Separation of Sugar Anomers by Aqueous Chromatography on Calcium- and Lead-Form Ion-Exchange Columns: Applications to Anomeric Analysis of Enzyme Reaction Products

Analytical useful separations of the α- and β-anomers of five economically important monosaccharides (glucose, xylose, galactose, mannose and arabinose) can be obtained by totally aqueous chromatography at 1.5°C on a commercially available calcium-form ion-exchange column, the Bio-Rad HPX-87C. Such analyses are expected to be very important in determining the mechanisms of action of different enzymes converting the polysaccharides found in woody biomass to monomeric units fermentable to fuel alcohol. Aqueous chromatography on a similar column having a different metal counterion, the lead-form Bio-Rad HPX-87P, separates the anomers of glucose but fails to separate the anomers of the other four sugars that are anomerically resolved by the calcium column. This counterion-dependence is shown to arise not from a lack of chromatographic selectivity by the lead-form column, but to the substantially higher rate of mutarotation of the sugars in the presence of the lead-form packing material. Analysis of the shapes of the elution profiles yields estimates of the effective rate constants for mutarotation of glucose on the calcium columns; the observed perturbation of the values of k_α and k_β with respect to those measured in water at the same temperature in turn suggest a possible mechanism for the anomeric separation. A third column, the calcium-form Bio-Rad HPX-42C, which has a more open resin structure, shows promise for the separation of the products of enzyme reactions converting polysaccharides to disaccharides (such as maltose and cellobiose) rather than monosaccharides.