Comparison of cholesterol/C18-immobilized core-shell microspheres in reversed-phase liquid chromatography

Development of cholesterol-functionalized core-shell silica microspheres for reversed-phase liquid chromatography. • Significant reduction in analysis time and improved selectivity for polar compounds compared to conventional C18 stationary phases. • Successful application in complex sample analysis of sweeteners with LC-MS demonstrated an enhanced separation performance. To address the limited selectivity of C18 stationary phase toward structurally similar and polar compounds in complex matrices, we developed a cholesterol-functionalized core-shell silica stationary phase (SiO 2 @SiO 2 -chol). Monodisperse core–shell silica microspheres were fabricated via a two-phase method, followed by surface modification using silane coupling to immobilize cholesterol ligands. For direct comparison, a C18-bonded core-shell material (SiO 2 @SiO 2 -C18) was prepared under identical conditions. The SiO 2 @SiO 2 -chol stationary phase could not only reduce analysis time for alkylbenzene separations, but also increase resolution for structurally similar compounds such as PAHs, compared to the SiO 2 @SiO 2 -C18. This improvement is attributed to multimodal interactions, including hydrophobic effects, hydrogen bonding and steric hindrance, which are particularly effective for resolving polar and structurally similar analytes. For instance, naphthalene was used as a probe, showing a column efficiency (N) of 131,760 N·m −1 on SiO 2 @SiO 2 -chol, nearly double that of the SiO 2 @SiO 2 -C18 (70,470 N·m −1 ). In the separation of active ingredients from traditional Chinese medicines (TCMs), the SiO 2 @SiO 2 -chol column offered baseline separation of four target analytes (quercetin, apigenin, methoxsalen, and curcumin), whereas satisfactory resolution could not be achieved using the C18 stationary phase. Furthermore, the SiO 2 @SiO 2 -chol column was successfully coupled with LC-MS for the quantification of sweeteners in complex matrices, demonstrating practical applicability for food safety analysis. Mechanistically, the cholesterol moiety introduces additional shape-selective interactions and steric hindrance, which are absent in C18 materials, thereby enhancing the retention and separation of analytes with subtle structural differences. Taken together, the cholesterol-functionalized core-shell stationary phase combines structural robustness with multimodal recognition, providing a versatile and efficient alternative for separation of various analytes. Download: Download high-res image (82KB) Download: Download full-size image