Nano-liquid Chromatography with Monolithic Stationary Phase Based on Naphthyl Monomer for Proteomics Analysis

Monolithic poly(2-vinylnaphthalene-co-divinylbenzene) columns were introduced, for the first time, and were evaluated as the separation media for nano-liquid chromatography (nano-LC). These columns were prepared by in-situ polymerization of 2-vinylnaphthalene (2-VNA) as the functional monomer and di-vinylbenzene (DVB) as the crosslinker in a fused silica capillary column of 50 mu m i.d. Various porogenic solvents, including tetrahydrofuran (THF), dodecanol and toluene were used for morphology optimiza-tion. Final monolithic column (referred to as VNA column) was characterized by using scanning electron microscopy (SEM) and chromatographic analyses. Alkylbenzenes (ABs), and polyaromatic hydrocarbons (PAHs) were separated using the VNA column while the column offered excellent hydrophobic and pi- pi interactions under reversed-phase conditions. Theoretical plates number up to 41,200 plates/m in iso-cratic mode for ethylbenzene could be achieved. The potential of the final VNA column was demonstrated with a gradient elution in the separation of six intact proteins, including ribonuclease A (RNase A), cy-tochrome C (Cyt C), lysozyme (Lys), beta-lactoglobulin (beta-lac), myoglobin (My) and alpha-chymotrypsinogen (alpha-chym) in nano LC system. The column was then applied to the peptide analysis of trypsin digested cytochrome C, allowing a high peak capacity up to 1440 and the further proteomics analysis of COS-7 cell line was attempted applying the final monolithic column in nano-LC UV system.(c) 2023 Elsevier B.V. All rights reserved.