Insights into the mechanism of separation of heparin and heparan sulfate disaccharides by reverse-phase ion-pair chromatography

Reverse-phase ion-pair high performance liquid chromatography (RPIP-HPLC) and ultra-performance liquid chromatography (RPIP-UPLC) are increasingly popular chromatographic techniques for the separation of organic compounds. However, the fine details of the RPIP separation mechanism are still being debated. Many factors including type and concentration of the ion-pairing reagent, mobile phase pH, organic modifier, ionic strength, and stationary phase all play a role in the overall efficiency and optimization of ion-pairing separations. This study investigates the role that competition between ion-pairing reagents with different steric bulk and hydrophobicity plays in the separation of structural isomers of heparin and heparan sulfate (HS) disaccharides. In addition to providing insights into the mechanism by which RPIP-HPLC can resolve closely related disaccharides, the use of competition between ion-pairing agents could lead to new methods for the separation of larger heparin and HS oligosaccharides. This approach should also be applicable to the analysis of other compound classes, and could lead to a general approach for the chromatographic resolution of mixtures of charged analytes having similar structures.