A novel aqueous two-phase system of polyethylene glycol (PEG)/fructose-1,6-bisphosphate (FBP) trisodiurn salt was employed to purify an at-amylase inhibitor (alpha-Al) from wheat flour after failure in use of the conventional aqueous two-phase systems of PEG/(NH(4))(2)SO(4) and PEG/dextran (DEX). Phase compositions including PEG molecular weight and concentration of PEG, fructose-1,6-bisphosphate trisodium salt concentration, as well as pH of the system affected the partition of alpha-Al. The inhibitor was obtained with a purification factor of 3.2 and recovery of 79% by using 11.7% (w/w) of PEG2000 and 19% (w/w) of fructose-1,6-bisphosphate trisodium salt at pH 7.0 in the top phase. (C) 2008 Elsevier Ltd. All rights reserved.