measurements. It has been found, that recoveries are practically identical for both enantiomers of a particular amino acid and varies in the range 75-99% depending on the type of amino acid. The effectivity of this sample preparation and GC method has been verified by preconcentration of amino acids from orange juice fortified by racemic mixture of some selected amino acids.     

Enantioselective trace analysis of amphetamine in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry

A method for the quantitative enantioselective analysis of amphetamine in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry (GC/NICI-MS) is presented. Five-fold deuterated analogues of both enantiomers were used as internal standard. Plasma sample preparation was performed by a rapid liquid-liquid extraction using n-hexane. Derivatization with (S)-(-)-N-(heptafluorobutyryl)prolyl chloride was accomplished directly in the n-hexane extract to avoid loss of amphetamine during sample concentration. The method was validated in the expected concentration range of 0.006 for a pharmacokinetic study. Calibration curves were linear within a range 0.006-50 ng/mL plasma. Precision and accuracy were acceptable over the entire calibration range. Baseline separation of the enantiomers was easily achieved on a 15-m nonchiral apolar column. The method is simple and robust, and has been applied to the batch analysis of amphetamine enantiomers.     

Novel chiral selectors anchored on polydimethylsiloxane as stationary phases for separation of derivatized amino acid enantiomers by capillary gas chromatography

Six chiral selectors of S-(-)-t-Leu-cyclopropylamide, S-(-)-t-Leu-cyclopentylamide, S-(-)-t-Leu-cyclohexylamide, S-(-)-t-Leu-cycloheptylamide, S-(-)-t-Leu-cyclooctylamide, S-(-)-t-Leu-cyclododecylamide have been prepared and anchored individually through amide bonding to a polydimethylsiloxane functionalized with 2,2,2-trifluoroethyl ester groups by way of nucleophilic displacement reaction. The resulting chiral polysiloxanes have been provided as stationary phases for the separation of amino acid enantiomers by capillary GC. Amino acids were derivatized into N(O)-trifluoroacetyl isopropyl esters. Especially, polydimethylsiloxane anchored with S-(-)-t-Leu-cyclooctylamide was found to be efficient for the separation of aspartic acid (Asp) enantiomers. The method was applied to the estimation of ages from the extent of Asp racemization in human dentines.     

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.     

Dynamics of interconversion of enantiomers in chiral separation systems: a novel approach for determination of all rate constants involved in the interconversion

When enantiomers separated by chromatography or capillary electrophoresis undergo interconversion reaction (enantiomerization) during the separation, it leads to a typical detection pattern: two individual peaks of the separated enantiomers are connected with a plateau consisting of a mixture of both separated enantiomers. We propose a separation method for determination of all individual rate constants (or inversion barriers) of the interconversion. The method enables to distinguish which part of interconversion takes place in the free (unbound) form of the analyte and which part in the complexed (bound) form. Further, we propose a complete dynamic model of capillary electrophoresis of interconverting enantiomers based on solving a complete set of continuity equations for all constituents of the separation system together with complexation and acid-base equilibria. This allows a simulation of both linear and nonlinear mode of separation and understanding all processes taking place in such enantioseparation systems. We demonstrate the applicability of the method on determination of the rate constants of interconversion of oxazepam enantiomers separated in systems with charged cyclodextrin chiral selectors.