Chiral Separations in Capillary Electrophoresis Using Human Serum Albumin as a Buffer Additive

This study examined the theory and mechanisms of chiral separations in capillary electrophoresis based on the use of proteins as buffer additives. Human serum albumin (HSA) was used as the model ligand; D,L-tryptophan and (R,S)- warfarin were used as the test analytes to be separated by this protein. Items examined in this work included the amount of HSA adsorbed to the capillary wall and the stability of this adsorbed protein layer. These were investigated by performing frontal analysis on the capillary with HSA and by injecting neutral markers through the capillary at different applied voltages before and after HSA treatment. The role of adsorbed HSA vs HSA in the buffer in determining the stereoselectivity of the CE system was also examined. Adsorbed HSA was the predominant agent involved in the separation of (R,S)-warfarin, while HSA in the buffer had the most significant effect in the resolution of D,L-tryptophan. Two distinct separation mechanisms were proposed to explain these differences. Good agreement was obtained between the results predicted by these mechanisms and the experimental data. Under optimized conditions, both pairs of enantiomers were separated with baseline resolution in less than 12 min.