Visualizing demulsification of oil-in-water nano-emulsion in LC-TEM

Abstract text (incl. figure legends and references)

Nano-emulsions are known as nano-sized dispersions of at least one immiscible liquid in another liquid, kinetically stabilized by emulsifying agent(s), with vast possible applications [1]. One particular interest for probably all application fields is the stability of nano-emulsions, since their stability goes hand in hand with their functionality. However, generally applied procedures are limited to averaging over a bulk volume or are static. Liquid-cell TEM (LC-TEM) is a door opener to reveal local insights to temporally resolved nano-emulsion stability [2].

The here presented work aims direct visualization of kinetically stabilized oil-in-water nano-emulsions using LC-TEM, evaluating their morphology, dynamics and mechanisms of demulsification.

Ultrasonication and homogenization is used to generate droplets with HFE-7500 as the oil dispersed in a Zonyl (surfactant) containing aqueous continuous phase. The average droplet size depends on the applied shear rate and is controlled by dynamic light scattering with a Malvern Panalytical Zetasizer Nano. Protochips" Poseidon LC-TEM holder was used for scanning (S)TEM investigations with Microwell E-chips. (S)TEM studies were performed on a JEOL ARM200F equipped with a cold FEG and Gatan"s Oneview camera.

Nano-droplets of the oil phase (initial average diameter approximately 120 nm, as prepared) are directly visualized in both TEM (homogeneous dose rate of 17·10⁶ Gy/s for observation of dynamics) and STEM (averaged dose rate 1·10⁴ Gy/s for overview images) mode. The more strongly scattering oil phase appears brighter in annular dark-field STEM excluding a possible confusion with gas bubbles. Moreover, in closely confined regions at the borders of the used LCs, a destabilization of initial nano-droplets towards a formation of larger ones (average diameter 340 nm) is recognized. Surface charges on the LC membranes may locally change the concentration of surfactants that leads to coalescence. In addition to those static observations, in-situ TEM reveals demulsification by coalescence (figure 1) with a rate of 97·10³ nm³/s. This determined demulsification rate is orders of magnitude higher than other reported values from indirect determination methods for similar nano-emulsions, where Oswald ripening was assumed to occur [3]. The difference in mechanism and rate might be caused by strong influences of our experimental conditions, such as electron beam damage of surfactants or electric field trap for surfactants at LC membranes, both decreasing the surfactant concentration in the dispersion and therefore destabilizing the nano-droplets leading to coalescence.

Our approach successfully enables studies of oil-in-water nano-emulsions by direct imaging. Demulsification is observed to take place because of a confinement effects in the LC and because of electron beam illuminating effects inducing changes of the surfactant concentration followed by coalescence of nano-droplets. Excluding or reducing those electron beam effects should help to broaden the knowledge on nano-emulsion stabilities and demulsification processes even further. [4]

[1] Anton N et al.; Pharm Res 28 (2011)

[2] Vratsanos, MA et al.; ACS Nano 16 (2022)

[3] Delmas T et al.; Langmuir 27 (2011)

[4] Max Planck Society is acknowledged.

Figure 1: TEM images of nano-emulsion dynamics. Nano-droplets can immediately identified (a) and merge by coalescence during further electron beam exposure (b)-(d).