Analytical Ultracentrifugation – Density gradient

This technique is used for the determination of particle densities and density distributions. In contrast to the density variation method, this an equilibrium technique. This experiment is exceptionally adequate for

  • measuring densities of one or multiple species in high resolution,
  • measuring densities, excluding transport processes.

The principle: A dense solvent or solute is added to the system. Exposed to a strong gravitational field, a gradient will be established, the more dense component accumulating towards the cell bottom. The dispersed particles or molecules will migrate to the radial position where their own density is matched. Their density and, to some extent, molecular masses can be calculated. Increasing angular velocity allows to zoom into the density range of interest. In this manner, a high resolution is achieved, reaching the fourth digit of density.

AUC density gradient sample a
Analyte redistributing along the radial coordinate as the density gradient is established
AUC density gradient sample b
Density distribution according to the local concentration of gradient material

The figure shows the analyte redistributing along the radial coordinate as the density gradient is established. It is separated into two species, both accumulating at the respective gradient position matching their own density.

An interesting aspect of density gradients is that multiple species are actually separated and can be characterized by their spectral properties. This provides an orthogonal access to their nature – in addition to their density. This is particularly effective for characterizing compounds, such as capsids loaded with cargo – the mass weighted density average and the sum of spectral properties provide redundant information on their composition.

The challenge of this experiment is that the gradient material must not interfere with the analyte. Fragile structures, such as micelles, might be affected by high ionic strength, as well as it might induce conformational changes for proteins. Various gradient materials are available, and some refinements are at hand in order to extend applicability – however, density gradients are a sophisticated technique requiring some consideration and experience.

Further theoretical background can be found on our scientific website under