Patterns of protein adsorption in chromatographic particles visualized by optical microscopy

A new method is presented to image transient patterns of protein adsorption in individual spherical chromatographic particles under strong binding conditions. The method takes advantage of the difference in refractive index between the protein-free and protein-saturated adsorbent matrix. When the particles are viewed with an ordinary microscope using white light illumination, the adsorption front appears as a bright ring that moves in time from the surface of the particle to its center. Experimental data are obtained for the proteins lysozyme and albumin with a commercial agarose-based cation exchanger. Sharp rings are observed in both cases confirming that protein mass transfer within the particles occurs via a shell-progressive diffusion mechanism. Quantitative analysis based on the shrinking core model provides an accurate and precise way of determining the intraparticle diffusivity for individual particles as a function of protein concentration and mobile phase composition.