The evolution of HB-networks across three regimes of X_EG is illustrated: (i) bulk-like water with a percolating HB network, (ii) a mixed HB phase balancing waterwater and EGwater interactions, and (iii) an EG-rich phase dominated by EG self-association and hydrophobic clustering, correlating with cryoprotective efficiency.

Abstract

Understanding the molecular mechanisms of cryopreservation is crucial for optimizing antifreeze formulations. In this study, we investigate the hydrogen bond (HB) configurations of aqueous ethylene glycol (EG) solutions using a combined approach of Fourier transform infrared (FTIR) spectroscopy and molecular dynamics (MD) simulations. Our results reveal that EG progressively integrates into the HB network, modifying the structural organization of water across different concentrations. At low EG content, water maintains its percolating HB-network, while at intermediate concentrations (X _EG ≈ 0.3–0.6), a mixed HB configuration emerges, balancing EG–water and water–water interactions. This structural transition correlates with the lowest freezing point and the most efficient cryoprotective behavior. Beyond X _EG > 0.6, EG self-association dominates, reducing water's HB connectivity and inducing hydrophobic clustering effects. The analysis of HB populations and tetrahedral order parameters (TOP) confirms that EG disrupts the extended tetrahedral HB framework of water, thereby delaying ice nucleation. These findings establish a direct correlation between local solvation structures and cryoprotective efficiency, highlighting the importance of mixed HB environments in tuning antifreeze functionality.