Selectivity and resolution were studied for the separation of seven corticosteroids by micellar electrokinetic capillary chromatography (MEKC) using a mixed micellar solution of sodium dodecyl sulfate (SDS) and sodium cholate (SC), buffered with 3-(N-morpholino)propanesulfonic acid (MOPS) or 3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropane sulfonic acid (AMPSO). The changes in selectivity were compared for the AMPSO-SDS-SC system by varying the pH and the concentrations of AMPSO, SDS and SC. The experimental design started with the central composite design and continued in a sequential manner. The optimum selectivity for the separation of the corticosteroids was calculated from the analyte migration times and the analyte velocities, by using empirical quadratic regression models. Satisfactory regression fits and coefficients of determination for prediction were obtained with cross-validated models. To optimize the resolution, the physical parameters of capillary length and analysis time were varied under the conditions optimal for the selectivity. In both the selectivity and the resolution, optimization the overall optimum was determined by using the desirability function technique. Analysis times were controlled by using 1,3-diaminopropane to influence the electroosmotic flow velocity (veo). The voltage was kept constant, which resulted in higher electric field strength in shorter capillaries. No changes in the selectivity were observed when 1,3-diaminopropane was used to control the electroosmotic flow velocity. Such an optimization technique, where the chemical and physical factors affecting the separation are treated independently, seemed to be effective for finding the best possible resolution for the corticosteroids.
The corticosteroids studied can be effectively separated by employing micellar electrokinetic capillary chromatography (MECC). The effect of pH, borate concentration and the sodium dodecyl sulfate (SDS) concentration on both the resolution and the selectivity was studied under 15 different experimental conditions. The experimental design was similar to the central composite design approach. Empirical quadratic regression models were derived for analyte migration time, band broadening and analyte velocity. Satisfactory regression fits and coefficients of determination for prediction were obtained with cross-validated models. The models for analyte migration time and analyte velocity were in good agreement with theory. Modeling of the band broadening seemed to be somewhat more complicated. Optimum conditions for resolution and selectivity were different. This is due to the fact that selectivity studies ignore the electroosmotic and band broadening properties of different electrolyte solutions. However, the study of the selectivity yielded good information about the suitability of the electrolyte systems for the particular separation problem. Although a high solubilizing power of SDS caused the corticosteroids to partition strongly into the SDS micelles, a good separation could be achieved at low SDS concentrations.
Because of the different physiological impact that stereoisomers may have, it is often vital to separate these forms from one another. Because of their structural similarity, the separation is usually difficult to achieve and zones may elute very close to each other. This is a particular problem in capillary electrophoresis, where the repeatability of absolute migration times is fairly poor, mainly due to the irreproducibility of the electroosmotic flow. The separation is usually repeatable, however, and when the disturbing effects are eliminated by using a migration index system incorporating two marker compounds the identification of the enantiomers becomes extremely good. Relative standard deviation (RSD) values less than 0.1% for the migration index of each enantiomer were obtained in both intra-day and day-to-day (6 days) studies. The best separation was achieved with the electrolyte solution made of 40 mM borate, 32 mM sodium dodecyl sulfate (SDS), 12 mM beta-cyclodextrin (beta-CD), and 6 mM alpha-cyclodextrin (alpha-CD) at pH 9.3.
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