Direct liquid chromatographic separation of enantiomers on immobilized protein stationary phases. IX. Influence of the cross-linking reagent on the retentive and enantioselective properties of chiral sorbents based on bovine serum albumin.

Three modifications of silica-bound, cross-linked bovine serum albumin (BSA) were evaluated as chiral sorbents for use in the liquid chromatographic separation of enantiomers. Glutaraldehyde, formaldehyde and di-(N-succinimidyl) carbonate were used as bifunctional reagents for the immobilization of BSA. The sorbents all contain the same loading of BSA (14.4 +/- 0.1%, w/w) and differ only with respect to the cross-linker used for immobilization. Despite their apparent similarity, the sorbents show very different chromatographic properties, not only with respect to retention of analyte enantiomers (k' and alpha), but also with respect to column efficiency (affecting Rs values). The data obtained indicate that the chemical structure of the cross-linking reagent affects to a large extent the accessibility of important chiral binding sites. Although the data obtained are difficult to interpret in any detail, certain generalizations concerning the different behaviour of the sorbents can be made.