Relationship between the Association Constant and Enantioselectivity on the Flavoprotein-Conjugated Chiral Stationary Phase for High-Performance Liquid Chromatography

The relationship between the enantioselectivity and association constants of enantiomers on high-performance liquid chromatography with a protein-conjugated chiral stationary phase (CSP) was investigated using a flavoprotein-conjugated CSP, with ketoprofen (KP) as a model drug. The association constants of the enantiomers for native flavoprotein were different; it has been found that they contribute to the chiral separation on a protein CSP. Equations including the association constants were developed to calculate, the capacity factors and enantioselectivities on protein CSPs; they were validated by means of experiments using a flavoprotein column in combination with variable numbers of non-specific columns in series. The capacity factors and enantioselectivities calculated with the developed equations showed reasonable agreement with those obtained by chromatography employing a conventional flavoprotein column (150 mmX4.6 mm i.d.) with a mobile phase containing methanol as an organic modifier. Our results imply that protein-conjugated CSPs are complex-stationary phases consisting of both a specific phase for chiral separation and a non-specific phase, and that the resolution of the enantiomers depends upon the balance of interactions of solute molecules with the two phases.