Applications of isopycnic measurements in supercritical fluid chromatography

Kinetic plots have gained increasing interest in the last decade because, contrary to van Deemter curves, they bring into account the relationship between plate height, column permeability, particle size and column length in one type of curve. Kinetic plots in SFC cannot be constructed in the same way as in HPLC because measuring the performance of an SFC separation at different flow rates on a fixed column length using a fixed back pressure results in varying retention factors (k) and diffusion coefficients (Dm) of the analytes. As a result, the measured values for plate height (H) do not truly represent the evolution of the performance with the mobile phase velocity because also the other parameters that influence H (like k and Dm) are not kept constant and extrapolation errors are made when applying the conventional kinetic plot method developed for HPLC to SFC. In this contribution, it is shown that measuring the performance of an SFC separation as a function of flow rate and correctly extrapolating this values to the kinetic performance limits is possible when the measurements are performed in an isopycnic way. This means that the average column density is fixed by decreasing the back pressure when the flow rate is increased. In this way, constant retention factors as a function of flow rate are observed and correct kinetic plots are constructed. The use of this isopycnic kinetic plot method is shown to be adequate to compare SFC separations with different pressure characteristics as SFC separations on columns packed with particles of varying size are compared. Correct comparisons between SFC and HPLC separations on all kind of columns are performed as well. The isopycnic approach also allows implementations of the stationary phase optimized selectivity methodology in SFC separations (SOS-SFC). In this approach, basic measurements on column segments with different stationary phases are performed after which a prediction is made of the retention factors of the compounds on all possible combinations of these column segments. This is followed by straightforward computerized prediction of the column combination generating the best separation. As the methodology relies on the predictivity of retention factors for the use of different column lengths at a fixed flow rate, the isopycnic methodology is introduced to ensure control thereof in SFC, allowing the development of SOS-SFC. This is practically accomplished by applying a lower back pressure when a longer column is used