Two polyurethanes of different molecular weights were prepared by the copolymerization of phenyl diisocyanate and diisopropyl tartrate. The polyurethanes having terminal isocyanate groups were reacted with 3-aminopropyl silica gel to afford two chiral stationary phases. The Mn of the two polyurethanes were 4057 g/mol and 6442 g/mol. The polyurethanes and the corresponding chiral stationary phases were characterized by FT-IR, 1H NMR and elemental analysis. The loading capacities of the polyurethanes on silica gel were 0.68 mmol units/g and 0.61 mmol units/g, respectively. The separation performance and the influence of additives, triethylamine and trichloroacetic acid, on the separation of chiral compounds were investigated by HPLC. The chiral stationary phase prepared from polyurethane with Mn of 4057 g/mol demonstrated better enantioseparation capability than that with Mn of 6442 g/mol. Additionally, it was found that the addition of triethylamine and trichloroacetic acid in the mobile phases significantly improved the enantioseparation for these two chiral stationary phases.