Abstract:

As the main components of cell membranes, lipids play important roles in organisms. Lipids have been proved to be closely associated with the occurrence and development of serious diseases, such as cancers and metabolic diseases. The development of novel separation materials for use in high-performance liquid chromatography (HPLC) is essential for high-efficiency lipid separation. Such materials can promote further in-depth research of the structure and biological functions of lipids. In this study, we focused on the preparation of an ionic-liquid-modified silica-bonded HPLC stationary phase and on its chromatographic retention mechanisms and separation performances for lipids. An imidazolium-based ionic liquid with C₁₂ side chain, viz. 1-vinyl-3-dodecylimidazole bromide (VDI), has shown good biocompatibility and has previously been used for the solubilization of membrane proteins. Thus, VDI was first exploited as the functionalized monomer for the HPLC stationary phase. It was grafted onto the surface of thiol-functionalized silica spheres by a one-step click reaction to afford a new VDI silica-bonded stationary phase (Sil-VDI). Fourier-transform infrared (FT-IR)spectroscopy and thermogravimetric analysis were used to prove the successful preparation of Sil-VDI and characterize its structure. The chromatographic retention properties of the column packed with Sil-VDI was first studied using hydrophobic alkylbenzenes. The results showed that the Sil-VDI column was a typical reversed-phase liquid chromatography retention column. Since Sil-VDI has a permanent cationic imidazole structure, it should demonstrate anion exchange retention. Thus, inorganic anions BrO₃ ^- , NO₃ ^- , and IO₃ ^- were selected to further investigate the retention mechanism of the Sil-VDI column. The results demonstrate that the Sil-VDI column also possesses ion-exchange retention mode. Thus, the Sil-VDI column has typical reversed-phase and ion-exchange mixed-mode retention characteristics. Based on the reversed-phase retention characteristics of the Sil-VDI column, hydrophobic alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs), basic anilines, and benzene derivatives were selected for testing the HPLC separation performances of the Sil-VDI column. The results demonstrate that this new column gave good separation selectivity with good peak shapes. For example, PAHs like diphenyl, o -terphenyl, m -terphenyl, and triphenylene were used to investigate the chromatographic performances of the Sil-VDI column. These four PAHs were baseline separated within 7 min with good peak shapes. In addition, the positional isomers o -terphenyl and m -terphenyl show good separation efficiency, with resolution as high as 3.26. Based on the ion-exchange retention characteristics of the Sil-VDI column, inorganic anions BrO₃ ^- , NO₃ ^- , and IO₃ ^- were selected to test the separation performance of the column for ionic compounds. Using 250 mmol/L KCl solution (pH 4.1) as the mobile phase, baseline separation of the three anions was achieved within 6 min. These results demonstrate that the Sil-VDI column has good potential for separation of ionic compounds. The separation performance of the Sil-VDI column was further verified based on the separation of lipids extracted from egg yolk and lung adenocarcinoma cells. Six main chromatographic peaks could be recognized within 7 and 5 min for the lipids extracted from lung adenocarcinoma cells and egg yolk, respectively. These results primarily demonstrate that the Sil-VDI column has good potential for the separation of lipid samples. In conclusion, a new ionic-liquid-based Sil-VDI stationary phase material was successfully fabricated via a simple synthesis method. The Sil-VDI column shows good separation performances for versatile samples. In future, further research will be performed on the separation ability of the Sil-VDI column for different biological samples.

Key words: ionic liquid, bonded stationary phase, reversed-phase/ion-exchange chromatography, lipid separation