Gas chromatographic separation of hydrogen isotopes on the combined column of 5 angstrom molecular sieve and Al2O3 at cryogenics has been investigated. This paper described the procedures for preparation and operation of the chromatographic combined separation column and discussed the separation effects. Experimental results showed that the purity of deuterium reached above 99.9% through the combined separation column. This system possesses some advantages such as simple construction, low operation cost, a less investment and so on. It is promising for engineering applications, particularly in some laboratories and departments where a small amount of high purity species of hydrogen isotope is required. Not only it is used for the separation of hydrogen and deuterium, but also used for the separation of deuterium and tritium. (c) 2006 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.