An open tubular capillary electrochromatography column covalently bonded with polystyrene sulfonate was prepared via in situ polymerization using functionalized Azo-initiator 4,4'-Azobis(4-cyanopentanoyl chloride). Scanning electron, fluorescence, and atomic force microscopy techniques showed the formation of a relatively rough layer of polymer. In addition, -CN and C = O stretching vibrations from infrared spectroscopy proved the successful immobilization of the azo-initiator through covalent bonding and X-ray photoelectron spectroscopy confirmed the elemental composition of the formed polymer layer. The prepared column was found to be appropriate for small and medium-sized molecules separation. Compared to bare fused silica capillary column higher selectivity and resolution were obtained for the separation of alkaloids, sulfonamides, and peptides as a result of the electrostatic and pi-pi stacking interactions between the small organic molecules and the coated column without compromising the electroosmotic flow mobility. Separation efficiency was also increased compared to the bare capillary for the separation of alkaloids (about 1.5 times). Moreover, intraday, inter-day, intra-batch, and inter-batch relative standard deviation values of retention time and peak area of peptides were within 2% and 10%, respectively, indicating good repeatability of the column preparation procedure. The developed method for the covalent bonding of polymers through a functionalized azo-initiator could represent a promising stable method for the preparation of an open tubular column.
Keywords: anionic column; electrochromatographic separation; functionalized azo-initiator; open tubular column; peptide separation.
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