Optimizing conditions in online RPLC × SFC for the analysis of complex samples containing neutral compounds: Solving injection issues.

The online combination of reversed-phase liquid chromatography and supercritical fluid chromatography (online RPLC × SFC) is an attractive technique for the characterization of complex samples containing neutral compounds as the two techniques are highly complementary, especially with a polar stationary phase in supercritical fluid chromatography (SFC). However, the setup is challenging due to the presence of hydro-organic solvents in RPLC, which become injection solvent in SFC. In this study, numerous key experimental parameters were identified and found to have a major effect on peak shape under RPLC × SFC conditions. These parameters included the organic modifier in reversed-phase liquid chromatography (RPLC), the co-solvent in SFC, the gradient conditions and the column ID in SFC, the configuration of the valve and finally, the injection volume in SFC that should be maximized. Acetonitrile (ACN) in RPLC, a mixture of ACN and methanol (MeOH) (50/50, v/v) in SFC, a minimum initial composition of 5% B in SFC, column IDs of 1.0 mm and 2.1 mm in RPLC and SFC respectively and flushing the interface loops with pure CO₂ while adding the co-solvent after the valve, are all conditions that have been identified as perfectly suitable for online RPLC × SFC. They were successfully applied to the online RPLC × SFC separations of microalgae bio-oil samples. Despite unusual injection conditions, the peaks were symmetrical over the entire chromatogram, leading to a high separation power.