Novel open‐tubular columns with highly hydrophobic stationary phase for peptide and protein separation by nano‐liquid chromatography

Abstract

AbstractIn this study, novel open‐tubular nano‐columns with highly hydrophobic stationary phase were prepared via the surface graft polymerization of polyhedral oligomeric silsesquioxane‐methacrylate and 2‐vinylnaphthalene in a narrow capillary (i.e., 20 μm i.d. In this preparation, polyhedral oligomeric silsesquioxane‐methacrylate was used as a reactive and stable monomer while 2‐vinylnaphthalene imparts hydrophobic character for the stationary phase. Toluene and dodecanol were used as the porogenic solvent. Scanning electron microscope images of the columns showed that polyhedral oligomeric silsesquioxane and 2‐vinylnaphthalene were bound to the surface of the capillary. The chromatographic characterization of the open‐tubular columns was performed using homologous alkylbenzenes, including toluene, ethylbenzene, propylbenzene, and butylbenzene. The satisfactory separation of alkylbenzenes could be obtained using the final open‐tubular column with 17.1% of 2‐ vinylnaphthalene. Theoretical plates number up to 34000 plates/m in the isocratic mode for ethylbenzene were achieved. The open‐tubular column with various lengths was used for the separation of intact proteins in nano‐liquid chromatography, allowing a notable change in the retention time of each intact protein, including ribonuclease A, Lysozyme, and α‐Chymotryp A. The final open‐tubular column was then applied to the peptide separation of trypsin‐digested cytochrome C. It was shown that the open‐tubular column yielded a high‐resolution analysis of peptides, providing a high peak capacity of up to 1000.