Automated Electrostatic Attraction/Repulsion Hydrophilic Interaction Liquid Chromatography Screening for Polar and Charged Analytes in Complex Matrices

Abstract Electrostatic repulsion hydrophilic interaction liquid chromatography (ERLIC) provides unique selectivity for highly polar and charged analytes, yet method development remains challenging. Careful optimization of organic solvent gradients and buffer concentrations is essential to achieve optimal selectivity. However, application of a pH gradient in addition to organic solvent and buffer concentration gradients can enable mixed-mode interactions beyond those governed solely by electrostatic repulsion. In this work, we introduce Electrostatic Attraction/Repulsion Hydrophilic Interaction Liquid Chromatography (EARLIC), a novel separation mode that extends the selectivity of ERLIC by leveraging both electrostatic attraction and repulsion through pH gradient modulation of analyte−stationary phase interactions. Fundamental retention mechanism differences between these separation modes are proposed while also outlining key chromatography practices for enhanced selectivity. In addition, an ERLIC/EARLIC screening platform integrating cation and anion exchange columns with twelve mobile phases to systematically modulate electrostatic and hydrophilic interactions is introduced. Automated column and mobile phase switching valves enabled rapid scouting and efficient assay development while also minimizing manual intervention. This workflow demonstrated broad applicability across nucleotides, N-glycans, synthetic precursors, intact glycoproteins, and aminoglycoside antibiotics. Compared to classical HILIC, EARLIC improved retention of highly hydrophilic species, delivered sharper peaks in high-salt matrices, and provided orthogonal selectivity for charged moieties. Coupling ERLIC/EARLIC to mass spectrometry streamlined sensitive quantitation of kanamycin in cell culture matrices, confirming compatibility with complex sample loads. This analytical framework establishes a systematic approach for selecting the optimal combination of stationary phase charge, buffer system, ionic strength, pH, and organic fraction, accelerating ERLIC/EARLIC adoption across both academic and industrial sectors.