Band broadening in fast gradient high-performance liquid chromatography: application to the second generation of 4.6...

The sources of band broadening in fast gradient elution chromatography include the contributions of the tubings placed upstream the column, the isocratic migration of the sample that takes place in the column until the moment when the front of the gradient catches up with it, dispersion and mass transfer resistance during band migration under gradient elution to the column outlet, and dispersion in the tubings downstream the column to the detector. The measurements of all these contributions is meticulously described, analyzed, and applied to assess the kinetic performance of monolithic columns of the second generation (KGaA Merck) in gradient elution, at the highest possible speed at which they can be operated (t(0)=25s at P=200 bar). These results are compared to those provided by several 4.6mm × 100mm columns packed with sub-3 μm core-shell particles (Kinetex, Phenomenex and Halo, Advanced Material Technologies) and by a 2.1mm × 100mm column packed with fully porous 1.7 μm BEH particles (Waters). A RPLC checkout sample (Agilent) containing nine alkanophenones (molecular weights between 120 and 204 g/mol) was used for this purpose. The mobile phase was a mixture of acetonitrile and water and the instrument, an optimized version of the 1290 Infinity HPLC system (Agilent). The results show that the overall peak capacity provided by this second generation of monolithic columns is nearly 30% smaller than that provided by commercial columns packed with sub-3 μm core-shell particles and 20% smaller than that provided by a narrow-bore column packed with sub-2 μm fully porous particles. After isolating the sole band broadening contribution due to the gradient migration of the analytes along the columns, the BEH column proved to provide the highest resolution power (apparent average plate height, of 2 μm), followed by the 2.6 μm Kinetex particles (3.0 μm), the Halo particles (3.2 μm), and the second generation of 4.6mm I.D. monolithic columns (6.5 μm). This work demonstrates that analysts have an urgent need for very high pressure liquid chromatographs having smaller dwell and extra-column volumes that those of the currently available instruments. The gradient elution peak capacity provided by the modern, narrow-diameter columns, which are short and highly efficient (plate heights of 3-4 μm), is barely half what the column alone could produce if it were fitted to a perfect instrument. The other half of the peak capacity is just wasted by the instrument.