To improve resolution power of chiral selector and enantiomeric peak efficiency in CE, single isomer negatively charged beta-CD derivatives, mono(6-deoxy-6-sulfoethylthio)-beta-CD (SET-beta-CD) bearing one negative charge and mono[6-deoxy-6-(6-sulfooxy-5,5-bis-sulfooxymethyl)hexylthio]-beta-CD (SMHT-beta-CD) carrying three negative charges, were synthesized. The structure of these two beta-CD derivatives was confirmed by H-1 NMR and MS. SET-beta-CD and SMHT-beta-CD successfully resolved the enantiomers of several basic model compounds. SMHT-beta-CD provided for a significantly greater enantioseparation than SET-beta-CD at lower concentrations. This appears to be due to the higher binding affinity of SMHT-beta-CD to the model compounds and the wider separation window resulting from an increased countercurrent mobility of the selector. Overall, the new chiral selectors provided enantioseparations with high peak efficiency while avoiding peak distortion due to polydispersive and electrodispersive effects. The information obtained from an apparent binding constant study suggested that the enantioseparation of the model compounds followed the predictions of charged resolving agent migration model and that the observed degree of enantioseparation difference were due to the magnitude of differences in both enantiomer-chiral selector binding affinities (Delta K) and the mobilities of the complexed enantiomers (Delta mu(c)).