Influence of buffer substances and urea on the β‐cyclodextrin‐mediated chiral separation of dipeptides in CE

Abstract

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.

Effect of urea on analyte complexation by 2,6‐dimethyl‐β‐CD in peptide enantioseparations by CE

“…Only few reports have been published on the effect of urea on the mobilities of the free analytes and the diastereomeric complexes. Although unchanged mobilities were found in the case of BOC-Phe and b-CD as chiral selector [9] decreased values were reported for the enantioseparation of thioridazine by b-CD [16]. Lower thermodynamic pK a values were reported in the presence of urea compared with pure aqueous solutions for weak acids and bases [17][18][19].…”

“…Furthermore, pH-dependent effects were observed. Although improved b-CD-mediated separations of the enantiomers of the dipeptides Ala-Phe and Ala-Tyr were observed at pH 2.5 in the presence of 2 M urea, addition of the compound resulted in a deterioration of the enantioseparations when the pH was raised to 3.5 [8] or 3.8 [9].…”

“…Therefore, the effect of urea on analyte complexation, mobility and protonation is of great interest. With regard to complex formation, weaker complexes in the presence of urea as expressed by reduced complexation constants between the analytes and the selector have been frequently observed [9,[11][12][13][14][15][16]. However, increased complexation constants have also been reported, for example, for 2-naphthoic acid in the presence of 8 M urea [14].…”

“…In a previous study, the effect of 2 M urea on the separation of the enantiomers of Ala-Tyr and Ala-Phe using b-CD as chiral selector has been investigated [9]. Both peptides displayed a pH-dependent reversal of the enantiomer migration order between pH 2 and 4 [22][23][24].…”

Effect of Urea on the Ion-Association Capillary Electrophoresis Separation of the Anionic Metal Complexes Using Hydrophobic Tetraalkylammonium as Ion-Association Agent

“…It has been shown that urea can be used to improve the separation selectivity in miceller electrokinetic chromatography 10 and electrokinetic chromatography with cyclodextrin. [11][12][13][14][15] Yet, there have been no reports about the use of urea for improving the IA-CE resolution by controlling the ion-association of analytes and the interaction reagent. The fine-tuning of the resolution of IA-CE separation of metal complexes was accomplished as a function of the urea concentration.…”