Sander Delahaye

Design and evaluation of various methods for the construction of kinetic performance limit plots for supercritical fluid chromatography

Supercritical fluid chromatography (SFC) is attributed many advantages over high performance liquid chromatography (HPLC). Next to the fact that SFC is greener than HPLC, which is especially important for preparative separations, SFC is claimed to be able to deliver faster separations at higher efficiencies (N) than HPLC. This is due to the higher diffusitivity of analytes in supercritical fluids compared to liquids (higher optimum mobile phase velocity) and to the lower viscosity of the mobile phases in SFC compared to HPLC, which results in smaller pressure drops allowing the use of longer columns and/or columns packed with smaller particles at higher velocities. In order to quantify this claimed kinetic performance advantage, it is essential to construct unbiased kinetic plots to make the comparison between HPLC and SFC. The high compressibility of the mobile phase in SFC however makes this problematic. A variable column length (L) kinetic plot method is therefore developed in this work. Because the pressure history in the column is kept constant for every data point in this method, this way of working definitely delivers exact values for the kinetic performance limits in SFC. It is shown that the traditional way of measuring the performance as a function of flow rate (fixed back pressure and column length) cannot deliver the same correct results as this variable L method. However, the isopycnic way of working on a fixed column length has also been proven to be a good alternative for the expensive and time consuming variable L method. Finally, isopycnic kinetic plots are used to compare SFC and HPLC performance in a quantitative way.

Application of the isopycnic kinetic plot method for elucidating the potential of sub-2 µm and core-shell particles in SFC.

In this work the isopycnic method to construct kinetic plots for SFC was used to investigate the performance limits of an SFC system when using sub-2 µm fully porous particles and sub-3 µm superficially porous (core-shell) particles. This isopycnic kinetic plot method for SFC was developed and tested earlier for SFC separations on native silica with pure CO2 as mobile phase. In the current work, octadecyl based reversed phase columns were used in combination with a mobile phase that contains 10% methanol as modifier in order to study the applicability of the described methodology to assess the kinetic performance limits of experimental setups in which SFC is used and will, according to all probability, be evolving. SFC and HPLC van Deemter and kinetic plots are constructed for columns packed with fully porous particles with various diameters and for a column packed with core-shell particles. The influence of the experimental kinetic performance limits of the particle diameter and morphology in SFC is shown to be the same as in HPLC. Additionally, kinetic plot predictions were constructed for separations on 1 µm and 0.5 µm particles using the data measured on the 5 µm, 3.5 µm and 1.8 µm fully porous particles. By doing this the potential applicability of 1 µm particles on the contemporary SFC and HPLC systems was demonstrated together with the irrelevance of the use of 0.5 µm particles in SFC.

Implementing Stationary-Phase Optimized Selectivity in Supercritical Fluid Chromatography

The performance of stationary-phase optimized selectivity liquid chromatography (SOS-LC) for improved separation of complex mixtures has been demonstrated before. A dedicated kit containing column segments of different lengths and packed with different stationary phases is commercially available together with algorithms capable of predicting and ranking isocratic and gradient separations over vast amounts of possible column combinations. Implementation in chromatographic separations involving compressible fluids, as is the case in supercritical fluid chromatography, had thus far not been attempted. The challenge of this approach is the dependency of solute retention with the mobile-phase density, complicating linear extrapolation of retention over longer or shorter columns segments, as is the case in conventional SOS-LC. In this study, the possibilities of performing stationary-phase optimized selectivity supercritical fluid chromatography (SOS-SFC) are demonstrated with typical low density mobile phases (94% CO2). The procedure is optimized with the commercially available column kit and with the classical isocratic SOS-LC algorithm. SOS-SFC appears possible without any density correction, although optimal correspondence between prediction and experiment is obtained when isopycnic conditions are maintained. As also the influence of the segment order appears significantly less relevant than expected, the use of the approach in SFC appears as promising as is the case in HPLC. Next to the classical use of SOS for faster baseline separation of all solutes in a mixture, the benefits of the approach for predicting as wide as possible separation windows around to-be-purified solutes in semipreparative SFC are illustrated, leading to significant production rate improvements in (semi)preparative SFC.