<p>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 (<i>D</i>_intra) was found to depend on protein sizes, whereas no significant pH dependence of <i>D</i>_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.</p> Graphic Abstract <p></p>