Insights into the Capillary Electrophoresis Separation of Heparin Disaccharides from Nuclear Magnetic Resonance, pKa, and Electrophoretic Mobility Measurements

Abstract

Determination of the pK(a) values of heparin disaccharide functional groups can provide insights into the nature of glycosaminoglycan (GAG)-protein interactions and prove useful for optimization of the charged-based separations typically used in GAG analysis. In order to gain a better understanding into the capillary electrophoresis (CE) separation process, the pK(a) values of the carboxylate and primary amine moieties of 11 heparin disaccharide standards were determined through (1)H NMR detected pH titrations. These pK(a) values were used to calculate the effective net charge of each disaccharide and compared to the electrophoretic mobilities measured by CE. Although a different migration order had been reported by other researchers, our results indicate a strong positive correlation between the two measurements, consistent with the migration order observed in our CE separations. The effect of mutarotation was also examined by (1)H NMR. Mutarotation equilibrium constants favored the alpha anomer over the beta conformation. pK(a) values determined for both anomers of the four disaccharide standards containing a GlcN primary amine indicated that the beta anomer of the GlcN residue was more acidic. Partial separation of these anomers was achieved in CE separations using either formic acid or phosphate buffer.