Mixed-mode hydrophilic interaction/ion-exchange liquid chromatography – Separation potential in peptide analysis

Abstract

In this work, three recently developed HILIC columns, i.e., HILIC-A containing silica gel, HILIC-N containing polyhydroxy phase and HILIC-B containing aminopropyl, were characterized in detail to compare their peptide retention, selectivity, and peak symmetry. The detailed study of chromatographic behavior of peptides revealed that a multimodal retention mechanism is present in systems with these stationary phases. Using three model analytes (caffeine, benzenesulfonic acid, and benzyltrimethylammonium chloride), we demonstrated that the HILIC-A column behaves as a HILIC/cation exchanger, and the HILIC-B column as a HILIC/anion exchanger depending on the mobile phase pH and buffer concentration. Furthermore, the pH (ammonium formate, pH 2.1 and 3.5; ammonium acetate pH 5.5) and buffer concentration (10–50 mM) of the mobile phase affect peptide retention and peak symmetry. Satisfactory retention and peak shape of peptides can be obtained by balancing ionic and hydrophilic properties of both analyte and stationary phases. Therefore, buffer concentration and pH optimization is an effective tool for manipulating retention and peak symmetry, thereby enhancing the potential of these columns in the analysis of diverse compounds. The potential application of these columns in the separation of biologically active peptides was confirmed by achieving baseline separation under optimized gradient elution.