Abstract: This paper represents a new high-performance liquid chromatography (HPLC) system which consists of butyl-alcohol as mobile phase and β-cyclodextrin stationary phase. The model of molecular interactions among tertiary-butyl alcohol, normal-butyl-alcohol, 2-butyl-alcohol and cyclodextrin has been built and verified via computer simulation and spectro-photometric approach. By choosing tert-butyl-alcohol as the mobile phase, five types of enantiomers have been separated successfully. This result suggests that the improvement of chiral selectivity of HPLC system might be attributed to the competitive interaction between butyl-alcohol and cyclodextrin.