Chromatography on a SP-Spherodex column was tried to purify pop corn inhibitor, a proteinase inhibitor from corn. Addition of organic solvent to mobile phase (acetonitrile, isopropanol or ethylene glycol) was tested as a method of improving selectivity. Thirty percent acetonitrile gave optimal results and allowed one to design a single-step purification procedure for the inhibitor. The efficacy of acetonitrile addition, most likely, is not due to an influence on eventual hydrophobic (or hydrophilic) interactions of the chromatographic support with proteins but to the induction of some subtle protein conformation changes of the proteins retained on the column. The effect of acetonitrile on protein conformation was studied by the sensitive technique of time-resolved fluorescence spectroscopy.