Analytical band centrifugation revisited

Abstract

Analytical band centrifugation (ABC) is a powerful tool for the analysis of macromolecules and nanoparticles. Although it offers several advantages over the sedimentation velocity (SV) experiment like a physical separation of the individual components and the possibility to perform chemical reactions, its analysis is still very restricted. Therefore, we investigated the integration of ABC data as an alternative approach, as this results in data similar to SV, which can then be evaluated by many established evaluation programs. We investigated this method using two different test systems, myoglobin as a biopolymer with significant diffusion and 100 nm polystyrene latex as a large particle with negligible diffusion, and found some limiting issues. These are namely, broadening of the initial boundary by diffusion of the sample, which can be taken into account and the dynamic density gradient between the solvent in the sector and the overlaid solution, which deforms the initial band upon movement through the gradient and is currently not taken into account. We show the influence these two factors have on the evaluation and show that it is possible to calculate the time-dependent change in solvent density and viscosity in the AUC cell using the integrated form of Fick's second law. We conclude that taking the dynamic density gradient into account will open ABC for the sophisticated methods based on the analysis of the whole sedimentation boundary and not just the determination of an average sedimentation coefficient.

Analytical band centrifugation for the separation and quantification of empty and full AAV particles

“…Analytical band centrifugation (ABC) is an alternative method to sedimentation velocity in AUC. 11 , 12 , 13 ABC uses special centerpieces that have two fine capillaries connecting to a reservoir. Samples are loaded in the reservoir during cell assembly, pressured through the capillary channel at low rotor speed, and overlaid as a thin layer on top of the bulk solution in the centerpiece.…”

“…They explored transforming ABC data to sedimentation boundary data and utilizing the sophisticated processing methods that had been developed for SV-AUC. 11 …”

“…Since then, there have been few other reported ABC applications in literature, mostly studying interacting systems. 11,17 Schneider et al 12 attributed the sparse adoption of ABC to the poor understanding of its initial condition from sample overlaying and the dynamic nature of solvent density and viscosity during sedimentation. They explored transforming ABC data to sedimentation boundary data and utilizing the sophisticated processing methods that had been developed for SV-AUC.…”

“…They explored transforming ABC data to sedimentation boundary data and utilizing the sophisticated processing methods that had been developed for SV-AUC. 11 In this study, we implement Vinograd's original separation methods on modern AUC instruments for pharmaceutical applications. We report using ABC separation as an alternative to SV-AUC for quantifying empty and full AAV particles.…”

Formation of Nanoclusters in Gold Nucleation

“…It seems like the fit tries to compensate for another issue by adjusting v, which is yet unknown to us. It could be related to the formation of the dynamic density gradient due to the overlaying of H 2 O onto D 2 O [18,38], which is not considered in the analysis [39]. This impact increases with the decreasing density of the species and therefore could influence the basis for the v determination, although the smaller particles are more impacted by shorter wavelengths.…”