All practical chromatographic processes involve the separation of complex mixtures by competitive adsorption. In this work, we employ single-molecule methods and a novel isocratic competitive mode of column chromatography to study the complex mechanistic details involved in protein chromatography. Using a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT), we present the direct mapping and kinetic characterization of individual functional sites on thin-film agarose ion-exchange matrices. By extracting single-protein adsorption and desorption kinetics at individual ligands, direct experimental evidence in support of the stochastic theory of chromatography is obtained. We also investigate the influence of ionic strength on the heterogeneity of protein ion-exchange functional adsorption sites, and therefore the heterogeneity of elution profiles. We observe that the number of functional adsorption sites is smaller at high ionic strength and these sites have reduced desorption kinetic heterogeneity; this is in accordance with our earlier reports based on equilibrium adsorption isotherm measurements with cytochrome b5. In an integrated study, we use single molecule spectroscopy and a novel isocratic competitive chromatographic operating regime to study a multicomponent, competitive chromatographic system. We observe that the competitor (insulin) does not change the single molecule kinetics of α-lactalbumin adsorption, but reduces the number of adsorption events on cationic spermine ligands. Overall, we have shown that single molecule super-resolution imaging can advance our understanding of multicomponent competitive ion-exchange adsorptive separations. The results of such studies could be useful in improving ion-exchange chromatographic separations of realistic multicomponent systems.

Single-molecule and isocratic protein competition on ion-exchange adsorbents

255th National Meeting (2018)

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