Aim: This study aimed to investigate the influence of the preparation process and composition on the microstructure of the O/W primary emulsions and the corresponding impact on the formation of oil-in-water-in-oil (O/W/O) multiple emulsions. Objectives: Multiple emulsions were prepared by a two-step emulsification method and the microstructure was characterized by the microscope. Methods: The primary emulsion was prepared by four kinds of preparation methods, which include both high-energy and low-energy emulsification, and then the primary emulsion was re-emulsified by stirring in the outer phase. Result: Through the theoretical investigation and the corresponding verification experiments of the interfacial film, the geometric reason for O/W/O multiple emulsion which was relatively difficult to prepare has been found. The microstructure of O/W particles was more obvious, and the particle size became smaller with the increase of the hydrophilic emulsifier amount beneficial to the formation and stability of O/W/O structures. However, the excess emulsifier that existed in the water phase could interfere the stability of the W/O interface. Moreover, the viscosity of inner oil phase had a large influence on the formation of O/W/O emulsion by affecting the particle size of the primary emulsion and the dynamic equilibrium between the inner and outer oil phase. Conclusion: It can be concluded that fine multiple emulsions were formed when the particle size of the primary emulsion was moderate since the large particles would break through the outer interface membrane and small particles would combine with the outer oil phase due to the Ostwald ripening.
