Equilibrium sedimentation

Differential centrifugation is a common procedure in microbiology and cytology used to separate certain organelles from whole cells for further analysis of specific parts of cells. In the process, a tissue sample is first lysed to break the cell membranes and mix up the cell contents. The lysate is then subjected to repeated centrifugations, where density separation causes a sediment to form known as "pellet". After each centrifugation the pellet is removed and the centrifugal force is increased. Finally, purification may be done through equilibrium sedimentation, and the desired layer is extracted for further analysis.

In general the equilibrium sedimentation profile is a function of particle-density alone; however, in a viscous fluid the rate of sedimentation is also a function of particle size. The reason for this is that (for a given radius of rotation and rotational velocity), the centrifugal force on a particle is proportional to the particle diameter cubed (for a given particle-density), whereas the force from viscous drag (or Stoke's force) is only proportional to the particle diameter squared. This means that larger particles sediment more quickly and at lower centrifugal forces, potentially causing a sedimentation profile that may be distorted by particle sizes and not quite in equilibrium with density. For this reason, multiple centrifugation steps are used in the separation process in order to obtain sedimentation profiles that are closer to having an equilibrium with density.

As an example, unbroken whole cells will pellet at low speeds and short intervals such as 1,000g for 5 minutes. Smaller cell fragments and organelles remain in the supernatant and require more force and longer durations to pellet. In general, one can enrich for the following cell components, in the separating order in actual application:

High g-force makes sedimentation of small particles much faster than Brownian diffusion, even for very small particles. When a centrifuge is used, Stokes' law must be modified to account for the variation in g-force with distance from the center of rotation.

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