Instrumentation

Centrifugation

Centrifuge

Centrifugation is a process which uses centrifugal force to separate and purify mixtures of biological particles in a liquid medium. It is a key technique for isolating and analyzing cells, subcellular fractions, supramolecular complexes and isolated macromolecules such as proteins or nucleic acids.

 

The particles are separated from a solution according to their size, shape, density, viscosity of the medium and rotor speed.

 

In other words it is a technique of separating substances which involves the application of centrifugal force or method to separate particles from mixture by spinning.

 

Centrifugation can be analytical as well as preparative. Analytical centrifugation is mainly concerned with the study of purified macromolecules or isolated supramolecular assemblies,

 

Preparative centrifugation methodology is devoted to the actual separation of tissues, cells, subcellular structures, membrane vesicles and other particles of biochemical interest.

 

Development of the first analytical ultracentrifuge by Svedberg in the late 1920s and technical
refinement of preparative centrifugation by Claude and colleagues in 1940s has made this procedure a important part of biomedical research along with biochemical, cellular and molecular biological studies..

 

Centrifugation is achieved by device centrifuge. A centrifuge is a piece of equipment that puts an object in rotation around a fixed axis (spins it in a circle), applying a potentially strong force perpendicular to the axis of spin (outward direction).

 

It works under sedimentation principle, where centripetal acceleration causes denser substances and particles to move outward in the radial direction.

 

Centrifugation process

 

Principle

The principle of the centrifugation technique is to separate the particles suspended in liquid media under the influence of a centrifugal field. Centrifugation is majorly dependent upon following rules:

 

  • More dense a biological structure is, faster it sediments in a centrifugal field.
  • More massive a biological particle is, faster it moves in a centrifugal field.
  • Denser the biological buffer system, slower the particle will move in centrifugal field.
  • Greater the frictional coefficient is, slower a particle will move.
  • Greater the centrifugal force is, faster will particle sediment.
  • Sedimentation rate of a given particle will be zero when density of particle and the surrounding medium are equal.

 

 

In a solution, particles whose density is higher than that of the solvent sink (sediment), and particles that are lighter than it float to the top.

 

Biological particles moving through a viscous medium experience a frictional drag, whereby the frictional force acts in the opposite direction to sedimentation and equals the velocity of the particle multiplied by the frictional coefficient. As sample moves towards bottom of a centrifuge tube, its velocity will increase due to the increase in radial distance. At the same time the particles also encounter a frictional drag that is proportional to their velocity & thus, gets sedimented.

 

Greater the difference in density, the faster they move. If there is no difference in density (isopycnic condition), particles stay steady. For employing even tiny differences in density to separate various particles in a solution, gravity can be replaced with the much more powerful centrifugal force provided by a centrifuge. At same time, objects that are less dense are displaced and move to the center.

 

In a laboratory centrifuge that uses sample tubes, the radial acceleration causes denser particles to settle to the bottom of the tube, while low- density substances rise to the top.

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