Size-exclusion chromatography performed in capillaries. Studies by liquid chromatography-mass spectrometry

Miniaturization of the chromatographic column led to increased sensitivity and shortened time of analysis. In our work we applied 300 microm I.D. capillaries packed with a novel stationary phase Superdex Peptide for the size-exclusion chromatography, capable of separating molecules within the mass range of 0.1-7 kDa. Here we proved that such capillary columns can operate effectively at high sensitivity. Several peptide mixtures were efficiently chromatographed and analyzed on line with electrospray ionization mass spectrometry as a detection technique. A CNBr peptide map, derived from human globin alpha subunit, was effectively separated using this method. These fragments are difficult to elute from the reversed-phase column at low pH, therefore, such approach can be considered as a complementary to other separation techniques, in particular for analyzing hydrophobic components and complex mixtures.