Enantioseparation by chromatographic and electromigration techniques using ligand-exchange as chiral separation principle

This article gives a short overview of the application of the principle of chiral ligand-exchange in HPLC, CE, and CEC. Since its introduction by Davankov, more than thousand articles have appeared in this field. Citing all these papers would extend the scope of this review—it would fill several books. Therefore only some milestones are mentioned in this article and it will focus on our own activities in this field. Some new developments are mentioned, and selected biochemical and biomedical application are presented.     

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.     

Investigation of enantiomeric separation of basic drugs by capillary electrophoresis using clindamycin phosphate as a novel chiral selector

A wide number of chiral selectors have been employed in CE and among them macrocyclic antibiotics exhibited excellent enantioselective properties toward plenteous racemic drugs. Different from macrocyclic antibiotics, the use of lincomycin antibiotics as chiral selectors has not been reported previously. In this study clindamycin phosphate belonging to the group of lincomycin antibiotics is first used as a novel chiral selector for the enantiomeric separations of several racemic basic drugs, which possess high separability, consisting of nefopam, citalopram, tryptophan, chlorphenamine and propranolol. Other basic drugs giving partial enantioseparation include tryptophan methyl ester, metoprolol and atenolol. Clindamycin phosphate possesses advantages such as high solubility and low viscosity in the water and very weak UV absorption. In the course of this work we observed that both migration time and enantioseparation were influenced by several parameters such as pH of the BGE, clindamycin concentration, capillary temperature, applied voltage and organic modifier. The optimum pH that was in the neutral or weak basic region but varied among drugs, a low capillary temperature and a clindamycin concentration of 60 or 80 mM are recommended as the optimum conditions for chiral separation of these drugs. Moreover, comparison of the influences of the studied parameters was further investigated by means of Statistical Product and Service Solutions in this paper.     

Evaluation of carbon nanostructures as chiral selectors for direct enantiomeric separation of ephedrines by EKC

Single-walled nanotubes and multi-walled nanotubes (MWNTs) have been evaluated as chiral selectors for the enantiomeric separation of ephedrines by using EKC with surfactant-coated carbon nanotubes. The analysed compounds were (+/-)-ephedrine, (+/-)-norephedrine and (+/-)-N-methylephedrine. The potential of those carbon nanostructures as chiral selectors has been evaluated by changing different experimental variables such as pH, addition of organic modifiers, potential and injection time. The capability of MWNTs to resolve enantiomeric mixtures was demonstrated by using partial filling of the capillary with concentrated surfactant-coated MWNTs. Differences in the enantioselectivity were discussed.     

Evaluation of clarithromycin lactobionate as a novel chiral selector for enantiomeric separation of basic drugs in capillary electrophoresis

Nowadays, macrocyclic antibiotics are presenting an increasing number of enantioseparation applications. The macrocyclic antibiotics used as chiral selectors in capillary electrophoresis (CE) include the ansamycins and the glycopeptides. The macrolides, another important class of macrocyclic antibiotics, have been reported as a new type of chiral selectors recently. In this study, clarithromycin lactobionate (CL), belonging to the group of macrolide antibiotics, was first investigated for its potential as a novel chiral selector in CE for enantiomeric separation of several basic drugs. As observed, CL allowed excellent separation of the enantiomers of metoprolol, atenolol, propranolol, bisoprolol, esmolol, ritodrine, and amlodipine, as well as partial enantioresolution of labetalol and nefopam. In addition, CL possesses advantages such as high solubility and low viscosity in the solvent and very weak UV absorption. In the course of this study, it was found that both migration times and enantioseparation of the basic drugs were influenced by several experimental parameters, e.g. selector concentration, the composition and pH of the BGE, the type and concentration of organic modifier, and applied voltage. Thus, the effects of these factors were systematically investigated, and satisfactory enantioseparations of the studied drugs were achieved at the buffer pH range of 7.3–7.5 using 12.5 mM borax buffer with 50% v/v methanol, 60 mM CL, and 20 kV applied voltage. Moreover, comparison of the influences of the studied parameters was further investigated by means of Statistical Product and Service Solutions (SPSS) in this article.     

