Influence of the mobile phase composition and pH on the chromatographic behaviour of polar neutral and ionized compounds in hydrophilic interaction chromatography

Abstract

The chromatographic behaviour of anionic, cationic and neutral analytes on the bare silica stationary phase in the hydrophilic interaction chromatography (HILIC) mode was investigated. The effect of various mobile phase parameters such as the nature and concentration of an organic solvent, pH and buffer concentration on the retention and efficiency was evaluated. The eluting strength of organic solvents follows the order: acetonitrile < isopropanol < methanol. For all analytes acetonitrile provided the highest peak efficiency. Minimal changes in the retention and efficiency of neutral compounds were observed upon changing pH (3–7) and ammonium acetate buffer concentration (0–10 mmol/L). The efficiency of charged analytes significantly increased with increasing pH and buffer concentration. The changes in the retention for cationic and anionic compounds with increasing pH and buffer concentration were in the opposite direction. The retention of anions increased, whereas an opposite trend was observed for cationic analytes.