The use of confocal laser scanning microscopy to study the transport of biomacromolecules in a macroporous support

Large-pore materials or supports resembling polymer conduits are used as packing material in chromatographic operations. Our ongoing research has shown that, when modified with peptides or ligands, chitosan beads that are 800 μm in diameter and have 3.5% solids can be used as matrices in bioseparations. The goal of the present study is to evaluate the transport properties of biomolecules in the modified chitosan beaded matrices. Batch uptake experiments with fluorescently tagged pure human IgG, human IgA and human IgM were conducted to visualize the distribution of binding sites throughout the bead as well as to evaluate restrictions to diffusion, if any, within the support. The chromatographic performance of the macrobeads was first assessed by the classical height equivalent of a theoretical plate HETP analysis. The independence of HETP on linear flow rates studied suggests that a likely mode of solute transport within the macrobeads may be a combination of convection and diffusion-convective components. By using fluorescent-tagged immunoglobulins, the penetration of the adsorbent particle at different times and different levels of saturation was visually observed. The profiles obtained from dynamic experiments were compared to the profiles obtained from finite bath experiments. With an increase in the incubation time, the degree of penetration increased and the bead interior was saturated with FITC immunoglobulins at the end point of the finite bath experiment. In the dynamic uptake experiment, the degree of penetration was found to be a function of the linear velocity and level of breakthrough. The penetration of the bead radius, at times lower than the predicted diffusion time, suggests that the mode of transport in the chitosan beads is governed by a combination of convective and diffusive forces.