Advances in chiral separation using capillary electromigration techniques

This review gives an overview of recent developments in CZE, EKC, and CEC covering the literature since the year 2004. Since there appeared a special issue on applications, this review focuses on the progress in electromigration techniques and new methodological developments. New techniques, new chiral selectors as well as new chiral stationary phases for CEC are discussed.     

Dispersive phenomena in electromigration separation methods

A review on dispersive effects and on peak broadening in electromigration separation methods (capillary electrophoresis and electrochromatography) is presented, mainly covering papers published between the beginning of 1997 and the beginning of 2000. Most attention is drawn to work dealing with nonlinear effects that cause anomalous electromigration dispersion in electrolyte systems with two or multiple coions. Further, topics cover the comparison of electroosmotic and pressure-driven modes in electrochromatography, dispersive effects due to nonhomogeneous velocity fields in packed electrochromatography columns, to nonuniform electroosmotic flow, to sorption of analytes (mainly proteins) at the column wall or the stationary phase, and due to the influence of the nonideal column geometry like coiling or irregularities in shape.     

Synthesis and application of dehydroabietylisothiocyante as a new chiral derivatizing agent for the enantiomeric separation of chiral compounds by capillary electrophoretic

A new chiral derivatizing reagent, dehydroabietylisothiocyante (DHAIC), was synthesized and used for the enantiomeric separation of chiral compounds in capillary electrophoresis (CE). The synthetic route to obtain DHAIC is described. The separation conditions for the chiral separation of several chiral compounds, such as protein amino acids and chiral drug DOPA were optimized. Best results for the chiral separation of DHAIC derivatized amino acids and DOPA were obtained in a running buffer consisted of 50 mM borate (pH 9.5), 5 mM sodium dodecyl sulphate (SDS) and 20% acetonitrile for amino acids and 60 mM Na₂HPO₄ (pH 8.0), 17 mM SDS and 25% acetonitrile for DOPA. Under the conditions studied, chiral separation of five amino acids including Ser, Val, Ala, Thr, Cys and a chiral drug DOPA as their diastereomeric DHAIC derivatives has been achieved by micellar electrokinetic chromatography (MEKC).     

Vancomycin degradation products as potential chiral selectors in enantiomeric separation of racemic compounds

This review discusses the use of vancomycin-related substances as potential chiral stationary phase that can be used as a packing material for the enantioselective separation of various racemic compounds in various modalities including HPLC, CE and also as chiral mobile phase additives. The chiral recognition mechanisms involved including the role of dimerization are presented.     

Synthesis of chemically bonded cellulose trisphenylcarbamate chiral stationary phases for enantiomeric separation

Cellulose trisphenylcarbamate is regioselectively bonded to 3-aminopropyl silica gel and underivatized silica gel, respectively, at the 6-position of the primary hydroxyl group on the glucose unit of cellulose with 4,4'-diphenylmethane diisocyanate (DPDI) as a spacer. Enantioseparations are evaluated on these prepared chiral stationary phases (CSPs) with several organic acids as the modifiers in the mobile phase by high-performance liquid chromatography. The influence of the amount of DPDI used on chiral resolution is investigated. Also, the corresponding coated-type phase is also prepared for the aim of comparison. It is observed that the bonded-type phase shows a lower chiral recognition power but a better column efficiency than the coated-type phase under the liquid chromatographic mobile phase with hexane-alcohol. However, the bonded-type CSPs are compatible with a wider number of solvents such as tetrahydrofuran (THF) or chloroform, which generally result in the solubility or swelling of the cellulose derivatives on the coated-type CSPs. The results obtained from this study indicate that the bonded-type CSP may provide complementary enantioselectivity over the coated-type phase by adopting THF as a component in the mobile phase.     

