The influence of buffering substances and urea on the beta-CD-mediated chiral separations of the dipeptides Ala-Phe and Ala-Tyr was studied in the pH range of 2.5-3.8. Only minor effects of the buffer substances on the chiral separation selectivity alpha were observed at a beta-CD concentration of 15 mg/mL. In contrast, the selectivity improved at pH 2.5 but decreased at pH 3.8 upon the addition of 2 M urea. Complexation by beta-CD resulted in a shift of the pK(a) values toward higher values which was more pronounced for the DD-enantiomers of both dipeptides than for the LL-enantiomers. Addition of urea further increased the pK(a) shift. The consequence of this pK(a) shift is an increase of the fraction of the protonated, positively charged form of the peptides which explained the improved chiral separation at pH 2.5 and the reduced selectivity at pH 3.8. A pK(a) shift by the addition of urea was also observed for N-tert-butyloxycarbonyl phenylalanine (BOC-Phe) as a model compound that is strongly complexed by beta-CD. This effect was not stereospecific. Addition of urea resulted in a decrease of the apparent complexation constants between beta-CD and the BOC-Phe enantiomers to the same extent but this did not affect the separation selectivity alpha. For chiral separations that display strong pH dependence such as peptide enantioseparations close to the pK(a) values of the compounds, urea may not solely be regarded as a solubility enhancer for beta-CD but may also influence the separation.

A separation selectivity model for capillary electrophoresis enantioseparations of weak bases in the presence of uncharged chiral selectors was described as a function of buffer pH and chiral selector concentration. On the basis of the selectivity at the extreme pH and selector concentration values, 15 principal cases could be distinguished describing the migration behavior of the analytes when increasing either pH or selector concentration. A pH-dependent reversal of the enantiomer migration order can be observed (1) when the complex mobility ratio is reversed due to an enantioselective complexation-induced pK(a)-shift, (2) in the case of an inversion of the ratio of the mobilities of the analyte-selector complex and the free analyte due to significantly weaker complexation of the neutral species, and (3) in the case of opposite chiral recognition of the protonated and uncharged species by the chiral selector. Reversal of enantiomer migration order as a function of the selector concentration at a fixed buffer pH is caused by the opposing effects of complexation constants and complex mobilities at the respective pH value. The model was applied to rationalize the enantioseparations of dipeptides, especially the pH-dependent reversal of the migration order of the LL- and DD-enantiomers of Ala-Tyr in the presence of heptakis-(2,6-di-O-methyl)-beta-cyclodextrin as the chiral selector based on the complexation constants of the protonated and zwitterionic forms of the analytes.

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A CE assay for the simultaneous determination of the impurities of levodopa listed in the European Pharmacopoeia including the (R)-enantiomer was developed and validated. The analysis was performed in a fused-silica capillary employing sulfated beta-cyclodextrin as chiral selector at an applied voltage of 20 kV and a temperature of 18 degrees C. The optimized background electrolyte consisted of 0.1 M sodium phosphate buffer, pH 2.0, containing 6 mg/mL sulfated beta-cyclodextrin. L-phenylalanine was used as the internal standard. The assay was validated in the range of 0.1-1.0% for the impurities at a concentration of levodopa of 2 mg/mL. The effect of different batches of sulfated beta-cyclodextrin was investigated using levodopa and L-tyrosine as critical pair. The method was applied to determine the purity of several samples of levodopa including the chemical reference substance of the European Pharmacopoeia.

The effect of pH on complex formation between beta-CD and the enantiomers of the dipeptides Ala-Phe, Ala-Tyr and Asp-PheOMe was investigated at 298.15 K by CE and calorimetry. Beta-CD displayed a higher enantioselectivity toward the protonated peptides compared to their zwitterionic forms. While stronger binding of the DD-enantiomers than the LL-stereoisomers were found by calorimetry regardless of the ionization state of the peptides, essentially equal complexation constants of the enantiomers were determined by CE for the zwitterionic species of the peptides. The reversal of the enantiomer migration order observed in CE was attributed primarily to a stereoselective complexation-induced pK(a) shift. In calorimetry, complexation of the protonated DD-enantiomers by beta-CD was accompanied by higher enthalpy and entropy changes resulting in more stable complexes compared to the LL-peptides. The enthalpy and entropy of complexation was affected by pH and peptide structure.