From a Protein's Perspective: Elution at the Single-Molecule Level

Column chromatography is a widely used analytical technique capable of identifying and isolating a desired chemical species from a more complicated mixture. Despite the method's prevalence, theoretical descriptions have not advanced to accommodate today's common analyte, proteins. Proteins are increasingly used as biologics, a term that refers to biological pharmaceuticals, and present new complexities for chromatographic separation. Large variations in surface charge, chemistry, and structure among protein analytes expose the limits in the current theoretical framework's ability to predict the efficiency of a column without empirical data. The bottleneck created by empirical optimization is a strong motivation for a renewed effort to achieve an in-depth understanding of the range of interactions that occur between a protein analyte and the stationary phase that together enable its selective separation from other constituents of a mixture. The physical and chemical processes that dictate the amount of time an analyte spends in the column are often abstracted by theory and treated as statistical distributions. Until recently, these distributions could not be mapped experimentally as traditional experimental techniques could not reveal underlying heterogeneity in structure, charge, and dynamics. Aligning the latest experimental and theoretical advances is thus a hurdle to be overcome so that significant progress can be made toward a predictive chromatographic theory. In this Account, we detail the work of the Landes Lab in developing single-molecule techniques that refine the stochastic theory of chromatography as a first step toward predictive chromatographic column design. We provide a brief review of the development of stochastic theory and establish a mathematical framework to put the discussed physical chemistry in context. We describe our investigations of three pertinent phenomena: mobile/stationary phase exchange, adsorption/desorption kinetics, and hindered diffusion. We highlight experimental evidence that points to nonuniform behavior. Then, we describe our work in developing single-molecule techniques that can evaluate these effects on a protein-by-protein basis. We highlight two developments: fast imaging via super temporal-resolved microscopy (STReM) and visualizing diffusion within pores via a combination of fluorescence correlation spectroscopy and super-resolution optical fluctuation imaging (fcsSOFI). Both methods offer new ways to study chromatographic elution at the single-protein level. Such methods can identify the rare heterogeneities that prevent efficient separations and advance the field closer to predictively optimized protein separations.