A Multiscale Assessment of Oil-in-Water, Water-in-Oil, and Bicontinuous Structures in 2-Butanol/Water/Citric Acid surfactant-free microemulsions

Surfactant-free microemulsions (SFMEs) are attracting growing interest as sustainable alternatives to conventional microemulsions, yet their structural organization remains incompletely understood. Here, we investigate a novel green SFME based on water, 2-butanol, and citric acid, combining dynamic light scattering, small-angle X-ray scattering, fluorescence spectroscopy, confocal laser scanning microscopy, and rheology to probe its organization from the nano- to the microscale. Samples located near the phase boundary exhibited a complex multiscale structure. Scattering measurements revealed the coexistence of nanometric and submicrometric-to-micrometric domains, while direct confocal observations confirmed the presence of micron-sized aggregates in optically transparent formulations. Fluorescence spectroscopy indicated that the local hydrophobic environment remained essentially unchanged across compositions, suggesting that structural evolution is mainly governed by changes in domain size and connectivity. As the oil-to-water ratio approached unity, the scattering behaviour deviated from that observed for the other formulations, indicating the emergence of a distinct structural regime. Combined scattering, microscopy, and rheological analyses identified this region as consistent with a bicontinuous organization already present below the macroscopic phase-separation temperature. Temperature-dependent experiments further revealed domain growth and enhanced viscoelastic contributions preceding visible phase separation. These findings demonstrate that transparent water/2-butanol/citric acid SFMEs can host pre-ouzo, ouzolike, and bicontinuous structures, providing new insight into the structural complexity of sustainable hydrotrope-based microemulsions.