Abstract: For high efficient hydrogen isotope separation in lab-scale application, a palladium/kieselguhr (Pd/k) column （2 m in length） was made. Ethanol was applied as liquid heat transfer medium to cool the Pd/k column below 273 K. TCAP total reflux experiments with different cycles were carried out to test the separation efficiency of the column. Hydrogen isotope mixtures (15% of total column content) were withdrawn both from the bottom and top end after each test. D concentration of the mixtures was tested by cryogenic chromatography. Results show that D concentration in the bottom section rises from 50% to 98.5% at the initial charge before reflux cycling. High purity (99.9%) deuterium is obtained after only five cycles (1.25 h). D concentration in the top section drops from 50% to 13.6% after 15 cycles. Through a simulated D distribution model, fast flow rate through the top section may obviously reduce separation efficiency. The lowest D concentration is found near the middle of the column, and the raffinate with high H concentration will be achieved in the middle position.