High-speed liquid chromatography by simultaneous optimization of temperature and eluent composition.

Abstract

Clearly, a major trend in liquid chromatography is to increase its speed to make it faster. Improving throughput for routine analysis of pharmaceutical release samples and stability assays are all key motivations for improving separation speed. Recent work has focused on the theoretical benefits of temperature on speed in liquid chromatography. We have shown that the 5-10-fold decrease in eluent viscosity that comes from a temperature increase of 175 degrees C over ambient, and the concomitant increase in analyte diffusitivity, act to dramatically decrease the time needed to generate a theoretical plate. Lower viscosities at elevated temperatures decrease the pressure drop across the column and allow the use of higher linear velocities as the pump pressure limit is approached. Simultaneously, faster analyte diffusion at higher column temperature improves efficiency at high eluent velocity conditions compared to the efficiency at lower temperatures at the same velocity. We find that higher temperature plays a central role in improving speed. In this work, we show that when the percent organic modifier in the eluent and column temperature are adjusted to keep retention factors fixed, highly efficient, subminute separations can be routinely achieved when a hot column is used at the maximum system back pressure. We find that the best way to facilitate such an optimization, assuming constant selectivity, is to use a very retentive column so that one can work at both high temperature and high volume fraction of organic modifier to achieve the lowest possible eluent viscosity. We have also analyzed the effect that key extracolumn contributions have on column selection and system design.