Rapid Separation of Peptides and Amino Acids in Glass Microchips by Reversed-Phase Electrochromatography

Abstract

We have developed microfabricated glass chips for reversed-phase separation followed by laser-induced fluorescence detection of peptides and amino acids. Hydrophobic acrylate-based porous polymer monoliths were cast in the channels by photopolymerization. Use of UV-light for initiation of polymerization allows for selective patterning of stationary phase in the channels for optimal design of injection and detection regions. Charged functionalities such as either sulfonic acids or quaternary amines were incorporated during polymerization for generation of electroosmotic flow. These monoliths can be cast in situ in less than 10 minutes, are very reproducible with respect to separation characteristics, and allow easy manipulation of separation parameters such as charge, hydrophobicity, and pore size. Moreover, the solvent used to cast the polymer is chosen so as to allow electroosmotic flow to flush the channels thereby obviating any need for application of high pressures for conditioning. Rapid (50 seconds) and efficient (up to 600,000 plates/m) separations of peptides and amino acids were achieved in the microchip.