Degradingdehydroabietylisothiocyanate as a chiral derivatizing reagent for enantiomeric separations by capillary electrophoresis

Abietic acid is a naturally occurring enantiomeric diterpenic acid. Its absolute optical purity and very stable stereochemistry structure makes it an excellent starting material for preparing chiral derivatizing reagents for chromatographic or electrophoretic applications. This paper describes the synthesis and evaluation of a novel chiral derivatization reagent, i.e., degradingdehydroabietylisothiocyanate (DDHAIC) derived from dehydroabietic acid. Its applicability for the enantioseparation of racemic amino acids by CE was demonstrated. DDHAIC reacted readily with amino acids at an elevated temperature (70 degrees C). The resulting derivatives were highly stable and separable by MEKC. Separation of amino acid-DDHAIC diastereomers was achieved with a running buffer consisting of 50 mM Na(2)HPO(4) (pH 9.0), 18 mM SDS, and 25% v/v ACN. Under the conditions selected, diastereomers formed from ten pairs of tested amino acid enantiomers including D/L-Asn, D/L-Met, D/L-Leu, D/L-Phe, D/L-Trp, D/L-Ser, D/L-Val, D/L-Ala, D/L-Thr, and R/S-vigabatrin were well resolved. The resolution values were in the range of 0.95-8.9.     

Histamine-modified cationic beta-cyclodextrins as chiral selectors for the enantiomeric separation of hydroxy acids and carboxylic acids by capillary electrophoresis.

The enantiomeric separation of alpha-hydroxy acids and carboxylic acids was successfully performed by using 6-deoxy-6-N-histamino-beta-cyclodextrin (CD-hm), a monosubstituted positively charged beta-cyclodextrin (beta-CD) bearing a histamine moiety linked to the C6 of a glucose unit in the upper CD rim via the amino group. Good results were obtained at a low selector concentration (1 mM). The number of positive charges on the upper rim may be modulated as a function of pH, because of the different pKa of the amino and the imidazolyl groups, and was found to affect both the enantioselectivity and resolution factors. With the analogous 6-deoxy-[4-(2-aminoethyl)imidazolyl]-beta-cyclodextrin (CD-mh) bearing the histamine moiety linked to the C6 via the imidazolyl group, very poor results were obtained, showing that the proximity of the positive charge to the cavity plays an important role in the enantiomeric recognition. The complexation mode was studied by electrospray ionization-mass spectrometry (ESI-MS) and two-dimensional nuclear magnetic resonance (2-D NMR) ROESY experiments: the recognition model is consistent with an inclusion complexation of the aromatic ring of the analyte within the CD cavity coupled to electrostatic interactions between the carboxylate and the protonated amino group of the cyclodextrin.     

Use of polystyrene nanoparticles to enhance enantiomeric separation of propranolol by capillary electrophoresis with Hp-beta-CD as chiral selector

We describe the use of polystyrene (PS) nanoparticles to manipulate chiral selectivity of propranolol analysis by capillary electrophoresis, by dispersing PS nanoparticles into the run buffer employing hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector. Distinct separational differences are observed between the buffer containing PS nanoparticles and buffer without, when changing separating conditions including PS nanoparticles concentration, pH, buffer concentration, HP-β-CD concentration and when adding an organic additive. Selectivity improvements are reflected by changes in the observed mobility as a result of interactions between the propranolol enantiomers and HP-β-CD governing the absorption process on the PS particles surface. The presence of PS nanoparticles increases the enantioseparation at low particle concentration in the presence of HP-β-CD as a chiral selector.     

A validated chiral LC method for the enantiomeric separation of AT13148 on polysaccharide-based chiral stationary phase

A sensitive and accurate LC method for the determination of AT13148 enantiomeric purity has been developed and validated. Baseline separation with a resolution higher than 1.8 was accomplished within 15 min using a Chiralpak AD-H column (250 × 4.6 mm; particle size 5 μm) and n-hexane: 2-propanol: diethylamine (85:15:0.1, v/v) as mobile phase at a flow rate of 1 mL min^?1. Eluted analytes were monitored by UV absorption at 254 nm. The effects of mobile phase components, temperature and flow rate on enantiomeric selectivity and resolution of enantiomers were investigated. Calibration curves were plotted within the concentration range between 7 and 500 μg mL^?1 (n = 11), and the recoveries between 98.24 and 100.99% were obtained, with relative standard deviation lower than 1.32%. LOD and LOQ for AT13148 were 2.46 and 7.38 μg mL^?1 and for its enantiomer were 2.54 and 7.49 μg mL^?1, respectively. It was demonstrated that the developed method was accurate, robust and sensitive for the determination of enantiomeric purity of AT13148, especially for the analysis of bulk samples.     

