Cholesterol-assisted lecithin-modified zirconia stationary phase enables pH-tunable retention and extraction of organic compounds.

Lecithin-modified zirconia (LMZ) stationary phases have attracted attention as pseudo-biomembrane materials for chromatographic separation; however, their practical application has been limited by poor intercolumn reproducibility caused by heterogeneous phospholipid immobilization. In this study, we prepared a cholesterol-assisted lecithin-modified zirconia (LCMZ) stationary phase to improve the uniformity and reproducibility of the lecithin immobilization. Co-immobilization of lecithin and cholesterol onto zirconia resulted in more consistent surface coverage (0.63 ± 0.12 wt.%) and markedly improved intercolumn reproducibility compared with LMZ, with the variation in retention times reduced from up to 37% to below 13%. Chromatographic investigations revealed that LCMZ exhibited pH-tunable retention behavior governed by a balance between electrostatic interactions with zirconium sites and hydrophobic interactions with phospholipid alkyl chains. The retention of charged and neutral analytes could be systematically controlled by adjusting the mobile phase pH, ionic strength, and organic modifier mass fraction. Mechanistic studies using amino acids and dipeptides demonstrated that electrostatic interactions dominated under low ionic strength conditions, while hydrophobic interactions became important as the organic solvent concentration increased. The incorporation of cholesterol improved the uniformity and reproducibility of lecithin immobilization while preserving the fundamental retention mechanism. Additionally, the LCMZ material exhibited effective adsorption and recovery of acidic dipeptides in flow-through solid-phase extraction, demonstrating its potential for combined separation and sample pretreatment applications. These findings provide insight into the design of stable pseudo-biomembrane stationary phases and highlight the potential of LCMZ as a reproducible platform for chromatographic separation of polar and amphiphilic compounds.