Realization and potential advantages of gradient separations performed under steady state temperature regime

•Gradients have 3 steps, ramping eluent concentration, washing and equilibrating the column.•During all three steps the temperature distribution is constant throughout the column volume.•The gradient timetable keeps constant the heat power dissipated through the column wall.•The largest variation of the temperature during the three steps of a gradient run is below 0.4 °C.•The RSDs of retention times and peak widths in fast HPLC are below 0.1% and 2.0%, respectively.

This paper describes a new method designed to perform gradient separations while keeping constant the distribution of temperature throughout the column during the three phases of a gradient run (the ramp up of the strong eluent concentration, the column wash, and the re-equilibration steps) in high pressure liquid chromatography (vHPLC). The method is based on the solution of the integrated heat balance equation along the column knowing the variations of the eluent specific heat, its thermal expansion coefficient, and its viscosity with the concentration of the strong eluent. This new method is called gradient under constant wall heat (cWH). Its value is demonstrated by the stability of the eluent temperature at the column outlet during a series of consecutive gradient runs. The potential advantages of cWH gradients over classical gradients execution mode at constant flow rate (cF) are illustrated in the example of two such gradients run in the same time. They include (1) a diminution by a factor 8 of the largest temperature variation during the sequence of injections (0.4 °C versus 3.2 °C); (2) a reduction of the baseline noise by a factor two (0.05 mAU versus 0.10 mAU); and (3) the achievement of an excellent injection-to-injection repeatability of the vHPLC system used to measure the first (average RSDs, 0.04%) and the second central (average RSDs ≃ 0.75%) moments of the peaks of an eight-compounds mix, baseline separated, from the first to the last injection.