Nanoscale Insights:Exploring Undersaturated Crystallizationof Flufenamic Acid with Liquid Phase Transmission Electron Microscopy

Pharmaceutical crystallization traditionally requires supersaturation to occur and is typically monitored through macroscopic characterization methods that can measure induction times and growth rates. However, detailed insights revealing the precise steps and mechanisms of crystal nucleation and growth dynamics are lacking. This study explores the crystallization of a nonsteroidal anti-inflammatory drug, flufenamic acid, in undersaturated ethanol and isopropanol solutions using liquid phase transmission electron microscopy (LPTEM). LPTEM is a technique that provides real-time observations of nanoscale processes. Continuous electron-beam irradiation was found to induce nucleation and crystal growth at concentrations far below saturation. LPTEM can extract key kinetic parameters such as nucleation induction times and growth rates that inform and refine mechanistic models of crystallization. Due to the undersaturated nature of the crystallization conditions, these findings suggest newly discovered nucleation pathways, alternative to classical nucleation theory, revealing previously inaccessible mechanisms in crystallization of organic molecules.