Innovative Water-Free Two-Phase Systems Based on Acetonitrile, Polymers, and Ionic Liquids as a Platform for Selective Separation of Lycopene and Ascorbic Acid Present in Tomatoes

Water-free two-phase systems (WFTPSs) represent a promising alternative for separating hydrophobic biomolecules. This study reports novel systems composed of acetonitrile, polymers (polyethylene glycol and polypropylene glycol with different molecular weights), and ionic liquids (IL) based on [C2mim]X (X = acetate, methanesulfonate, and chloride). Phase diagrams were constructed and well described by the Merchuck equation, with high correlation coefficients (R2 = 0.985–0.999). Among the systems investigated, PPG-4000 + [C2mim][OAc] + ACN exhibited the largest biphasic region and the best performance for selective separation of tomato-derived biomolecules. At 308.15 K, lycopene preferentially migrated to the polymer-rich phase with an extraction efficiency of 96.0 ± 3.4%, while ascorbic acid accumulated in the IL-rich phase with 80.7 ± 0.5% recovery, yielding a maximum selectivity of 77.9. Thermodynamic analysis revealed that lycopene transfer was spontaneous (ΔtrG°m < 0), exothermic ΔtrG°m < 0 at 308 K, corresponding to the highest EELYC value (ΔtrH°m = −25.9 kJ mol–1), and entropy-driven (ΔtrS°m = 106 J mol–1 K–1), whereas ascorbic acid migration followed an endothermic and enthalpy-driven mechanism. These findings demonstrate that WFTPSs can be rationally designed for efficient separation of hydrophobic and hydrophilic biomolecules, providing a reproducible, low-cost, and versatile platform for bioseparations. Furthermore, such systems offer potential applications in the food, pharmaceutical, and nutraceutical industries, particularly for the sustainable recovery of antioxidant compounds from agro-industrial residues.