Effects of column back pressure on supercritical fluid chromatography separations of enantiomers using binary mobile phases on 10 chiral stationary phases

The most obvious difference of packed column supercritical fluid chromatography (pSFC) from HPLC is the use of CO2 with an elevated column back pressure. However, how back pressure affects SFC separations using methanol/CO2 binary mobile phase has neither been thoroughly studied experimentally, nor well understood in theory. We studied the effects of back pressure for the most widely applied SFC enantiomeric separation. The impacts of back pressure on dead time (t0), solute retention (k), selectivity (α), efficiency (N) and resolution (Rs) were studied on 10 chiral columns using 11 pairs of drug-like enantiomers covering the common retention time range using the mobile phase of 20/80 methanol/CO2. Increased apparent t0 was observed at an increased column back pressure. Careful analysis of the experimental data indicated that t0 depends not only on the relative density change along the column length, but also on the adsorption of modifier (methanol) onto the stationary phase. The measured k over pressure was found to follow a linear relationship. Moreover, a linear correlation was derived between the k and the rate of retention change with pressure, dk/dP. The resulting empirical equation not only is universally applicable for all 10 tested columns, but also successfully explains the constant selectivity observed with column back pressure changing from 100 to 200 bar under the mobile phase condition. The observed slight efficiency loss due to pressure increase was found to be statistically significant. The combined effects of pressure from all the SFC attributes (t0, k, α and N) on resolution were confirmed by the experimental results from 48 enantiomeric resolutions. As the column back pressure increased from 100 to 200 bar, resolution decreased only slightly, on average 6%, due mainly to the retention and efficiency decrease. The importance and possible application of our study results will also be discussed.

► We systematically study pressure effects on chiral SFC separations.
► We discuss in detail the pressure effects on t0, k, α, N and Rs.
► We observe, for the first time, a linear relationship between dk/dP and k itself.
► Based on 48 enantio-separations, the α is generally independent on back pressure.
► Resolution decreases at higher back pressure due to retention and efficiency loss.