Centrifugation is a process that involves use of centrifugal force for separation of particles from mixture with the use of centrifuge. Depending upon the medium of suspension in which the separation is carried out It is of two types:
- Density Gradient
Density Gradient centrifugation can be further divided into rate zonal and isopycnic.
Differential centrifugation: Separations carried out in a homogenous medium are known as differential centrifugation. It is most common type of centrifugation employed.
Rate of particle sedimentation depends mainly on its size and the applied g-force. Most commonly used method for isolation of intracellular organelles from tissue homogenates because of its relative ease, convenience and time economy.
Poor resolution and recovery because of:
- Particle size heterogeneity.
- Preparations obtained are never pure.
Particles starting out at rmin have furthest to travel but initially experience lowest RCF. Smaller particles close to rmax have only a short distance to travel and experience the highest RCF.
- Tissue are homogenized in a sucrose solution that contains buffer.
- The homogenate is placed in a centrifuge and spun at constant centrifugal force at constant temperature.
- After sometime, sediment forms at the bottom of centrifuge called pellet and overlying solution called supernatant.
- The overlying solution is then placed in another centrifuge tube which is then rotated at higher speeds in progressing steps.
Density Gradient Centrifugation: Centrifugation carried out in suspending medium such as sucrose or cesium chloride having density gradients. It permits separation of multicomponent mixture of macromolecules and measurement of sedimentation coefficient.
The separation under centrifugal field is dependent upon buoyant densities of the particles. It is mainly used to purify viruses, ribosomes, membranes etc.
- A sucrose density gradient is created by gently overlaying lower concentrations of sucrose on higher concentrations in centrifuge tubes
- Particles of interest are placed on top of the gradient and centrifuge in ultra-centrifuges.
- Particle travel through gradient until they reach a point at which their density matches with density of surrounding sucrose.
- Fraction is removed and analyzed.
Rate-Zonal: It is also known as band or gradient centrifugation. The gradient used has maximum density below that of least dense sedimenting particle.
This involves careful layering of a thin layer of the sample solution on top of a preformed liquid density gradient whose density continuously increases towards the bottom of the sample tube.
Components of mixture sediments/separates according to shape, size and density. Useful for separating particles which differ in size but not in density, separation of RNA-DNA hybrids and ribosomal subunits.
Isopycnic: Also called buoyant or equilibrium separation. Components are separated solely on the basis of their density & size only affects the rate of movement of molecules.
The density of gradient medium must be greater than density of particles to be separated. In this method, components never sediment to the bottom of tube.
Upon centrifugation, particles of a specific density sediment until they reach a point where their density is same as gradient media (i.e. equilibrium position). This is to say the sample molecules move to the region where their density equals to the density of gradient.
The gradient is then said to be isopycnic and particles are separated according to their buoyancy. It is a true equilibrium procedure since it depends on buoyant densities, not velocities
Since the density of biological particles is sensitive to the osmotic pressure of the gradient, isopycnic separation may vary significantly depending on the gradient medium used.
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