Centrifugation Technique

INTRODUCTION

A centrifuge is a device that separates particles from a solution based on their size, shape, density, medium viscosity, and rotor speed.

This instrument is used and based on centrifugal forces. With the help of an electric motor, it rotates a container around the center axis. Different types of rotors, such as angle head and swinging bucket types, are available for cooling centrifuges, high-speed centrifuges, and ultracentrifuges. The sample in the angle type rotor is kept at an angle of about thirty degrees to the horizontal, whereas the sample in the latter is kept horizontal during spinning.

Clinical centrifuges are the most popular form of the centrifuge. The spun rotors provide force under vacuum to reduce friction in this case. Particles with a higher density than the solvent sink [sediment] in a solution, while those with a lower density float to the top. The greater the difference in density, the faster they move. The particles stay constant if there is no difference in density [isopycnic circumstances].

CENTRIFUGATION

The centrifugal force is used to separate macromolecules through a liquid medium. The viscosity of the medium, particle size or molecular weight, rotor speed, gravitational force, pressure, and temperature are all factors.

Gravity can be replaced by the far more powerful ‘centrifugal force’ generated by the centrifuge to take advantage of even slight changes in density to separate various particles in a solution. This approach is important in biochemistry and biotechnology since it is a required step in every procedure used in biological research, from the separation of cell organelles to sophisticated investigations including the separation of sub-cellular fractions.

TYPES OF CENTRIFUGES AND THEIR USES

There are four major types of centrifuges. They are;

  • Small bench centrifuges
  • Large capacity refrigerated centifuges
  • High- speed refrigerated centifuges
  • Ultracentrifuges

SMALL BENCH CENTRIFUGES

They are used to collect small amounts of quickly sedimenting material such as yeast cells, erythrocytes, etc. They have a maximum relative centrifugal field of 3000-7000g.

LARGE CAPACITY REFRIGERATED CENTRIFUGES

They have a refrigerated rotor chamber and the ability to change rotor chambers to accommodate various sizes. They can weigh up to 6500g and are used to sediment or collect the substances that sediment rapidly such as erythrocytes, yeast cells, nuclei, and chloroplast.

HIGH SPEED REFRIGERATED CENTRIFUGES

They can generate a speed of about 60000g. They are used to collect micro-organisms, cellular debris, larger cellular organelles, and proteins precipitated by ammonium sulfate.

ULTRA CENTRIFUGES

They can generate 65000RPM. They have a limited lifetime. It is expensive and required special rotors. It is used very carefully and used in research applications.

PREPARATIVE CENTRIFUGATION

● It is used for the separation of micro molecules and organelles.

● It can accommodate a large volume of samples.

● There is no optical system is present.

It is of two types;

  • Differential centrifugation
  • Density centrifugation

DIFFERENTIAL CENTRIFUGATION

The components of a mixture are separated using this procedure based on their size, shape, and density. Larger molecules typically reach the lower part of the column before smaller molecules settle. Differential centrifugation techniques are used to separate particles of the same size based on their differences in density, whereas differential centrifugation techniques are used to separate particles of the same density. In this procedure, a homogenous solution of a component mixture is taken and centrifuged for a fixed duration with a specified centrifugal field. After some time, it is found that some of the components are sediment and form a supernatant solution. The pellet part is removed and again centrifuged for a fixed duration at a specific centrifugal field.

Because the first centrifugation does not produce a pure pellet, the pellet portion must be centrifuged again to obtain a pure pellet. The supernatant fluid is separated from which the particle having the lowest sedimentation rate can be separated. As a result, the supernatant fluid has lost all large, medium, and small particles that were present at the top of the tube. As a result, the various components of the mixture are separated.

DENSITY CENTRIFUGATION

The sample is placed in a dense solution with a low concentration and a high diffusing property. The mixture is spun in a centrifuge, however, the sample must be homogenous before beginning the procedure. The solution then generates a density gradient, and the sample components occupy the density gradient places that correspond to their density. The bands of the sample formed are separated by puncturing the tube.

IT IS FURTHER DIVIDED INTO TWO PARTS:

  • Zonal centrifugation
  • Isopycnic centrifugation

ZONAL

The sample in the solution is centrifuged to separate the components of the mixture. Sedimentation takes place in a column containing a solution. As we move down the column, the density of the solution increases. The solution should be inert, resulting in the formation of a gradient. This sample mixture is then placed in a column that allows the sample to separate into different bands based on the rate of sedimentation. The component with the highest rate of sedimentation is found at the bottom of the column. The size and shape of the molecule also affect the sedimentation, as the sedimentation coefficient is a function of the mass of the particle. The bands are formed at various positions which can be separated by puncturing the tube, bands are collected separately.

ISOPYCNIC CENTRIFUGATION

Particles are separated based on their density in isopycnic separation. It is also known as buoyant or equilibrium separation. Particle size only affects the rate at which particles move until their density equals that of the gradient medium around them. The gradient medium’s density must be higher than that of the particles to be separated. No matter how long the centrifugation time is, particles will never sediment to the bottom of the tube with this procedure. It is commonly used to separate different kinds of nucleic acids.

ANALYTICAL ULTRA CENTRIFUGATION

Analytical ultracentrifugation is a versatile and powerful method for analyzing macromolecules in solution quantitatively. AUC has a wide range of applications in the research of biomacromolecules in a variety of solvents and at a range of solute concentrations. The AUC has three optical systems that allow for precise and selective sedimentation observation in real-time. The fluorescence system, in particular, offers a new technique to expand the scope of AUC to investigate the behavior of biological molecules in complex mixtures with high solute concentrations.

The movement of solute in high centrifugal forces is interpreted using hydrodynamic theory to characterize the size, shape, and interactions of macromolecules in sedimentation velocity. Sedimentation equilibrium is a thermodynamic method for determining molecular mass, assembly stoichiometry, association constants, and solution non-ideal by analyzing equilibrium concentration gradients at lower centrifugal forces. Researchers can utilize sedimentation velocity to determine the homogeneity of a sample and identify whether it undergoes concentration-dependent association reactions using specialized sample cells and modern analysis software.

REFERENCES

● Graham, John, 2001(Biological centrifugation).

● Regel, Liya and William R.Wilcox, 2001 (processing by centrifugation).

● Ballou, David P.; Benore, Marilee; Ninfa, Alexander J. (2008). Fundamental laboratory approaches for biochemistry and biotechnology (2nd ed.). Hoboken, N.J.: Wiley. p. 43.

Article By:

Anuwanshi Sharma

Ph.D. Scholar

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