Superhydrophobic fluorinated microporous microsphere stationary phase for liquid chromatography separation of highly polar pyrimidine compounds under water mobile phase

Fluorous chromatography traditionally relies on large volumes of toxic organic solvents as mobile phase, conflicting with sustainable development principles. Here, we introduce a superhydrophobic fluorinated microporous microsphere stationary phase (SFMMS) for separating highly polar pyrimidine compounds under aqueous mobile phases. The SFMMS exhibited a high specific surface area of 497.7 m²/g and ultrahigh water repellency (contact angle > 150 ° across diverse aqueous conditions), enabling drastically reduced back pressure compared to commercial columns. Baseline separation of cytosine, uracil, and 5-fluorouracil was achieved under highly aqueous systems containing minimal acetic acid or a ChCl-EG deep eutectic solvent (DES) as green mobile phases. Thermodynamic analyses revealed negative Gibbs free energy changes (ΔG < 0) and exothermic enthalpy changes (ΔH < 0), indicating spontaneous and enthalpically driven adsorption. Density functional theory (DFT) calculations confirmed binding energies of up to -3.05 kcal/mol for 5-fluorouracil, supporting a multi-interaction mechanism involving fluorine-fluorine (F-F) and fluorine-π (F-π) interactions. The high surface area provided abundant binding sites, contributing to prolonged retention of polar analytes on the fluorinated column. Column reproducibility was demonstrated by relative standard deviations (RSDs) of retention times ranging from 0.44% to 0.56% for intra-day tests and from 0.61% to 1.18% for inter-day tests. Spike-and-recovery results verified mean recoveries for 5-fluorouracil in urine samples ranging from 80.9% to 98.5%, with RSDs between 0.54% and 1.80%. This work offers a novel strategy for the separation of polar pyrimidines under water mobile phases on a fluorinated column, aligning with the principles of green liquid chromatography.