SummaryThe enzyme amyloglucosidase was immobilized on oxidized DIOL silica and used to separate enantiomers of amino alcohols. The influence of pore size on enantioselectivity was studied and an optimum in the separation factors was found using 500 A DIOL silica as the starting material. About twice the amount of the protein could be immobilized on the 500 .~ DIOL silica material as on the 300 and 1000 ]~ materials. The immobilization procedure was easy to reproduce and no significant difference in the chromatographic behavior was observed between two amyloglucosidase columns produced in-house. The effect of solute structure on enantioselective retention was studied using a set of 10 closely related amino alcohols. High separation factors (~ > 2) were obtained and the efficiency of the amyloglucosidase columns was greater than 25 000 plates/m based on the last eluted enantiomer.

Key WordsColumn liquid chromatography Glucoamylase Enantioselective retention and resolution Effect of operating parameters Ionic strength SummarySeveral mobile phase parameters were investigated for controlling enantioselective retention and resolution on a chiral stationary phase made in-house. The chiral selector was the enzyme amyloglucosidase, which was immobilized onto a silica support via reductive amination. The influences of the mobile phase pH, concentration and type of uncharged organic modifier, ionic strength and column temperature on enantioselectivity were studied. The analysis time for resolving enantiomers could be adjusted with only a minor decrease in enantiosetectivity by using a high ionic strength mobile phase buffer. This indicated a retention mechanism involving ion-exchange interactions. It was further confirmed by the decreasing enantioselectivity of amines when using a mobile phase pH below the isoelectric point of the native protein. Interesting effects were observed when the organic modifier concentration was increased and also when the column temperature was raised. Both retention and enantioselectivity increased with increasing concentration of 2-propanol in the mobile phase. Examples are given where both enantioselectivity and retention increased with increasing column temperature. Thermodynamic studies were performed to calculate the entropy and enthalpy constants. The results showed that, depending on mobile phase composition, the enantioselective retention may be caused by differences in entropy or enthalpy.