Ion-pair Capillary Electrophoresis Research Articles

Direct chiral resolution of tartaric acid by ion-pair capillary electrophoresis using an aqueous background electrolyte with (1R,2R)-(-)-1,2-diaminocyclohexane as a chiral counterion.

Chiral resolution of native DL-tartaric acid was achieved by ion-pair capillary electrophoresis (CE) using an aqueous-ethanol background electrolyte with (1R,2R)-(-)-1,2-diaminocyclohexane (R-DACH) as a chiral counterion. Factors affecting chiral resolution and migration time of tartaric acid were studied. By increasing the viscosity of the background electrolyte and the ion-pair formation, using organic solvents with a lower relative dielectric constant, resulted in a longer migration time. The optimum conditions for both high resolution and short migration time of tartaric acid were found to be a mixture of 65% v/v ethanol and 35% v/v aqueous solution containing 30 mM R-DACH and 75 mM phosphoric acid (pH 5.1) with an applied voltage of -30 kV at 25 degrees C, using direct detection at 200 nm. By using this system, the resolution (Rs) of racemic tartaric acid was approximately 1. The electrophoretic patterns of tartaric and malic acids suggest that two carboxyl groups and two hydroxyl groups of tartaric acid are associated with the enantioseparation of tartaric acid by the proposed CE method.

Optimization of the separation of β-blockers by ion-pair capillary electrophoresis in non-aqueous media using univariate and multivariate approaches

The separation of four β-Blocking drug substances (atenolol, sotalol, betaxolol, and metoprolol) selected as model basic analytes has been investigated in non-aqueous capillary electrophoresis (NACE) using the principle of ion-pair formation. Camphorsulphonate was selected as the counter-ion in a background electrolyte (BGE) made up of formate buffer in a mixture of acetonitrile/methanol or acetonitrile/ethanol. The influence on resolution of the concentration of the counter-ion, the nature and proportion of the organic solvents, as well as the concentration of the ionic components of the BGE was first studied by a univariate approach. An experimental design was then applied to estimate possible quadratic effects and first-order interactions. To identify the most important factors affecting the separation of the four β-blockers, a two-level fractional factorial design with 16 experimental points was applied as a screening test. A three factor Box-Behnken design with 12 experimental points was used to predict and optimize the selectivity. Finally the optimal conditions obtained by the univariate and the multivariate approaches were compared.

Electrolyte and additive effects on enantiomer separation of peptides by nonaqueous ion-pair capillary electrophoresis using tert.-butylcarbamoylquinine as chiral counterion.

Nonaqueous ion-pair capillary electrophoresis separations of N-protected (all-R)/(all-S) alanine peptide enantiomers with up to six amino acid residues using tert.-butylcarbamoylquinine as selector and employing the partial filling technique are presented. The effects of various conditional parameters on separation were studied, namely chemical nature of the capillary wall, solvent composition of the background electrolyte (BGE), acid-base-ratio (equivalent to apparent pH), ionic strength and selector concentration. The influence of the solvent composition (methanol-ethanol ratios) on resolution turned out to be rather complex. The separation of the peptide enantiomers was strongly altered by small changes in pH and ionic strength. An increase of the selector concentration was found to offer an easy way for enhancing enantioselectivity, although some drawbacks, e.g., elongation of run times, have to be considered. A method was developed that allowed the separation of N-3,5-dinitrobenzoyl oligoalanine enantiomers containing 1-6 amino acid residues in one run. Like in a recent high-performance liquid chromatography (HPLC) study, separation selectivity thereby decreased from 1.541 (Ala), 1.340 (Ala(2)), 1.054 (Ala(3)), 1.029 (Ala(4)), 1.024 (Ala(5)) to 1.020 (Ala(6)). In addition, all four stereoisomers of N-2,4-dinitrophenyl- and N-3,5-dinitrobenzyloxycarbonyl-protected alanylalanine could be baseline-resolved.

Evaluation of enantioselective nonaqueous ion-pair capillary electrophoresis as screening assay in the development of new ion exchange type chiral stationary phases

This study evaluates how enantioselectivity values for the separation of chiral N-derivatized amino acids achieved in non-aqueous ion-pair CE with cinchona alkaloid derived chiral counter-ions are related quantitatively to those obtained in enantioselective anion-exchange chromatography with the same selectors immobilized on silica supports. Three different setups of CE experiments, viz. a conventional total filling technique (TFT), a countercurrent technique (CCT), and a partial filling technique (PFT), were investigated and were tested in terms of the goodness of fit of correlations between apparent electrophoretic and chromatographic a values. Both selectands and selectors have been varied. Correlation coefficients higher than 0.95 are commonly obtained with all three CE techniques, yet even better fits are found for TFT-CE and CCT-CE. The common premise for the good correlations is the equal molecular recognition mechanism in ion-pair and ion-exchange modes, both involving ionic interactions as driving force for selectand-selector association, and the fact that separation selectivity in both techniques is primarily determined by the difference of the binding constants of (R) and (S) enantiomers with the chiral selector, i.e. the intrinsic or thermodynamic enantioselectivity. Factors related to the separation technique, which also influence apparent enantioselectivity and possibly contribute to a deterioration of the fit, are discussed. The good correlations prove that enantioselective ion-pair CE may be used as screening method in the development and optimization of ion-exchange type chiral stationary phases.

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.