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. Du…
Centrifugation is a method of separating suspended particles from a liquid by spinning in a centrifuge at a high number of revolutions per minute.
The particles sediment at a rate proportional to the centrifugal force they experience.
After centrifugation, the particles settle at the bottom, while the supernatant liquid can be decanted.
When the components are of equal density, the heavier particles sediment first. However, among particles with equal mass, the denser particles sediment first.
In differential centrifugation, heavier particles are eliminated first at lower speeds. The centrifugation speed is increased stepwise until the target particles are isolated.
In density gradient centrifugation, the density of the solution increases toward the bottom of the tube.
Upon centrifugation, each component stops moving when it reaches a layer with equal density, resulting in separate bands.
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Q1: How does centrifugal force separate particles in a centrifuge?
Centrifugal force acts differentially on particles based on their density and mass. Particles sediment at rates proportional to the centrifugal force they experience. Heavier particles sediment first among equal-density components, while denser particles sediment first among equal-mass components. After spinning, the pellet settles at the bottom and the supernatant liquid can be decanted for further processing.
Q2: What is the difference between differential centrifugation and density gradient centrifugation?
Differential centrifugation separates particles by incrementally increasing speed until target particles isolate, working best when particles have similar densities. Density gradient centrifugation uses a preformed or developing density gradient where each component stops moving when reaching a layer with equal density, resulting in separate bands. This method is more sophisticated and effective for complex mixtures like cell lysates.
Q3: Why do denser particles sediment faster than lighter particles of the same size?
Sedimentation rate depends on particle density and mass. Among particles of equal size, denser particles experience greater centrifugal force, causing them to sediment faster. Larger particles tend to be heavier and sediment first, but when size is similar, density becomes the determining factor for sedimentation order.
Q4: What happens to particles when they reach a layer of equal density in a density gradient?
When a particle's density equals the solution density at a particular layer, the centrifugal force acting on it becomes zero, and sedimentation ceases. The particle remains isolated in that layer, creating distinct bands of separated components. This equilibrium principle allows precise separation of analytes with different densities in a single centrifugation run.
Q5: How is the supernatant used after centrifugation?
After centrifugation, the supernatant liquid is decanted from the pellet. Depending on the application, either the pellet or supernatant is retained for further purification. Sometimes the supernatant undergoes additional rounds of centrifugation and downstream processing, such as extraction or precipitation and co-precipitation methods.
Q6: Why might complex mixtures not yield distinctly separated particles despite density differences?
Complex mixtures such as cell lysates contain numerous components with overlapping densities and sizes, making simple separation difficult. Particles may not form distinct bands due to similar physical properties or interactions between components. Researchers use sophisticated centrifugation techniques like density gradient centrifugation to overcome these challenges and achieve better resolution.
Q7: What determines whether larger or denser particles sediment first during centrifugation?
When particles have equal density, larger particles sediment first because they are typically heavier. When particles have equal mass, denser particles sediment first due to higher sedimentation rates. The relative contributions of size and density determine sedimentation order, with centrifugal force acting proportionally on each particle's mass and density.