Analytical Ultracentrifugation - sedimentation equilibrium

Sedimentation equilibrium is applied for the determination of molecular masses. This is a classical technique in Analytical Ultracentrifugation application; in biochemistry countless molecular masses of protein have been measured with this experiment for decades. Nowadays, alternative and often less pricy methods are favoured, especially SEC, and in many cases, these methods are quicker as well.

Nevertheless, the following reasons still make Analytical Ultracentrifugation an attractive alternative in some cases:

  • In chromatographic methods, you encounter problems of column-sample interactions.
  • No feasible standard is available, of calibration is inefficient, calling for an absolute method.
  • No column for the specific solvent is available.
  • Particle geometries might produce misleading results with other methods.
  • You are interested in interparticular interactions.
  • ...

The accuracy of Analytical Ultracentrifugation equilibrium results (3% error) is unattained by other methods. Knowledge of particle density is required, otherwise, this property is provided by the density variation technique. The sample volumes needed for this experiment are also extremely small.

concentration profile in sedimentation-diffusion-equilibrium
Figure 1: concentration profile in sedimentation-diffusion-equilibrium

During a sedimentation equilibrium experiment, a dynamic equilibrium of sedimentation and diffusion is attained inside the sample cell. At first, the centrifugal field causes the material to sediment towards the cell bottom, increasing the local solute concentration. This causes back diffusion (diffusion is concentration dependent) to increase, opposing the centrifugal field. After sufficient time (this can take several days), a stationary condition is reached. The figure shows a typical exponential concentration profile in sedimentation-diffusion-equilibrium. From this profile, a weight-averaged molecular mass can be calculating, involving particle density and experimental conditions.

Molecular masses can also be obtained from the sedimentation velocity experiment, which is much faster and has the advantage of yielding molecular mass distributions rather than mass averages. This evaluation, however, requires knowledge of the frictional properties of the particle, as transport processes are involved.

To put it mathematically: Two transport properties are required to solve the corresponding equation: the sedimentation constant and the diffusion constant. Their errors culminate in the result for the molecular mass, making the equilibrium experiment, where transport processes are eliminated, more reliable.

For molcular mass distributions, the weight average is obtained, but numerical and z-averages are also accessible. This is a question of evaluation, and countless approaches have been documented in the literature. We use the M*-function developed by Creeth and Harding for evaluation. This is a rather sophisticated approach, requiring no model and providing high statistical certainty.

More detailed information on the theoretical background are compiled on our scientific website (in German).