An update on the progress in fundamental understanding of hydrophilic interaction liquid chromatography.

With expanding applications, Hydrophilic Interaction Liquid Chromatography (HILIC) has made significant progress in theoretical and methodological developments in the past decade. Research efforts to elucidate its complex retention mechanisms and kinetic mass transfer characteristics continue to gain more insights. Central to this progress is a refined understanding of the adsorbed water layer on the polar packing surface, which functions as the de facto stationary phase for hydrophilic partitioning. Recent advances, including molecular dynamic simulation and quantitative measurement of phase ratio, provide more clarity on the structure, formation, and chromatographic relevance of the adsorbed water layer. Concurrently, new theoretical and experimental approaches have emerged to dissect the contributions of various mechanisms including partitioning, surface adsorption, and electrostatic interactions to solute retention in HILIC, enabling solute-specific and system-specific mechanistic insights. These developments represent significant advances in the research on retention mechanisms and provide direct evidence linking the retention contributions to analyte properties (e.g., partitioning coefficients), stationary phase and mobile phase composition. Electrostatic effects for ionized analytes on retention are better understood in quantitative terms. In addition, reversed-phase and HILIC dual retention behaviors on moderately polar stationary phases have been studied extensively and may lead to alternative approaches to the separation of extremely polar compounds. Kinetic performance, particularly with regard to intra-particle diffusion has been linked to the adsorbed water layer. With improved knowledge of the retention mechanisms, the widely used selectivity tests are being re-evaluated. This will further our understanding of the selectivity principles in HILIC. This review synthesizes recent theoretical and experimental findings to provide a cohesive update on the current understanding of HILIC and outlines key questions that remain for future exploration.