Reversed-phase electrochromatography of amino acids and peptides using porous polymer monoliths

Efficient and rapid separation of minute levels of amino acids and bioactiv e peptides is of significant importance in the emerging field of proteomics as well as in the clinical and pharmaceutical arena. We have developed nov el UV-initiated acrylate-based porous polymer monoliths as stationary phase s for capillary- and chip-electrochromatography of cationic, anionic, and n eutral amino acids and peptides, followed by absorbance or laser-induced fl uorescence detection. The rigid monoliths are cast-to-shape and are tunable for charge and hydrophobicity. For separations at low pH, monoliths contai ning quaternary amine moieties were used to achieve high electroosmotic flo w, and for high pH separations monoliths with acidic sulfonic acid groups w ere employed. Efficient and reproducible separations of phenylthiohydantoin -labeled amino acids, native peptides, and amino acids and peptides labeled with naphthalene-2,3-dicarboxaldehyde (NDA) were achieved using both negat ively- and positively-charged polymer monoliths in capillaries. Separation efficiencies in the range of 65 000-371 000 plates/m were obtained with cap illary electrochromatography. Buffer composition and the degree of column h ydrophobicity were studied systematically to optimize separations. The mono liths were also cast in the microchannels of glass chips and electrochromat ographic separation followed by laser-induced fluorescence detection of thr ee NDA-labeled bioactive peptides was obtained. (C) 2001 Elsevier Science B Y All rights reserved.