Unraveling the Anomalous Physicochemical Properties of Ethanol-Water Binary Solutions via Hydrogen-Bond-Driven Self-Assembly of Ethanol Clusters.

Conventional solvents such as water and ethanol exhibit anomalous physicochemical properties in binary ethanol-water solutions, including negative relative partial molar volumes and maxima viscosity, which challenge classical equilibrium thermodynamics. While prior studies have attempted to explain these phenomena through hydrogen-bonding network behavior, experimental evidence remains insufficient. This study investigates structure characteristics of ethanol self-assembly clusters driven by hydrogen bonding, employing ethanol dehydrogenase assays, electrochemical voltammetry, electrospray ionization-mass spectrometry (ESI-MS), and micro-oxygen bomb calorimetry to verify structural features across self-assembling ethanol molecules. Results demonstrate that the distribution of electrochemically oxidizable and enzyme-accessible monomeric ethanol molecules aligns with trends in the partial molar volumes. Specifically, the anomalous inflection point at 50% ethanol concentration arises from the predominance of tetrameric ethanol clusters, as validated by ESI-MS and calorimetric data. By integration of structural chemistry principles, this work systematically elucidates the molecular origins of ethanol-water anomalies, establishing a foundational framework for theoretical interpretation.