Membranes made from 84% deacetylated chitosan biopolymer were cross-linked by a novel method using 2,4-toluylene diisocyanate (TDI) and tested for the separation of t-butanol/water mixtures by pervaporation. The unmodified and cross-linked membranes were characterized by Fourier transform infra red (FTIR) spectroscopy, X-ray diffraction (XRD) studies and sorption studies in order to understand the polymer-liquid interactions and separation mechanisms. Thermal stability was analyzed by differential scanning calorimetry (DSC) and thermo gravimetric analysis (TGA) while tensile strength measurement was carried out to assess mechanical strength. The membrane appears to have good potential for breaking the aqueous azeotrope of 88.2 wt% t-butanol by giving a high selectivity of 620 and substantial water flux (0.38 kg m(-2) hr(-1)). The effects of operating parameters such as feed composition, membrane thickness and permeate pressure on membrane performance were evaluated. (c) 2005 Society of Chemical Industry.