High peak capacity separation of peptides through the serial connection of LC shell-packed columns

High peak capacity was obtained for the separation of a HSA tryptic digest through the serial connection of three LC columns packed with shell material (C18, 15 cm x 4.6 mm, 2.7 um particle size). A major benefit of these particles, consisting of a 1.7 um solid core and a 0.5 um porous shell, is the small diffusion path, which reduces axial dispersion of solutes and minimizes peak broadening allowing lot higher resolving power to be obtained. Increase ill the temperature decreases the viscosity and the backpressure oil the columns and in this way allowed to couple multiple columns. Single and multiple column systems were first evaluated for the separation of a standard sample mixture in the isocratic mode; afterwards three columns could be coupled at 60°C, generating more than 90000 effective plates (200000 plates/meter) for the separation of HSA in gradient mode on a conventional HPLC instrument. Peak capacity ratios were found in good accordance with the theoretical gain in resolution, i.e values of 1.37 and 1.67 were measured, when doubling and tripling the length of stationary phases, respectively (the highest measured peak capacity on three coupled columns was 367).