Tithonia diversifolia is a tropical plant composed of several compounds with medicinal properties. This study aimed to design a sequential process for the extraction of tagitinin C from T. diversifolia using organic solvents (acetone, acetonitrile, methanol, and ethanol) and purification in the ethanolic two-phase system (ETPS) formed by protic ionic liquids (PILs), polypropylene glycol 2000 (PPG-2000) and ethanol, reported here for the first time. The extraction process variables such as type of solvent, solid–liquid ratio, temperature, and process time were evaluated. Ethanol was the best tagitinin C extracting agent (702.1 µg/mL). Phase diagrams were constructed using PPG-2000, PIL, and ethanol at 298.15 K. The sequencing with the purification process occurred by adding the other constituents directly into the ethanol extract. Tagitinin C preferentially migrated to the top phase (rich in PPG-2000), while phenolic compounds (contaminants) were partitioned mainly to the opposite phase (PIL-rich phase). ETPS formed by [2HEA][Ac] (15.52 wt%) + PPG 2000 (49.91 wt%) + ethanol (34.57 wt%) at 298.15 K presented the highest selectivity (S = 18.3), extraction efficiency in the top phase (EE% = 86 %) and purification factor (3.4-fold), demonstrating the ability of ETPS to selectively and sequentially extract and purify tagitinin C.
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