In this study, 6-azido-2,3-di(p-chlorophenylcarbamoylated) cellulose was synthesized and bonded onto aminized silica gel to obtain a new chiral stationary phase. Enantioselectivity of the chiral stationary phase and Chiralcel OF suggested promising chiral separation ability of the new cellulose chiral stationary phase. In addition, the effect of trifluoroacetic acid, diethylamine on enantioselectivity and retention factors on the chiral stationary phase in high performance liquid chromatography was investigated. Experimental results revealed that resolution increased as the trifluoroacetic acid concentration increased to 0.3% while resolution declined as the diethylamine concentration increased. Therefore, the optimal concentrations of trifluoroacetic acid and diethylamine were determined to be 0.3 and 0.1%, respectively. In most cases, trifluoroacetic acid shortened the retention of the first eluted enantiomer while it increased the retention of the other. For acidic compounds, with the existence of diethylamine in the mobile phase, the retention of both enantiomers decreased. But for basic compounds, the retention of both enantiomers increased.