Comprehensive two-step supercritical fluid extraction for green isolation of volatiles and phenolic compounds from plant material

Extraction of compounds with different physicochemical properties from a complex matrix usually involves several individual steps and requires large volumes of organic solvents. In this pioneering study, we propose a comprehensive two-step supercritical fluid extraction using carbon dioxide, ethanol, and water. This novel approach allows the extraction of non-polar and polar analytes within one run in two consecutive steps. Indeed, the first step with a dominant amount of CO 2 with only 2% cosolvent allowed the selective extraction of non-polar volatile terpenes only in 20 min. The conditions were then automatically switched. Increasing the cosolvent volume in the extraction solvent up to 44% (v/v) resulted in the extraction of more polar compounds, including flavonoids and phenolic acids, in 60 min. Importantly, switching the supercritical fluid extraction (SFE) conditions does not require any manual intervention but results in two separate fractions containing target compounds with distinctly different physicochemical properties. The novel method was verified in terms of repeatability, accuracy, precision, and greenness. Two-step SFE was applied to seven plant species differing in volatile terpenes and phenolic profiles. The results proved that this concept is suitable for the analysis of complex plant samples. In addition, it enables a reduction in the toxic solvents consumption, extraction time, and manual intervention required for traditional extraction approaches when isolating different groups of metabolites. A comprehensive two-step supercritical extraction using green solvents allows the extraction of non-polar and polar analytes within one run in two consecutive steps.