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

Abstract

In 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.

Open tubular liquid chromatography: Recent advances and future trends

“…For example, a column composed of a 20 μm capillary produced with the polymerization of polyhedral oligomeric silsesquioxane-methacrylate and 2-vinyl naphthalene (2-VNA). In isocratic mode, an efficiency higher than 34 000 plates/m was obtained using ethylbenzene [107]. The content of 2-VNA affected the phase thickness; using 17.1% of 2-VNA resulted in a thick layer, improving the mass loadability [107].…”

“…In isocratic mode, an efficiency higher than 34 000 plates/m was obtained using ethylbenzene [107]. The content of 2‐VNA affected the phase thickness; using 17.1% of 2‐VNA resulted in a thick layer, improving the mass loadability [107]. Furthermore, in gradient mode, the optimized version of this column was able to generate a peak capacity of up to 1000 for the separation of trypsin‐digested cytochrome C [107].…”

“…The content of 2‐VNA affected the phase thickness; using 17.1% of 2‐VNA resulted in a thick layer, improving the mass loadability [107]. Furthermore, in gradient mode, the optimized version of this column was able to generate a peak capacity of up to 1000 for the separation of trypsin‐digested cytochrome C [107]. The retention mechanism can be attributed to π‐ π interactions [107].…”

“…Furthermore, in gradient mode, the optimized version of this column was able to generate a peak capacity of up to 1000 for the separation of trypsin‐digested cytochrome C [107]. The retention mechanism can be attributed to π‐ π interactions [107]. Columns with 10 μm of i.d.…”