Displacement chromatography of binary protein mixtures is carried out in the hydrophobic interaction (HIC) mode for the first time. Sharp displacement; separations of a mixture of ribonuclease (RNase) and lysozyme (LYS) are achieved on a TSK-Butyl NPR column using bovine serum albumin (BSA) as the displacer in a carrier containing 2.3 M ammonium sulfate. On the other hand, chromatography of a BSA and LYS mixture on a micropellicular polystyrene based HIC sorbent at 23 degrees C shows evidence of selectivity inversion upon going from analytical conditions to displacement with a-chymotrypsinogen A (CHY) as the displacer, and the separation is not good. Measurement of the adsorption behavior of LYS, BSA, and CHY by frontal analysis on the polystyrene-based sorbent shows that whereas the BSA and CHY isotherms are concave downward, the LYS isotherm is S-shaped, indicative of a cooperative adsorption mechanism. In addition, the LYS and BSA isotherms intersect; The selectivity reversal and the resulting chromatographic behavior are adequately explained with the help of a stability analysis. For the purpose of the analysis, the ideal adsorbed solution theory is invoked to describe the multicomponent LYS and BSA isotherm. Operation at 40 degrees C is found to mitigate the effects of Selectivity inversion, and the displacement separation is accomplished.