Xylitol, a sugar substitute, is a high value product for pharmaceutical and food industries and its purification being of commercial importance. In the present study, the purification of xylitol obtained through Candida tropicalis by fermentation using synthetic xylose and corn cob hemicellulosic hydrolysate as substrates were studied for liquid-liquid extraction (21.72 g/l xylitol extracted in 1:5 (v/v) of ethyl acetate) and precipitation (67.44% xylitol recovery along with certain impurities). By this method xylitol recovery is difficult and expensive for large scale processes. Therefore, activated charcoal treatment followed by vaccum concentration and crystallization method for xylitol extraction was evaluated. The optimized conditions obtained for activated charcoal treatment followed by vaccum concentration and crystallization method were 15.0 g/l of charcoal concentration at 30 degrees C for 1 h with 10 times super saturation of initial concentration and crystallization temperature of -20 degrees C for initiation and then at 8 degrees C yielding 43.97%. After 4 cycles of crystallization, 76.20% and 68.06% xylitol crystallization yield was obtained in 50 ml and 5.01 of the synthetic xylose fermentation broth by adapted strain of C. tropicalis respectively. The effect of solvents on the crystalline structure of xylitol showed prismatic structure in the presence of ethanol and orthorhombic needles in the presence of tetrahydrofuran. The purity of the xylitol was characterized using C-13 and H-1 nuclear magnetic resonance, mass spectroscopy, and optical rotation, confirming 98.99% purity in a pure crystallized form. (C) 2011 Elsevier B.V. All rights reserved.