Optimization of experimental parameters for packed column supercritical fluid chromatography

The present research is a study of experimental parameters that affect the resolution of a test mixture of estrogens by packed column supercritical fluid chromatography. The parameters that were evaluated included fluid flow rate, effect on resolution and retention times of the type and concentration of four organic modifiers, namely methanol, ethanol, isopropanol, and acetonitrile. Also, the effect of column type on the resolution of a mixture of estrogen metabolites was studied. Two packing materials cyanopropyl silica and Betasil diol silica were selected for this study. The following percentages of each of the packing materials were used in columns connected in series for the separation of the test mixture:100.0% CPS, 37.5% CPS/62.5% Diol, 50% CPS/50% Diol, 62.5 CPS/32.5% Diol, and 100.0% Diol. The results indicated that connecting two columns having the same dimensions (a ratio of one-to-one CPS/Diol) in series gave the best resolution. There was no effect on retention times or resolution when the two columns were reversed, i.e., column order has no effect. Also, retention times were a function of the organic modifier; methanol gave the shortest retention times, with approximately the same separation factor as the other three modifiers.