The aim of the present study was the investigation of the effect of urea on analyte complexation in CD-mediated separations of peptide enantiomers by CE in the pH range of about 2-5. pH-independent complexation. and mobility parameters in the absence and presence of 2 M urea were obtained by three-dimensional, non-linear curve fitting of the effective analyte mobility as a function of pH and heptakis-(2,6-di-O-methyl)-beta-CD concentration. Urea led to decreased binding strength of the CD towards the protonated and neutral analyte enantiomers as well as to decreased mobilities of the free analytes. In contrast, mobilities of the fully protonated. enantiomer-CD complexes as well as the pK(a) values of the free and complexed analytes increased. The effect of urea on separation efficiency varied with pH and CD concentration. In the case of Ala-Tyr and Ala-Phe, separations improved in the presence of urea at pH 2 2. In contrast, separations were impaired by urea at pH 3.8 and low concentrations of the CD. Decreased separation efficiency was noted for Asp-PheOMe and Glu-PheNH(2) at low CD concentrations when urea was added but separations improved at higher CD concentrations over the entire pH range studied. The effect of urea on analyte complexation appeared to be primarily non-stereoselective. Furthermore, the pH-dependent reversal of the enantiomer migration order observed for Ala-Tyr and Ala-Phe can be rationalized by the complexation and mobility parameters.