cytochrome-c-t and divinyl-benzene

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 divinylbenzene (DVB) as the crosslinker in a fused silica capillary column of 50 µm i.d. Various porogenic solvents, including tetrahydrofuran (THF), dodecanol and toluene were used for morphology optimization. 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 π-π interactions under reversed-phase conditions. Theoretical plates number up to 41,200 plates/m in isocratic 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), cytochrome C (Cyt C), lysozyme (Lys), β-lactoglobulin (β-lac), myoglobin (My) and α-chymotrypsinogen (α-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.

Topics: Chromatography, Liquid; Cytochromes c; Proteomics; Vinyl Compounds

A strong cation-exchange separation material has been prepared from monodisperse divinylbenzene particles modified by a "grafting to" approach, utilizing as anchoring points epoxy groups introduced onto the surface of the particles via oxidation of residual vinyl groups. The grafted chains consisted of thiol-terminated telomers of sulfopropyl methacrylate prepared by iniferter mediated polymerization, and grafting was performed by reaction of the corresponding thiolate anion with the surface epoxy groups. Attachment through epoxy moieties that were subsequently converted into 2,3-propanediol groups increased the hydrophilicity of the polymeric particles and incubation experiments showed no signs of the proteins denaturing on the column during an extended contact time of 1 h at room temperature. The performance of the grafted material was demonstrated by the chromatographic separation of cytochrome C, lysozyme, myoglobin, and ribonuclease A, in a cation-exchange mode.

Topics: Chromatography, Ion Exchange; Cytochromes c; Ion Exchange Resins; Methacrylates; Microscopy, Electron, Scanning; Muramidase; Myoglobin; Particle Size; Polymers; Proteins; Ribonuclease, Pancreatic; Sulfhydryl Compounds; Surface Properties; Vinyl Compounds