Use of immobilized amyloglucosidase as chiral selector in chromatography. Control of enantioselective retention and resolution in liquid chromatography

Several mobile phase parameters were investigated for controlling enantiose lective retention and resolution on a chiral stationary phase made in-house . The chiral selector was the enzyme amyloglucosidase, which was immobilize d onto a silica support via reductive amination. The influences of the mobi le phase pH, concentration and type of uncharged organic modifier, ionic st rength and column temperature on enantioselectivity were studied. The analy sis time for resolving enantiomers could be adjusted with only a minor decr ease in enantioselectivity by using a high ionic strength mobile phase buff er. This indicated a retention mechanism involving ion-exchange interaction s. It was further confirmed by the decreasing enantioselectivity of amines when using a mobile phase pH below the isoelectric point of the native prot ein. Interesting effects were observed when the organic modifier concentrat ion was increased and also when the column temperature was raised. Both ret ention and enantioselectivity increased with increasing concentration of 2- propanol in the mobile phase. Examples are given where both enantioselectiv ity and retention increased with increasing column temperature. Thermodynam ic studies were performed to calculate the entropy and enthalpy constants. The results showed that, depending on mobile phase composition, the enantio selective retention may be caused by differences in entropy or enthalpy.