Separation systems with a zirconia‐based polystyrene HPLC column were characterized by different approaches, which allowed the recognition of interactions participating in the separation environments. Zirconia‐based HPLC columns as an alternative to silica‐based ones offer unique interaction mechanism based on Lewis acid–base theory. Besides hydrophobic interactions with the modified surface of the zirconia carrier it includes ion‐exchange and ligand‐exchange interactions that are helpful in the separation of many bioactive compounds. Three distinct approaches were applied for description of the complex separation mechanism. General chromatographic tests by Walters, Engelhardt and Galushko were applied to evaluate the fundamental properties of the systems – hydrophobicity and polarity. The complex model of linear free energy relationship described the interactions from the qualitative and quantitative points of view more in detail. Application of a set of basic compounds revealed the contribution of ion‐exchange interactions participating in the separation systems.