Accurate measurements of frontal analysis for the determination of adsorption isotherms in supercritical fluid chromatography

The implementation of the traditional FA method is difficult with classical supercritical fluid chromatography (SFC) instruments. The instrument mixer and other sources of extra-column volumes are large and significantly broaden the fronts of injected plugs, which diminishes the precision and accuracy of the FA method. An SFC instrument was modified to permit more accurate determinations of adsorption isotherm data. The sample, the modifier, and CO2 are separately pumped via small volume connection tubes into a small volume mixer (250 μL), where they are mixed into a homogeneous fluid fed to the column. The extra-column volumes and the column hold-up volume were accurately measured at each back pressure from the retention times of tracers. This modified instrument was used to measure the adsorption isotherm of S-naproxen by frontal analysis (FA) on a (R, R)-Whelk-O1 column, using a mixture of methanol (20%, v/v) and CO2 as the mobile phase. Its performance is studied at several different back pressures from 100 to 210 bar. In all the experiments, the total flow rate was kept to a low value (1 mL/min) in order to minimize the variation of the equilibrium constant along the column. Although a suitable breakthrough curve could not be obtained at low back pressures (<150 bar) due to the closeness to the critical point pressure of the methanol/CO2 mixture, excellent results were obtained at higher back pressures (>150 bar), conditions remote from the critical point and breakthrough curves with very sharp front shocks are obtained. The RSDs of the profiles recorded at each back pressures are excellent, better than 1%.