Supercritical fluid chromatography with fluorescence detection for tocochromanol profiling in oils and seed ethanol extracts : a comparative study with NPLC

This study focused on the development of a method designed to simultaneously separate four tocopherol (T), four tocotrienol (T3) homologues and plastochromanol-8 (PC-8) using supercritical fluid chromatography (SFC) with fluorescence detection (FLD). We examined the selectivity and resolution behaviour of tocochromanols on eight different stationary phases and investigated the retention factor of PC-8 (kPC-8) as a function of mobile phase composition, as well as the injection volume's impact on peak shape. The final SFC-FLD method developed involved a separation with two amino (NH2) columns connected in series (4.6 mm ID; 3 μm dp, 150 mm and 250 mm in length, respectively), at a flow rate of 2.5 mL/min, thermostated at 40 °C and a back-pressure regulator value of 10.0 MPa, injection volume of 10 μL and a gradient comprising of A:CO2 and B:MeOH. The sensitivity of SFC-DAD, SFC-FLD, NPLC-DAD and NPLC-FLD were compared and found to be 58.6-160.9, 5.9-51.9, 19.7-63.5 and 0.42-4.3 pmol, respectively. The SFC-FLD method developed was successfully applied to the analysis of seed ethanol extracts and plant oils and found to be suitable for the routine analysis. Furthermore, the SFC approach serves as a sustainable alternative compared to NPLC, due to the reduced human and environmental exposure and consumption of hazardous solvents.