Recently we described the use of riboflavin binding protein extracted from quail egg-white, as a new HPLC chiral stationary phase. In this study we show the further results obtained with the use of high-performance affinity chromatography to provide a better understanding of the chiral recognition mechanism for the observed enantioselectivity and to gain a deeper knowledge into the binding site that has been recently characterised by X-ray crystallography for chicken egg-white. High-performance affinity chromatography provides information on the potential protein structural changes occurring upon its immobilisation and enables competitive binding studies as well as the assessment of binding constants through frontal analysis experiments.