Hydrophilic Partitioning or Surface Adsorption? A Quantitative Assessment of Retention Mechanisms for Hydrophilic Interaction Chromatography (HILIC)

Retention mechanisms in HILIC have been investigated and reported in literature. However, the current understanding of retention mechanisms is qualitative and lacks quantitative details. Previously, mechanism elucidation was based on indirect evidence, and unambiguous assignment of retention mechanisms has not been reported based on direct data. This study aims to quantitatively determine the contributions of two major retention mechanisms in HILIC, hydrophilic partitioning and surface adsorption to the overall retention of neutral compounds. Using the methodologies we developed previously, the phase ratio for adsorbed water layer and distribution coefficients were measured and used to calculate the retention factors contributed by hydrophilic partitioning. The methodology allows the determination of the contribution of surface adsorption simultaneously. The evaluation of five test compounds demonstrates that the retention may be controlled by hydrophilic partitioning, surface adsorption or both depending on compound characteristics. Quantitative assessment of retention mechanisms also makes it possible to better understand the effect of acetonitrile on retention in HILIC.