Comparison of ultra-high performance supercritical fluid chromatography and ultra-high performance liquid chromatography for the analysis of pharmaceutical compounds

Abstract

Currently, columns packed with sub-2μm particles are widely employed in liquid chromatography but are scarcely used in supercritical fluid chromatography. The goal of the present study was to compare the performance, possibilities and limitations of both ultra-high performance liquid chromatography (UHPLC) and ultra-high performance supercritical fluid chromatography (UHPSFC) using columns packed with sub-2μm particles. For this purpose, a kinetic evaluation was first performed, and van Deemter curves and pressure plots were constructed and compared for columns packed with hybrid silica stationary phases composed of 1.7 and 3.5μm particles. As expected, the kinetic performance of the UHPSFC method was significantly better than that of the UHPLC. Indeed, the hmin values were in the same range with both strategies and were between 2.2 and 2.8, but uopt was increased by a factor of >4 in UHPSFC conditions. Another obvious advantage of UHPSFC over UHPLC is related to the generated backpressure, which is significantly lower in the presence of a supercritical or subcritical fluid. However, the upper pressure limit of the UHPSFC system was only ∼400bar vs. ∼1000bar in the UHPLC system, which prevents the use of highly organic mobile phases at high flow rates in UHPSFC. Second, the impact of reducing the particle size (from 3.5 to 1.7μm) was evaluated in both UHPLC and UHPSFC conditions. The effect of frictional heating on the selectivity was demonstrated in UHPLC and that of fluid density or decompression cooling was highlighted in UHPSFC. However, in both cases, a change in selectivity was observed for only a limited number of compounds. Third, various types of column chemistries packed with 1.7μm particles were evaluated in both UHPLC and UHPSFC conditions using a model mixture of acidic, neutral and basic compounds. It has been shown that more drastic changes in selectivity were obtained using UHPSFC columns compared to those obtained by changing UHPLC columns. In addition, there was a good complementarity between the two separation modes. Finally, by combining the use of small particles with supercritical fluids as a mobile phase, it was possible to achieve the analysis of pharmaceutical compounds in less than 1min or to attain a peak capacity of more than 250 in approximately 40min, both with a high degree of repeatability.