Objective To develop a high performance liquid chromatography (HPLC) chiral separation method for the enantiomers A and B of tropane derivatives by the normal phase chiral chromatographic column. Methods The chiral columns of Chiralpak AD and Chiralcel OD-H were employed under the normal phase condition. Effects of the composition of mobile phase, flow rate, and temperature on the chiral separation of enantiomers were observed. Thermodynamic parameters of the interaction of enantiomers and stationary phase such as the enthalpy difference and entropy difference were calculated. Results The optimal enantiomeric separation condition for compound A was obtained on the Chiralpak AD column. The optimal enantiomeric separation condition for compound B was obtained on the Chiralcel OD-H column. The lnα was linear with 1/T between 15 ℃ and 35 ℃. The chiral separation was controlled by enthalpy. Conclusion This HPLC chiral separation method can be used for the analysis and identification of optical purity of tropane derivatives A and B.