Over the last decade the pharmaceutical industry has been experiencing a scientific revolution, resulting in more biological-based therapeutics, also called biologics. Biologics are largely proteins or peptides, which have created a bottleneck in optimizing their purification processes. The major problem resides in the inability to predict and apply empirical processes to protein separation and purification. One ubiquitous method of protein purification and separation is chromatography. Despite its wide industrial usage and intensive development, there is still no detailed molecular-scale picture of protein dynamics during chromatographic separation. The lack of a predictive chromatographic theory is rooted in the absence of an in-depth understanding of interactions occurring inside a chromatographic column. To advance our understanding of underlying phenomena in a chromatographic column, 2D and 3D single-molecule techniques were utilized. We uncover the differences in protein motion in mobile phases, depending on salt concentration, and correlated the results to an ensemble chromatogram. We also demonstrate the importance of the combined influence of surface properties on adsorption-desorption kinetics of proteins to the stationary phase. Overall, we have shown that single-molecule methods can uncover the details of protein dynamics and transport at the nanoscale and relate them to ensemble chromatography and apply them to protein purification at-scale.