Intraparticle diffusion of proteins in diol group-functionalized porous silica particle revealed by fluorescence correlation spectroscopy.

Size exclusion chromatography (SEC) is widely employed for the purification and separation of macromolecules; however, its underlying separation mechanism remains poorly understood. In this study, we investigated the intraparticle diffusion of proteins-lysozyme (Lz), myoglobin (Mb), hemoglobin (Hb), and horseradish peroxidase (HRP)-within diol-functionalized silica particle, which serve as the stationary phase in SEC, using fluorescence correlation spectroscopy (FCS). The intraparticle diffusion coefficient (D) was found to depend on protein sizes, whereas no significant pH dependence of D was observed. These findings suggest that the steric hindrance between the proteins and pore walls governs intraparticle diffusion, while electrostatic interactions play a negligible role. Consequently, our results demonstrate that the intraparticle diffusion of Mb and Lz observed via FCS primarily reflects bulk-like diffusion within the pore without any interactions between the proteins and particle surface. In contrast, surface diffusion is evident for HRP and Hb.