Knowledge of the nanoscale distribution of proteins in chromatographic resins is critical to our mechanistic understanding of separations performance. However, the nano- to mesoscale architecture of these materials is challenging to characterize using conventional techniques. Small-angle neutron scattering was used to probe (1) the nano- to mesoscale structure of chromatographic media and (2) protein sorption in these media in situ with protein-scale resolution. In particular, we characterize the effect of the architecture of cellulose-based and traditional and dextran-modified agarose-based ion-exchange resins on the nanoscale distribution of a relatively small protein (lysozyme) and two larger proteins (lactoferrin and a monoclonal antibody) at different protein loadings. Traditional agarose-based resins (SP Sepharose FF) can be envisioned as comprising long, thin strands of helical resin material around which the proteins adsorb, while higher static capacities are achieved in dextran-modified resins (SP Sepharose XL and Capto S) due to protein partitioning into the increased effective binding volume provided by the dextran. While protein size is shown not to affect the underlying sorption behavior in agarose-based resins such as SP Sepharose FF and XL, it plays an important role in the cellulose-based S HyperCel and the more highly cross-linked agarose-based Capto S, where size-exclusion effects prevent larger proteins from binding to the base matrix resin strands. Based on the data, we propose that entropic partitioning effects such as depletion forces may drive the observed protein crowding. In general, these observations elucidate the structure and point to the mechanism of protein partitioning in different classes of chromatographic materials, providing guidance for optimizing their performance.