Liquid-liquid Phase Seperation in Multicomponent Polymer Systems

Liquid-liquid phase separation (LLPS) is a fundamental thermodynamic process that drives self-organization in multicomponent polymer systems, with critical importance in both cell biology and materials science. While the phase separation of simple two component systems is understood, there has been less work on the effect of additional components on the kinetics and thermodynamics of the separation. Here, we investigate the mechanism by which small molecule additives tune the phase behavior of the canonical Polyethylene glycol (PEG)-Dextran aqueous two-phase system (ATPS). Using a combination of quantitative phase contrast microscopy and Differential Dynamic Microscopy (DDM), we systematically mapped the phase boundary of the PEG-Dextran system as a function of temperature and additive concentration. To distinguish the molecular interactions, we compared the effects of additives with varying hydrogen-bonding capabilities.  Our results show that small amounts of additives such as urea, formamide, and tetramethylurea (TMU) all significantly lower the critical phase separation temperature. Crucially, Liquid Chromatography-Mass Spectrometry (LC-MS) experiments provided direct evidence that urea preferentially binds to PEG, confirming that the phenomenon is driven by a specific polymer-additive interaction rather than a general solvent effect. These findings indicate that LLPS is governed by a complex interplay of specific, non-linear interactions including hydrogen bonding and hydrophobic effects, and that small amounts of additives can have significant effects on the thermodynamics of the separation.