A novel on-line two-dimensional system was developed for peptide and protein mapping.The system combines capillary isoelectric focusing (cIEF) with pressurized capillary electrochromatography (pCEC) using a micro-inj ection valve as the interface.Sample fractions, which were focused and separated in the first dimensional cIEF based on their differences in isoelectric points (pIs), were electrically mobilized and further successively resolved by their differences in size, hydrophobicity, and electrophoretic mobility in the second dimensional pCEC.Therefore, this coupling system actually involves three dimensional separation mechanisms, namely isoelectric focusing, chromatographic partitioning and electrophoretic mobility.