Studies of ovomucoid-, avidin-, conalbumin- and flavoprotein-conjugated chiral stationary phases for separation of enantiomers by high-performance liquid chromatography

Abstract The effects of organic modifiers, buffer salts and pH on the retention and chiral separation of four protein-immobilized chiral stationary phases (CSPs), an ovomucoid-CSP, an avidin-CSP, a conalbumin-CSP and a flavoprotein-CSP, were investigated. Both retention and enantioselectivity were affected by alteration of the mobile phase conditions, and it was elucidated that the hydrophobic and ionic interactions between enantiomers and chiral recognition moieties in immobilized protein molecules were important to the chiral separation of each CSP. The protein bindings of enantiomers for four native proteins were also examined with chiral separation chromatography using a commercial ovomucoid-CSP (Ultron ES-OVM). The racemate which showed significant differences in protein binding abilities among its enantiomers was excellently resolved by chromatography.