The multiscale events critical to chromatographic bioseparations mean that measurement of all the parameters necessary to create mechanistic models is challenging. Several parameters such as tortuosity may have to be assumed based on correlations. The experimental techniques we do have are typically ensemble or summative measurements based on the chromatographic behaviour of tracer molecules and are indirect in their nature. We use techniques capable of measurement across 6 orders magnitude to directly investigate; 1) the structure of the packed bed, 2) single bead pore geometry, 3) adsorbed proteins at the solid-liquid interface. In addition to the increased assurance these techniques give for model development they also enable us to resolve heterogeneity across this range of scales. Three dimensional X-ray tomography measurements of packed beds are performed on two different resin materials requiring optimisation of column imaging to be performed for each. The results produce three dimensional representations at 3µm resolution. The effect of packing upon the bead structure and the visualisation of wall effects are among the heterogeneities that can be observed. The same technique in a different configuration was used to probe bead internal structure. By using settings that provided a trade-off between voxel-size and field of view, the whole bead was imaged at a pixel size of 63nm and an internal volume of interest at 32nm. To measure the structure of the adsorbed layer we use neutron scattering techniques to directly measure adsorbed monoclonal antibodies. Neutron reflectivity is used to characterise IgG bound to protein A attached to a silica surface to produce a configuration representative of an affinity chromatography resin. Adsorbed IgG structures extended up to 230 Å away from the surface and are suggestive of two IgG molecules bound to protein A with a somewhat skewed orientation in close proximity to the silica surface.

From bed to bead and below: Multiscale measurements of chromatography

253rd National Meeting (2017)

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