Comparison of the gradient kinetic performance of silica monolithic capillary columns with columns packed with 3 μm porous and 2.7 μm fused-core silica particles.

The kinetic-performance limits of a capillary silica C18 monolithic column and packed capillary columns with fully-porous 3 μm and fused-core 2.7 μm silica C18 particles (all 5 cm long) were determined in gradient-elution mode for the separation of peptides. To establish a kinetic plot in gradient-elution mode, the gradient time to column dead time ratio (t(G)/t₀) was maintained constant when applying different flow rates. The normalized gradient approach was validated by dimensionless chromatograms, obtained at different flow rates and gradient times by plotting them as a function of the retention factor. The separation performance of the different column types was visualized via kinetic plots depicting the gradient time required to achieve a certain peak capacity when operating at a maximum system pressure of 350 bar. The gradient steepness (applying t(G)/t₀=10, 20, and 40) did not significantly affect the gradient performance limits for low (< 250) peak-capacity separations. For high peak-capacity separations the peak capacity per unit time increases when increasing the t(G)/t₀ ratio. The C-term contribution of the porous 3 μm and fused-core 2.7 μm was comparable yielding the same gradient kinetic-performance limits for fast separations at a column temperature of 60 °C. The capillary silica monolithic column showed the lowest contribution in mass transfer and permeability was higher than the packed columns. Hence, the silica monolith showed the best kinetic performance for both fast and high peak-capacity gradient separations.