In this work the purification of biohydrogen was attempted in mixed gas measurements using a commercial polyimide membrane module. The impact of several process variables (gas composition, temperature, ratio of retentate and feed flows) on the real separation efficiency was statistically studied applying a 3-level full-factorial experimental design. The results showed that all the factors examined could significantly affect the achievable selectivity and it was observed that the theoretical and real separation factors were remarkably different. It was also found that - in comparison with other commercially available membranes - the module reflected potential for hydrogen enrichment. Considering the design boundaries, the highest H-2/CO2 gas selectivity (1.62) could be achieved at feed pressure, separation temperature and recovery value of similar to 2.2 bar, 55 degrees C and 0.6, respectively, using a 65 vol% hydrogen/35 vol% carbon-dioxide binary mixture. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.