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

Abstract

Miniaturization of the chromatographic column led to increased sensitivity and shortened time of analysis. In our work we applied 300 μm 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.