Interconversion of gradient and isocratic retention data in reversed-phase liquid chromatography: Effect of the uptake of eluent modifier on the retention of analytes

The experimental technique of mass spectrometric tracer pulse chromatography was used to study the effect of the sorption of eluent components by a C₁₈-bonded silica RPLC packing on the retention of a series of test analytes during isocratic and gradient elution experiments. The analytes of interest were a substituted phenol, a substituted nitroaniline, an anti-malaria drug, tetrahydrofuran, and methanol. The eluent used was a mixture of acetonitrile and water. The solutes and isotopically labeled eluent components were injected at fixed time intervals during each gradient run. The mass specific detector allowed the assignment of individual analyte peaks even when there was overlap in the chromatograms from successive injections. Thus, the retention time of each analyte could be determined as a function of gradient slope and initial eluent composition at the time of each injection. Experimental gradient retention time data were then compared with the calculated results from two theoretical models. The first model assumed the velocity of the mobile phase and eluent were equal. The second and most realistic model assumed the velocity of the eluent was less than the velocity of the mobile phase due to the uptake of eluent by the stationary phase. Gradient retention times predicted by the two models were reasonably accurate with the sorption model giving slightly more accurate values. Inverse calculations, i.e., calculation of isocratic retention factors from gradient elution data were also carried out with very similar results. That is, the model allowing for the uptake of eluent was slightly more accurate than the model assuming no eluent-stationary phase interaction.