BACKGROUNDHydrophobic interaction chromatography (HIC), as a typical separation technology, often suffers from low selectivity because it has a single interaction mode. Herein, a novel HIC resin (Octyl@BSA) with imprinted layer was synthesized and used for protein adsorption. Bovine serum albumin (BSA) and bovine serum γ‐globulin (bIgG) were used as model competitive proteins in this study. The effects of pH, salt type, concentration, and mass ratio were then investigated.RESULTSThe data from the static and dynamic adsorption experiments indicated that the introduction of spatial recognition effectively reduced the adsorption of competitive proteins, leading to significant improvements in selectivity. In comparison with raw HIC resin, Octyl@BSA retained a high binding capacity for BSA but rejected other proteins through spatial recognition. The maximum selectivity factors (α) of this resin may be close to 5. The adsorption kinetics and frontal adsorption experiments further confirmed the improvement in selectivity in batch and column mode. In addition, the recyclability of Octyl@BSA was tested using ten continuous BSA adsorption–desorption cycles.CONCLUSIONThis study demonstrated that the introduction of spatial recognition was an effective strategy for enhancing the advantage of target proteins during competitive adsorption, leading to an improvement in selectivity for HIC. © 2020 Society of Chemical Industry