| Limitations of mass transfer in chromatographic bioseparations employing traditional packed particles have fuelled the inception and development of alternative stationary phases with improved performance characteristics. This work investigates case studies in two categories of these alternative media, namely polymer-modified packed particles and continuous monolithic phases, for insight into their enhanced properties. Specifically, high-resolution microscopy techniques and image-based analysis algorithms were implemented to extract morphology information for these materials, in an attempt to elucidate the relation between microstructure and performance. For the monolith, mesoscopic simulation methods were also employed for a more rigorous analysis of the flow and dispersion behavior.;Scanning and transmission electron microscopy images of the commercial polymer-modified, agarose-based particle Sepharose XL were compared to those for its unmodified counterpart, Sepharose FF. Local regions in the composite dextran-agarose Sepharose XL particles were noted to exhibit a denser network of fibers and smaller pore sizes overall, compared to those in the traditional Sepharose FF particles. Images of particles equilibrated with high concentrations of protein revealed a significant difference in protein localization patterns, with the stained protein in XL occupying a markedly higher area fraction of the images. This suggests a higher volume available for adsorption and provides visual clues into how the consistently higher static capacity of these polymer-modified particles is manifested. Treatment of the XL particles with dextranase, an enzyme that breaks down dextran, resulted in a reduction of protein coverage, providing evidence that it is indeed the dextran that is responsible for the improved static capacity in this polymer-modified stationary phase.;Imaging and image analysis techniques were also used to analyze the commercial CIMTM disk monolith. Two- and three-dimensional pore size distributions obtained for the CIM disk using image processing algorithms were found to deviate significantly from the manufacturer-reported experimental mercury intrusion results, the difference being attributed to the local nature of the image-based methods or assumptions and limitations inherent in the experimental mercury intrusion method. A probe placement algorithm was introduced to estimate solute capacity from the explicit geometry of the monolith.;To enable a precise description of both flow and the geometry for a rigorous analysis of dispersion, a three-dimensional sample block for the CIM disk was reconstructed using serial imaging and sectioning, and the flow and mass transfer were simulated using a lattice-Boltzmann method and a particle-based random-walk method, respectively. Flow simulations hinted at the partitioning of flow into high- and low-velocity regions and the dispersion simulations obtained on top of the velocity field revealed artifacts in particle trajectories due to the symmetry of the lateral flow with respect to the periodic boundaries. Constraining the simulation length to reduce the effect of this symmetry yielded dispersion behavior suggestive of channeling, hinting that the sample geometry might not be representative of the macroscopic structure. Simulations of the local behavior of finite particles predicted the experimentally observed entrapment behavior, as well as the increase of the entrapment level with flow rate. Analysis of trajectories provided support for a previously hypothesized mechanism for entrapment. |
