Two-dimensional capillary liquid chromatography based on microfractionation

Abstract

A two-dimensional capillary liquid chromatography (LC) method is described which is based on microfractionation, automated reinjection, and rechromatography using an automated microcolumn switching setup to separate complex peptide 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 separation mechanisms. The first-dimension separations were carried out by either reversed-phase chromatography or strong anion exchange chromatography. The second-dimension separation was in all cases reversed-phase capillary LC. In case of two-dimensional reversed/reversed-phase separations, some degree of orthogonality was achieved by using either two different sets of mobile phase solvents and one reversed-phase capillary LC column or a single set of mobile phases and two capillary LC columns packed with different-reversed-phase stationary phases. Electrospray ionization mass spectrometry was conducted to confirm the orthogonality of the developed two-dimensional capillary LC technique. The analysis of tryptic digests of cytochrome C and fetuin and a major histocompatibility complex (MHC) class I peptide mixture are described