Gradient chromatography under constant frictional heat: realization and application

A new mode of gradient elution in liquid chromatography is proposed. It is derived from simple theoretical considerations and is particularly suitable for applications to fast, very-high pressure gradients. It is designed to improve the injection-to-injection repeatability of chromatographic runs at either constant flow (cF, cooling case scenario) or constant pressure (cP, heating case scenario). The purpose of this original gradient mode is to minimize the variations of the temperatures and the pressures across and along the column during the gradient time. These variations are caused by the heat generated in the column due to the friction of the eluent percolating the bed. Any temperature fluctuation affects to some extent the precision of the measurements of retention times and bandwidths of eluted compounds. The minimization of this effect was achieved by maintaining constant the frictional heat power (i.e., the product of the flow rate by the column pressure drop) generated during the gradient run, the washing step, and the re-equilibration time. The eluent temperature was recorded at the column outlet. One useful application of gradient chromatography at constant frictional heat (cFH) is illustrated for a 50-100% volume gradient of acetonitrile in water using a 4.6mm × 150 mm column packed with 3.5 μm BEH-C18 particles and operated with an Agilent 1290 Infinity liquid chromatograph. The reproducibility (eleven consecutive injections) of the retention times and peak variances of nine small molecules (RPLC check-out sample mixture) using cF, cP, and cFH gradients were compared for the same amount of heat produced (403 J) during each run time. The RSDs of the retention times and the peak variances for four consecutive injections were systematically below 0.035 and 0.50% in constant frictional heat gradient chromatography, after three initial injections. These RSDs were markedly higher for cF (0.050 and 0.90%) and cP (0.070 and 1.80%) gradients.