Quantitative Assessment of Retention Mechanisms for Ionized Compounds in Hydrophilic Interaction Chromatography (HILIC).

Abstract

The retention mechanisms for polar compounds in HILIC are only qualitatively understood to include hydrophilic partitioning, surface adsorption, and electrostatic interactions if both the analytes and stationary phases are charged. However, the main retention mechanism may be different for different compounds under different chromatographic conditions, and it is difficult to identify the main retention mechanism based on the existing knowledge and methods. We previously developed a methodology to quantitatively determine the retention contributions of hydrophilic partitioning and surface adsorption for nonionized compounds in HILIC. In this study, the methodology has been expanded to include the retention contribution of electrostatic interactions for the ionized compounds on charged stationary phases. When electrostatic interactions are sufficiently shielded at high salt concentrations, the partitioning coefficient of ionized compounds is determined using the same method for nonionized compounds. Then, the retention contributed by partitioning and adsorption is calculated. The retention contribution by electrostatic interactions (both attractive and repulsive) is determined by subtracting the retention contributed by partitioning and adsorption from the observed retention at each salt concentration. This is the first study that evaluated the retention contributions of hydrophilic partitioning, surface adsorption, and electrostatic interactions for ionized compounds. Quantitative information on retention mechanisms will be helpful to better understand selectivity in HILIC and facilitate the development of retention models.