4.4: Centrifugation
Centrifugation is a separation technique based on differences in density or size. It is commonly used to separate solids from aqueous interferents. During centrifugation, the sample is placed in centrifugation tubes and spun at high angular velocity, which allows centrifugal force to act differentially on the different densities or masses of the components. After spinning, the supernatant liquid is decanted. Depending on the specific application, either the pellet or the supernatant is retained for further purification. Sometimes, the supernatant is subjected to additional rounds of centrifugation and downstream processing.
In principle, centrifugation separates particles based on differences in size and density. Larger particles tend to be heavier and tend to sediment first. If particles are similar in size, denser particles tend to sediment first due to higher sedimentation rates. In practice, complex mixtures such as cell lysates may not yield distinctly separated particles despite differences in density or size. To address this issue, researchers have devised a variety of centrifugation techniques.
The simplest centrifugation technique is differential centrifugation. Here, the particles to be separated have similar densities, so larger particles will sediment at lower speeds. The speed is increased stepwise until the target particles are isolated.
A more sophisticated method is equilibrium density gradient centrifugation. In this technique, the analyte is placed in a solution with a preformed density gradient or a solution that forms a density gradient during centrifugation. Here, the density of the solution increases towards the bottom of the tube, so the sedimentation rate of each analyte component decreases as it moves toward the bottom. When a component's density equals that of the solution, the centrifugal force acting on it becomes zero, and sedimentation ceases. As a result, each component is isolated in a layer equal to its density.
