One-pot synthesis of a poly( styrene-acrylic acid) copolymer- modified silica stationary phase and its applications in mixed.mode liquid chromatography

As modified ligands with a wide range of sources, abundant functional groups, and good biocompatibility, polymers have been widely used in the development of silica-based chromatographic stationary phases. In this study, a poly( styrene.acrylic acid) copolymer.modified silica stationary phase ( SiO2@ P( St. b.AA)) was prepared via one-pot free-radical polymerization. In this stationary phase, styrene and acrylic acid were used as functional repeating units for polymerization and vinyltrimethoxylsilane ( VTMS) was used as a silane coupling agent to link the copolymer and silica. Various characterization methods, such as Fourier transform in frared ( FT-IR) spectroscopy, thermogravimetric analysis ( TGA), scanning electron microscopy ( SEM), N-2 adsorption-desorption analysis, and Zeta potential analysis, confirmed the successful preparation of the SiO2 @ P( St. b. AA) stationary phase, which had a well-main- tained uniform spherical and mesoporous structure. The retention mechanisms and separation performance of the SiO2@ P( St-b-AA) stationary phase in multiple separation modes were then evaluated. Hydrophobic and hydrophilic analytes as well as ionic compounds were selected as probes for different separation modes, and changes in the retention of the analytes under various chromatographic conditions, including different methanol or acetonitrile contents and buffer pH values, were investigated. In reversed. phase liquid chromatography ( RPLC) mode, the retention factors of alkyl benzenes and polycyclic aromatic hydrocarbons ( PAHs) on the stationary phase decreased with increasing methanol content in the mobile phase. This finding could be attributed to the hydrophobic and p.p interactions between the benzene ring and analytes. The retention changes of alkyl benzenes and PAHs revealed that the SiO2 @ P( St-b-AA) stationary phase, similar to the C18 stationary phase, exhibited a typical reversed. phase retention behavior. In hydrophilic interaction liquid chromatography ( HILIC) mode, as the acetonitrile content increased, the retention factors of hydrophilic analytes gradually increased, and a typical hydrophilic interaction retention mechanism was inferred. In addition to hydrophilic interaction, the stationary phase also demonstrated hydrogen.bonding and electrostatic interactions with the analytes. Compared with the C18 and Amide stationary phases prepared by our groups, the SiO2@ P( St-b-AA) stationary phase exhibited excellent separation performance for the model analytes in the RPLC and HILIC modes. Owing to the presence of charged carboxylic acid groups in the SiO2 @ P( St-b-AA) stationary phase, exploring its retention mechanism in ionic exchange chromatography ( IEC) mode is of great importance. The effect of the mobile phase pH on the retention time of organic bases and acids was further studied to explore the electrostatic interaction between the stationary phase and charged analytes. The results revealed that the stationary phase has weak cation exchange ability toward organic bases and electrostatically repels organic acids. Moreover, the retention of organic bases and acids on the stationary phase was influenced by the analyte structure and mobile phase. Thus, the SiO2 @ P( St-b-AA) stationary phase could provide multiple interactions, as demonstrated by the separation modes described above. The SiO2 @ P( St-b-AA) stationary phase showed excellent performance and reproducibility in the separation of mixed samples with different polar components, indicating that it has promising application potential in mixed-mode liquid chromatography. Further investigation of the proposed method confirmed its repeatability and stability. In summary, this study not only described a novel stationary phase that could be used in RPLC, HILIC, and IEC modes but also presented a facile " one-pot" preparation approach that could provide a new route for the development of novel polymer. modified silica stationary phases.