THEORY OF CENTRIFUGATION :-
- When a solid-liquid suspension is rotated in a cylindrical container (bowl) the suspension is subject to a centrifugal force in the radial direction.
- Centrifugation is a process by which solid particles are sedimented and separated from a liquid using centrifugal force as a driving force.
- Depending on the rotational speed and distance from the axis of rotation, the centrifugal force can be many times greater than the force of gravity, allowing even very small particles or particles slightly denser than the fluid to settle.
- Centrifugation: Any object moving in a circle at a steady angular velocity is subject to an outward directed force (F). The magnitude of this force depends on the angular velocity in radians (ω), and the radius of rotation (r).
F= gravitational force and also referred to as the relative centrifugal force (RCF)
- Sedimentation of a molecule influenced by
a. Properties of the molecules (Size, shape, density)
b. Properties of the solvent, or the gradient material(density, viscosity, temperature)
c. Interactions between the solute molecules and the solvent gradient molecule
-As the rotor spins, centrifugal force is applied to each molecule in the sample:
M= Mass (molecular weight),
ω=angular velocity (radians/sec),
r= distance from the axis of the rotation
TYPES OF CENTRIFUGATION :-
- Preparative :
- Used to separate organelles and molecules
- Can handle larger liquid volumes
- No optical read-out
- Separation methods used in preparative ultracentrifugation:
- Differential Centrifugation- pelleting,
- Density Gradient Centrifugation
- I Differential Centrifugation
- Based on the size of the particles.
- Used for simple pelleting, for the separation of sub cellular organelles and macromolecules.
- First sample must be homogenized.
- Sedimentation depends on mass, shape and partial specific volume of a macromolecule, as well as solvent density, rotor size, rate of rotation.
- Usually uses a fixed angle rotor.
- II Density gradient Centrifugation
- Method to purify subcellular organelles and macromolecules.
- Density gradients generated by placing layer after layer of gradient media.
- Density gradient centrifugation classified into two:
- Use of continuous density gradient of solvent such as sucrose.
- Density increases towards the bottom of the tube.
- Sample layered on the top molecules form discrete bands after centrifugation.
- Separation based on size of the molecules.
- For Example: – Swinging bucket rotors.
- Based on the density of the molecules
- The word “isopycnic” means “equal density“.
- Mix gradient material with the sample molecule (CsCl)
- Molecules move to the position where their density is same as the gradient material (isopycnic position).
- In order to generate a gradient, we select a CsCl concentration that will give a range of densities that includes the range of molecules that have to be separated.
- Analytical :
- Uses small size samples.
- Built-in optical system.
- Uses relatively pure sample.
- used for the separation of DNA.
– Swinging bucket or fixed angle rotor.