Two-dimensional capillary liquid chromatography based on microfractionation

A two-dimensional capillary liquid chromatography (LC) method is described which is based on microfractionation, automated reinjection, and rechromato graphy using an automated microcolumn switching setup to separate complex p eptide mixtures from different origin. Different types of separations modes , i.e., hydrophobic and charge separation mechanisms, have been applied to increase the selectivity and peak capacity and to approach orthogonal separ ation mechanisms. The first-dimension separations were carried out by eithe r reversed-phase chromatography or strong anion exchange chromatography. Th e second-dimension separation was in all cases reversed-phase capillary LC. In case of two-dimensional reversed/reversed-phase separations, some degre e of orthogonality was achieved by using either two different sets of mobil e phase solvents and one reversed-phase capillary LC column or a single set of mobile phases and two capillary LC columns packed with different-revers ed-phase stationary phases. Electrospray ionization mass spectrometry was c onducted to confirm the orthogonality of the developed two-dimensional capi llary LC technique. The analysis of tryptic digests of cytochrome C and fet uin and a major histocompatibility complex (MHC) class I peptide mixture ar e described. (C) 1999 John Wiley & Sons, Inc.