Abstract: The cryogenic cycling chromatography(CCC) is an effective way for hydrogen isotopes separation. The separation study of H2/HD system has been performed on the cryogenic chromatographic device after reform and upgrade. It was concluded that the deuterium abundance of pure hydrogen can reach 1.173×10^-3 by four flows from the original abundance 1.4×10^-4. In order to acquire the best chromatographic column efficiency, the injection volume of CCC must be regulated with capacity factor of ingredient peak decreasing 10%. Dual columns of CCC can serve as the desorption column and receipt column each other. Under same conditions of columns structure and temperature programmed, the discrepancy of separation result for dual columns may be caused by the choice of the sample injection point and different sample injection pressures, but not by the sample injection time.