Centrifugation method is a process of separating different components or the suspended particles from the homogenous solution, by the practice of centrifugal force. It separates particles based on their physical properties like size, shape, density etc. and chemical properties like molecular weight and viscosity.
A natural gravitational force can also concentrate particles but in an indefinite time so to increase its efficiency, a centrifugal force function to separate particles by their sedimentation property. The rate of sedimentation increases and decreases as per the increase in gravitational force and viscosity of the medium respectively.
Content: Centrifugation Method
Definition of Centrifugation
Centrifugation method can define as a process of concentrating suspended particles from the aqueous medium, where the particles segregate by the formation of discrete bands and pellet depending on their size and density. A centrifuge is an apparatus that aptly cause partition of two definite phases, namely supernatant aqueous phase and suspended solid phase. A rotor inside the centrifuge generates a central force that facilitates the separation of solid and aqueous phase according to the sedimentation rate, which commonly refers to as “Centrifugal force”.
Based on the type of separation molecules, there are two significant types of centrifugation:
- Preparative centrifugation: It is a technique concentrating one component from the other.
- Analytical centrifugation: It is a method that measures physical, chemical and hydrodynamic properties of suspended molecules.
Principle of Centrifugation Method
A centrifugation method depends on the gravitational movement of suspended particles, which cause deposition of high-density particles towards the bottom. It is important to note that higher is the difference between the densities of particles, easy will be the separation. If the density difference is low, then the separation becomes tough, and in such case, gravitational force must be accelerated.
Therefore, a centrifuge accelerates the rate of sedimentation by increasing the relative centrifugal force (RCF) measures by the rotations per minute (rpm). If RCF is equal to 100X g, then it is equivalent to the centrifugal force that is a hundred times greater than the force of gravity.
A centrifugal force is a kind of force that causes circular motion of the particles and can express as:
Centrifugal force = mω2r
Where, m= Mass of particle
ω= Angular velocity in radians per second
r= Radius of a particle from the axis of rotation
ω2r= Centrifugal acceleration
In terms of gravitation force, the equation will represent as,
Gravitational force (G)= mg
Where g= Gravitational constant
Other than, centrifugal force, there are some other interacting forces, namely buoyant and frictional force.
A buoyant force is an exerted force that causes movement of particles upwards and develops as a result of force applied to the particles by the fluid. The opposing buoyant force is smaller for a dense particle and can express as:
Buoyant force (Fb)= ρgV
Where ρ= Fluid density
g= Gravitational or centrifugal acceleration
V= Volume of fluid displaced
A frictional force is a kind of force that restricts the movement of dispersed particles in the solution. The frictional coefficient is smaller for a minute particle than the dense particle.
Frictional force (Ff )= fv
Where f= Frictional coefficient
v= Sedimentation velocity
The net force acting on the particle that represents as,
Net force= Centrifugal force – Buoyant force
= mω2r – ω2rρV (ω2r= Centrifugal acceleration, thus g= ω2r)
In a steady-state, the frictional force is equal to the net force.
fv= mω2r – ω2rVρ
= mω2r – ω2rmv̅ρ
= mω2r (1– v̅ρ)
The mass of 1mole of particles represented by,
M=mN (Where N is the Avogadro’s number)
fv= Mω2r (1– v̅ρ)/N
Now, the sedimentation coefficient (S) equals to the ratio of velocity to the gravitational acceleration.
= M/N. (1– v̅ρ)/f
A sedimentation coefficient is measured by the units of a second or the Svedberg unit (1S= 10-13second).
Types of Centrifugation Method
Density gradient Centrifugation
It is another kind of centrifugation method, where the sample containing different molecules mix with the high-density solution. Density gradient centrifugation makes the use of substrates (sucrose, glycerol etc.) to prepare a solution for the development of a density gradient. For the separation of different concentrates, the solution where the sample has to be mixed should have low concentration and high diffusing property. Before centrifugation of the sample solution, it should be stored in the uniform mixture for some time.
On centrifugation, the different molecules of different density will occupy the place in a density gradient respective to their mass. Finally, a concentrated layer of the particles is isolated by puncturing the centrifuge tube. It is of two types, namely:
- Rate zonal centrifugation: In this kind, a sample is centrifuged in a pre–established density gradient.
- Isopycnic centrifugation: Here, a self-generating gradient forms during the centrifugation of the sample.
Rate zonal Centrifugation
It also refers as “Velocity centrifugation” where the particles in a solution separate based on their sedimentation rate that directly depends upon the particle mass or size. The particles’ shape also influences the rate of sedimentation.
It makes the use of high-density sucrose solution of 5-20% to create a density gradient. The sedimentation of dense particles takes place at the bottom of the centrifugal tube, and the light particles remain at the top. Therefore, zonal centrifugation develops discrete bands or zones respective to the density of the particles present in the sample.
For the separation of bands, a centrifuge tube is prickled from the bottom. The particle size or mass may affect the efficiency of sedimentation, as sedimentation coefficient is proportional to the mass of a particle. The sedimentation coefficient and the mass of the molecule of interest can be determined by this technique.
It also refers as “Equilibrium density gradient centrifugation” where the particles are concentrated based on their buoyant density that is independent of particle size and shape. The time taken for centrifugation of particles does not affect the rate of sedimentation.
This method is employed for concentrating such particles those possessing the same size but different density. The sedimentation of biomolecules will not occur until the buoyant density does not equal to the density of the gradient. In Isopycnic centrifugation, 20-70% of sucrose solution develops a gradient to separate different molecules.
It is a kind of centrifugation method depends upon the differences in the sedimentation rate that directly influences the separation of different components according to their relative mass, shape and density. Generally, it separates the subcellular components. In differential centrifugation, the cells are blended, and the ruptured cell components are poured into a centrifuge tube that preserves the integrity of the cell components.
Then the suspended components in a solution are subjected to initial centrifugation at low centrifugal force for a defined time. The initial centrifugation causes sedimentation of larger molecules at the bottom (as a pellet) and supernatant at the top.
For the sedimentation of different cellular components, the supernatant is repeatedly decanted after centrifugation under a high centrifugal force. Therefore, the centrifugal force plus the centrifugal time increases successively after each step.
This method increases the earth’s gravitational force by the use of centrifugal force (1,000,000 times) greater to the gravitational force. Ultracentrifugation can sediment particles as small as 10kDa. In a homogenous mixture, the dense particles will appear as a pellet at the solvent sink, and the lighter particles will appear as supernatant at the solvent top. Ultracentrifugation uses a dense solution of sucrose or caesium chloride to concentrate particles.
Uses of Centrifugation
Following are the applications of centrifugation technique in different areas of research like:
- Environmental science: In the treatment of wastewater.
- Molecular science: Helps in the extraction of biomolecules like DNA, RNA, protein etc.
- Medical research: Helps in the separation of different components from urine, blood serum etc.
- Chemical science: Helps in the process of uranium enrichment.
- Food science: Helps in the production of skimmed milk by removing fat.
Therefore, we can conclude that the centrifugation technique has broad applicability in the field of research biochemistry, molecular biology, cellular biology and medical science.