Enzymatic digestion and liquid chromatography in micropillar reactors: hydrodynamic versus electro-osmotic flow

Enzymatic digestionand peptide separation are basic steps for preparation of protein samples prior to their analysis by mass spectrometry. Micro-pillar reactors for digestionand reverse phase liquid chromatography were designed and constructed using semiconductor technologies. The performances of the micro-machined reactors were evaluated: complete Cytochrome C digestionwas achieved in 6 min for a concentration up to 25 pmol.μl -1 and the separation micro-column was seen to exhibit separation capabilities and capacity close to those obtained with a commercial column. Furthermore, a comparative study between hydrodynamic and electroosmotic driven flows was performed for each peptide of a Cytochrome C digest. It was demonstrated that parasitic electrophoretic phenomena disturbed peptide mobility but not protein identification.