Cryogenic Electron Microscopy Study of Nanoemulsion Formation from Microemulsions Han Seung Lee, † Eric D. Morrison, ‡,∥ Chris D. Frethem, § Joseph A. Zasadzinski, † and Alon V. McCormick* ,† † Department of Chemical Engineering and Materials Science , University of Minnesota, Minneapolis, Minnesota 55455, United States ‡ Ecolab Food and Beverage Division, 655 Lone Oak Drive, Eagan, Minnesota 55121, United States § Characterization Facility, University of Minnesota, Minneapolis, Minnesota 55455, United States * S Supporting Information ABSTRACT: We examine a process of preparing oil-in-water nanoemulsions by quenching (diluting and cooling) precursor microemulsions made with nonionic surfactants and a cosurfactant. The precursor microemulsion structure is varied by changing the concentration of the cosurfactant. Water-continuous microemulsions produce initial nanoemulsion structures that are small and simple, mostly unilamellar vesicles, but microemulsions that are not water-continuous produce initial nanoemulsion structures that are larger and multilamellar. Examination of these structures by cryo- electron microscopy supports the hypothesis that they are initially vesicular structures formed via lamellar intermediate structures, and that if the lamellar structures are too well ordered they fail to produce small simple structures. ■ INTRODUCTION Finely dispersed oil-in-water emulsions have drawn consid- erable interest for use in personal care products, food products, and pharmaceuticals. 1−10 The term nanoemulsion 11 is usually used to imply that the oil droplets are in the range of 20−500 nm, 12 kinetically stable in a low viscosity aqueous dispersion. To prepare nanoemulsions mechanically, it is necessary to provide a great deal of power, overcoming increasing Laplace pressure to create droplets small enough to achieve kinetic stability. 13−15 Alternatively, there is increasing interest in low- energy chemical methods that create nanodroplets using paths in the phase diagram that induce high interfacial curvature. 16 When the path is created by temperature change, these methods are usually termed phase inversion temperature (PIT) methods,; 17−19 when the path is created by a composition change, they are variously termed spontaneous emulsifica- tion, 8,12,20,21 phase inversion concentration, 12,22,23 emulsion inversion point, 24−26 catastrophic phase inversion, 27 or self- emulsification methods. 8,28 Wadle et al. showed with such methods that the structure of the precursor emulsion can affect the stability of nanoemulsion, 29 and Kü hnle and co-workers suggested that the smallest nanodroplets may form if the composition path traverses regions of lamellar or micro- emulsion-like structures. 24 Solans and co-workers 17 and Dong and co-workers 30 noted the merit of simply beginning with a deliberately prepared, stable microemulsion as precursor, then diluting that microemulsion to make a kinetically stable nanoemulsion. Pons et al. 31 showed that, with such an approach, larger amounts of water in the microemulsion can result in smaller nanoemulsion structures. A modification of this last approach is what we will undertake in this work; we will examine the preparation of nanoemulsions by quenching microemulsion precursors, adding cooled water to warm microemulsions both to quickly dilute and to cool to room temperature. The microemulsions are made with n- hexadecane, water, and nonionic alkylphenolethoxylate surfac- tants. 32 Varying amounts of alkylphenol cosurfactant was used to afford the opportunity to examine the use of differently structured precursor microemulsions (cf. Salager and co- workers 33,34 ). Like previous works, 23,35−38 in this paper, conductivity and visual transparency are used to detect the microemulsion temperatures, and light scattering is used to determine the average size of the structures in the quenched nanoemulsion (well before any ripening or separation). We also directly image the structures with cryo-electron microscopy (cryo-EM), seeking to rationalize their structure and formation mechanism. Previously Saupe and co-workers 39 showed the usefulness of cryo-SEM to investigate the effect of compositional variables in a nanoemulsion preparation, and Heunemann et al. 40 used cryo-TEM images to support a hypothesis of a bicontinuous intermediate structure. Received: June 5, 2014 Revised: August 13, 2014 Published: August 20, 2014 Article pubs.acs.org/Langmuir © 2014 American Chemical Society 10826 dx.doi.org/10.1021/la502207f | Langmuir 2014, 30, 10826−10833