Computational chemistry applied to the design of chiral stationary phases for enantiomeric separation

A general computational scheme for the rational design of chiral stationary phases for the chromatographic separation of enantiomers has been established. The developed scheme was based on applying different interaction models (force field methods versus semi empirical quantum chemical methods), different docking algorithms (systematic grid search methods versus interactive methods guided by rules based on binding modes) and different levels of approximations (rigid versus flexible docking) to a representative test problem containing the 3,5-dinitrobenzoyl group. The computational methods in use covered the most sophisticated methods which could presently be applied to problems of such a size (about 80 atoms). It has been shown that the current computational approaches using rigid body approximations for the docked molecules and simple molecular mechanics (not taking pi-“effects” into account) are invalid in view of the required predictive precision of about 1–2 Kcal/mole for the differential binding energy. Another surprising result was the failure of the commonly used systematic search methods in determining the most favorable binding modes. Based on our calculations on the representative test problem we propose a new arrangement for the most stable complexes without parallel stacking of the aromatic pi-donor and the 3,5-dinitrobenzoyl pi-acceptor systems.     

On-line enantiomeric analysis using high-performance liquid chromatography in chiral separation by simulated moving bed

An automated on-line enantiomeric analysis system comprising an analytical HPLC set-up with two UV detectors sharing the same light source has been employed to monitor the internal composition profile in chiral simulated moving bed chromatography. This monitoring scheme does not use a polarimeter. Using a sampling interface placed between two SMB columns, effluent samples are directed onto a high-efficiency analytical column at a sampling rate faster than the overall dynamics of the preparative unit to achieve on-line enantiomeric analysis of the composition profile. The other UV detector is placed in the SMB loop before the fraction collector to provide instantaneous measurement of the total enantiomeric concentration. The feasibility and effectiveness of the on-line enantiomeric monitoring scheme were assessed experimentally on the separation of Tr?ger's base racemate, using Chiralpak AD as stationary phase and methanol as eluent. It was found that robust monitoring of the concentration profiles of the individual enantiomers is best achieved when the enantiomeric purity obtained from the peak areas of the on-line enantiomer analysis chromatograms is combined with the on-line UV measurement of total enantiomeric concentration. The accuracy and robustness of the proposed on-line enantiomeric monitoring system open up promising perspectives for process control and dynamic optimization of the SMB.     

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.     

Recent developments in the HPLC enantiomeric separation using chiral selectors identified by a combinatorial strategy

Two kinds of chiral stationary phases (CSPs) can be distinguished: (i) the "conventional" CSPs for which the selectivity is not pre-determined and (ii) the CSPs which are characterized by a predictable elution order, depending on the target enantiomer used for the selection of the chiral selector. At the present time, three general methodologies have been described to create chiral selectors specifically designed against the racemate to resolve: the molecular imprinting technology, the production of antibodies and the combinatorial approach. The latter methodology involves two categories of procedures to develop CSPs: an approach from a small library of low-molecular weight selectors and an approach from a very large library of single-stranded oligonucleotides (DNA and RNA aptamers). In this review, the recent advances in the HPLC applications of the chiral selectors identified through these two combinatorial procedures are addressed.     

Cyclodextrins as chiral selectors in capillary electrophoresis: a comparative study for the enantiomeric separation of some beta-agonists.

A comparative study for the enantiomeric separation of terbutaline, clenbuterol, salbutamol and dobutamine was performed by capillary electrophoresis using cyclodextrins and their derivatives as chiral selectors. Several parameters such as buffer composition and temperature were studied. Simple, fast and reliable enantioseparations were achieved for all drugs studied, especially when the isomerically pure sulfated beta-cyclodextrin derivatives were used as chiral selectors.