A theoretical study of the optimization of gradients at elevated temperature

In order to achieve fast chromatographic separations of unknown entities, very rapid gradients need to be performed. We have studied the influence of temperature on such rapid gradient separations carried out under reversed phase conditions. The tool by which the quality of a gradient separation is measured is the peak capacity. Elevated temperature has two major influences. The first one is a reduction in viscosity, which reduces the backpressure for a given set of operating conditions. The second effect is an improvement in the diffusivity of the analytes, which speeds up mass transfer. We have assessed the influence of flow rate, gradient duration, and temperature on peak capacity using columns with different particle sizes and column lengths. The beneficial effect of temperature on both the viscosity and the diffusivity of the sample results not only in an improvement of the speed of the separations but also in a reduction of the back pressure under optimal operating conditions. At reasonable pressures, good separation power can be achieved in as little as one minute or less.