Mixed-mode open tubular column for peptide separations by capillary electrochromatography.

Abstract

Mixed-mode chromatography open tubular column has been developed for peptide separation in electrochromatography. A column with 92cm effective length and 50 μm internal diameter is fabricated internally with a copolymer sheet of restricted thickness. Catalyst facilitated binding of the coupling agent 3,5-bis (trifluoromethyl) phenyl isocyanate has been carried out at the interior surface of the column. The initiator sodium diethyldithiocarbamate was bound to the coupling agent. A small amount of N-[2-(acryloylamino) phenyl] acrylamide was used along with methacrylic acid and styrene in the monomer mixture to induce a little polar character in the stationary phase fabricated inside the column. Twenty-three peptides have been separated from a chemically digested protein mixture present in cytochrome C in capillary electrochromatography, in addition to the separation of six commercial peptides. We achieved an average plate count of over 1.5 million/m with the column of current study both for the digested protein components and commercial peptides using 70/30% v/v (acetonitrile/20mM ammonium formate) at pH 6.5. In addition, the column resulted in baseline separation of all the peptides with very good resolution, enhanced peak capacity, and better retention time span.