The main goal of this paper was to check experimentally that in affinity chromatography, when porous particles are used, the enzymes inside the particles are partly retained instead of being adsorbed. Trypsin and Asparaginase were separated by affinity chromatography using a combined system with batchwise adsorption and columnwise desorption. The data obtained on specifically and non-specifically adsorbed enzymes were compared with those previously obtained from equilibrium studies. Langmuir-type behaviour was observed for the adsorption of asparaginase. From the isotherms obtained, the adsorption equilibrium constants were determined taking into account the partition coefficient. These constants were compared with those experimental determined from equilibrium studies. The mass balances carried out from the elution process revealed that asparaginase is adsorbed by specific bonds (73% of total bonds), non-specific bonds (27% of total bonds) and partly occluded (38% of the initial concentration). By contrast, trypsin was not adsorbed by specific bonds, but a small amount of occluded enzyme and non-specific bonded enzyme inside the particles was observed. It may thus be concluded that affinity chromatography affords perfect separation of these enzymes. (C) 2002 Elsevier Science Ltd. All rights reserved.