A L18 (21 × 37) OAD was employed to investigate the effects of SFC parameters on the asymmetric factor of a basic compound on C18 columns. The effect of active sites of silanol group on SFC could be much more significant than that of HPLC due to the low viscosity and high diffusivity of the supercritical fluid. Three Waters C18 columns with different silanol activities were employed to examine the effect of the SFC parameters on the asymmetric factor of 5‐(α‐phenylethyl)‐semioxamazide. The OAD data were evaluated by ANOVA to determine significant parameters of SFC on the asymmetric factor. The results of ANOVA indicate that the silanol activity is the most dominant factor affecting the asymmetric factor. The influence of other parameters on the asymmetric factor follows the order of: modifier type > modifier concentration > additive type > pressure > temperature ∼ additive concentration > injected sample concentration.