Supercritical and unified chromatography with variable flow rate. I. Estimation of apparent retention factors

Unified chromatographic methods based on modern SFC instrumentation merge supercritical, subcritical and liquid states of mobile phase within a single instrument and a single analytical run, thus providing a high-performance platform for separation of tremendously wide range of analytes. Usually, a gradient of mobile phase starting with almost pure CO₂ proceeds to 100% of methanolic-aqueous modifier complemented with various additives. The increased viscosity of the liquid mobile phase limits the applicable flow rates towards the end of the gradient, especially when utilizing modern sub-2 µm particle stationary phases (e.g. UHPSFC columns). In order to stay within the instrument pressure limits and to adjust the mobile phase velocity optimal for chromatographic efficiency, a flow gradient and/or a backpressure regulator gradient can be employed. However, varying flow rate during separation precludes straightforward calculation of retention factors directly from retention times. To address this issue, our paper introduces a procedure for calculating apparent retention factors based on pumped mobile phase volumes continuously corrected for compressibility/expansion. The usefulness of the proposed approach is demonstrated for column screening and comparison of chromatographic selectivity in unified chromatography employing simultaneous gradients of mobile phase composition, mobile phase flow rate, backpressure regulator and makeup liquid flow rate.