Understanding the Effect of the Counterion on the Reverse-Phase Ion-Pair High-Performance Liquid Chromatography (RPIP-HPLC) Resolution of Heparin-Related Saccharide Anomers

Reverse-phase ion-pair high-performance liquid chromatography (RPIP-HPLC) is an increasingly popular chromatographic technique for the separation of charged compounds, including oligosaccharides derived from the glycosaminoglycans (GAGs) heparin and heparan sulfate (HS). This family of heparin disaccharides has been shown to be useful compounds to probe the details of the RPIP-HPLC separation mechanism, the aspects of which are still being debated. In this manuscript, the effects of ion-pairing reagent (IPR) concentration, counterion, and mobile phase pH on the quality of the RPIP-UPLC separation were examined with particular emphasis on how these factors impact the separation of the disaccharide anomers. These results highlight the role of the IPR counterion and demonstrate that the resolution of the disaccharide anomers can be minimized by conducting the separation at low pH, simplifying chromatographic analysis and improving resolution. The results presented herein can also provide insights into strategies for developing more sensitive and efficient reverse-phase separations for other charged analytes including larger GAG oligosaccharides.