Abstract

Aqueous three-phase systems (ATPS) have emerged as versatile platforms for biomolecule extraction due to their mild conditions and high selectivity. Among the various phenomena influencing ATPS behavior, the triple solutingout effect stands out for its significant thermodynamic implications and practical applications. This phenomenon, characterized by the formation of a third phase upon addition of a solute, has garnered attention for its ability to enhance biomolecule partitioning and purification. In this study, we delve into the thermodynamics underlying the triple soluting-out effect in ATPS and explore its practical implications in biomolecule extraction. Through comprehensive theoretical analysis and experimental validation, we elucidate the role of solvent composition, temperature, and molecular interactions in driving phase separation and biomolecule partitioning. Furthermore, we highlight the diverse applications of the triple soluting-out effect in extracting various biomolecules, including proteins, enzymes, and nucleic acids, from complex biological matrices. The synergistic combination of thermodynamic insights and practical applications presented in this study offers new perspectives for optimizing biomolecule extraction processes and advancing biotechnological applications.