In biochemistry and cell biology, differential centrifugation (also known as differential velocity centrifugation) is a common procedure used to separate organelles and other sub-cellular particles based on their sedimentation rate. Although often applied in biological analysis, differential centrifugation is a general technique also suitable for crude purification of non-living suspended particles (e.g. nanoparticles, colloidal particles, viruses). In a typical case where differential centrifugation is used to analyze cell-biological phenomena (e.g. organelle distribution), a tissue sample is first lysed to break the cell membranes and release the organelles and cytosol. The lysate is then subjected to repeated centrifugations, where particles that sediment sufficiently quickly at a given centrifugal force for a given time form a compact "pellet" at the bottom of the centrifugation tube.
After each centrifugation, the supernatant (non-pelleted solution) is removed from the tube and re-centrifuged at an increased centrifugal force and/or time. Differential centrifugation is suitable for crude separations on the basis of sedimentation rate, but more fine grained purifications may be done on the basis of density through equilibrium density-gradient centrifugation. Thus, the differential centrifugation method is the successive pelleting of particles from the previous supernatant, using increasingly higher centrifugation forces. Cellular organelles separated by differential centrifugation maintain a relatively high degree of normal functioning, as long as they are not subject to denaturing conditions during isolation.
Theory
In a viscous fluid, the rate of sedimentation of a given suspended particle (as long as the particle is denser than the fluid) is largely a function of the following factors: Gravitational force Difference in density Fluid viscosity Particle size and shape
Larger particles sediment more quickly and at lower centrifugal forces. If a particle is less dense than the fluid (e.g., fats in water), the particle will not sediment, but rather will float, regardless of strength of the g-force experienced by the particle. Centrifugal force separates components not only on the basis of density, but also of particle size and shape. In contrast, a more specialized equilibrium density-gradient centrifugation produces a separation profile dependent on particle-density alone, and therefore is suitable for more fine-grained separations.
High g-force makes sedimentation of small particles much faster than Brownian diffusion, even for very small (nanoscale) 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.
where
D is the minimum diameter of the particles expected to sediment (m) η (or μ) is the fluid dynamic viscosity (Pa.s) Rf is the final radius of rotation (m) Ri is the initial radius of rotation (m) ρp is particle volumetric mass density (kg/m3) ρf is the fluid volumetric mass density (kg/m3) ω is the angular velocity (radian/s) t is the time required to sediment from Ri to Rf (s)
Procedure
Differential centrifugation can be used with intact particles (e.g. biological cells, microparticles, nanoparticles), or used to separate the component parts of a given particle. Using the example of a separation of eukaryotic organelles from intact cells, the cell must first be lysed and homogenized (ideally by a gentle technique, such as Dounce homogenization; harsher techniques or over homogenization will lead to a lower proportion of intact organelles). Once the crude organelle extract is obtained, it may be subjected to a varying centrifugation speeds to separate the organelles:
Ultracentrifugation
The lysed sample is now ready for centrifugation in an ultracentrifuge. An ultracentrifuge consists of a refrigerated, low-pressure chamber containing a rotor which is driven by an electrical motor capable of high speed rotation. Samples are placed in tubes within or attached to the rotor. Rotational speed may reach up to 100,000 rpm for floor model, 150,000 rpm for bench-top model (Beckman Optima Max-XP or Sorvall MTX150 or himac CS150NX), creating centrifugal speed forces of 800,000g to 1,000,000g. This force causes sedimentation of macromolecules, and can even cause non-uniform distributions of small molecules.