Centrifugation separation techniques

Centrifugation separation techniques

The sedimentation rate of the analyte particles in a suspension is dependent upon following properties:

·         Size of the particle

·         Medium

·         External forces applied

Stokes’s law is applicable whenever the centrifugal force of ωr² is used to carry out sedimentation. Here ω represents the rotational angular velocity and r is representing the distance from the diameter of the analyte particle to the axis of rotation.

dr/dt = 2r²_p (ρ_p - ρ_m) ω²r/ 9η

In this equation dr/dt is the particle’s velocity; η is the viscosity of the medium, r_p is the particle’s radius and (ρ_p - ρ_m) difference in the density of the particle and the density of the medium.

There are various techniques which involve the use of centrifugal force for the separation of particles based upon their rates of sedimentation. These subtypes of the Centrifugal FFF are given below:

·         Differential centrifugation
Rate zonal centrifugation
Isopycnic centrifugation
Centrifugal elutriation

Differential centrifugation

In this technique a homogenous solution containing cell suspension is allowed to sediment. This causes the spherical particles to take less time in settling than that of the asymmetric particles as a result the asymmetric particles settle more slowly. The comparative low resolution is the major drawback of this technique. Because when centrifugal force is applied to pellet the particles larger in size this also causes the pelleting of the smaller analyte particles contaminating the fractions.

Rate zonal centrifugation

This technique upon its completion causes the separation of particles differing in their sizes in the form of band because this technique involves the arrangement of a thin layer of suspension on the top of density gradient. Rate zonal centrifugation technique is mostly used for the separation of vesicles, proteins and lysosomes and the separation is based upon the size, density and shape of the particles.

Isopycnic centrifugation

This technique is mostly used for those particles which can be directly loaded on the density gradient. Isopycnic centrifugation is different from the rate zonal centrifugation on two bases:

1.      It is an equilibrium technique unlike rate technique

2.      The maximum density in the gradient is higher than the density of the analyte particles

Separation is not affected by the increased time of centrifugation as long as the gradient is constant. Optimal conditions for centrifugation and better selection of the density gradient can increase the resolution. The gradients used can be continuous and discontinuous, normally continuous gradients are used because discontinuous gradients can affect the separation and make it difficult. These types of gradients are generally used for the separation of the red blood cells from the white blood cells.

Centrifugal elutriation

Sanderson and Lindahl these two scientists explained the concept of separation based upon the stability between the two opposite forces that are the centripetal flow force and the centrifugal force. The separation of the particles can be achieved by increasing the flow rate or by decreasing the speed of the rotors i.e centrifugal force. The following equation explains the flow velocity

V=F/A

Here V shows the sedimentation velocity and another equation more explaining the situation is as follows

SV = 2r²_p (ρ_p – ρ_m) ω²r/ 9η – F/A

The sedimentation velocity is dependent upon the size and density of the particle. But as shown in equation radius or size of the particle is more important. So the cells or particles having smaller difference in their size will have large difference in their sedimentation velocities. This technique is capable of separating the cells with wide size range and provides fast elutriation.