Mechanistic study on metastable and stable liquid-liquid phase separation explored by molecular simulation and thermodynamic analysis

Liquid-liquid phase separation (LLPS) has been proven to play a key role in molecular self-assembly both in vivo and in vitro, and it is of great significance for life science and materials science. Comparative elucidation and research are essential for comprehensive understanding of different types of LLPS. Here, citicoline sodium was taken as model molecule to study stable and metastable LLPS. Process analytical technologies (PAT) were applied to characterize the process. Ternary phase diagrams and the solubility parameters of LLPS region were determined and discussed. Moreover, a combination of molecular simulations and spectroscopy was applied to analyze the mechanism of stable and metastable LLPS. Spectroscopy analyses showed that the solute molecules in metastable liquid intermediate were in a metastable state. And the MD simulation results indicated that LLPS was stable when the system was in the solvation-enhanced state, while metastable when the system was in the desolvation state.