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_intra) was found to depend on protein sizes, whereas no significant pH dependence of D_intra 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.