Tuesday, April 28, 2009

What is Centrifugation Process for isolation of molecules

Biomolecules can be isolated and purified by applying different techniques, which are based on various chemical and physical properties. The main physical and chemical properties that can be exploited for their separation and characterization of biomolecules are molecular weight and size, interaction with electromagnetic radiations or spectroscopic properties, solubility, molecular charge, and polarity.

Centrifugation is a technique based on size and weight of the molecules.

A centrifuge is a device for separating particles from a solution according to their sedimentation rate, which depends on factors like size, shape, density, viscosity of the medium, and centrifugal force (rotor speed). This process of separation of particles based on its sedimentation rate is called centrifugation. In biology, the particles are usually cells, sub-cellular organelles, viruses, and large molecules such as proteins and nucleic acids. The rate of sedimentation will be directly proportional to the molecular weight or size, if all other factors are constant. To simplify mathematical terminology we will refer to all biological material as spherical particles.

Following are the ways to classify centrifugation:

1) Analytical and Preparative Centrifugation:

The two most common types of centrifugation are analytical and preparative; the distinction between the two is based on the purpose of centrifugation. Analytical centrifugation involves measuring the physical properties of the sedimenting particles such as sedimentation coefficient or molecular weight. Optimal methods are used in analytical ultracentrifugation. Molecules are observed by an optical system during centrifugation, to allow observation of macromolecules in the solution as they move in the gravitational field. The samples are centrifuged in cells having windows that lie parallel to the plane of rotation of the rotor head. As the rotor turns, the images of the cell (proteins) are projected by an optical system onto film or a computer. The concentration of the solution at various points in the cell is determined by absorption of light of the appropriate wavelength (Beer's law is followed). This can be accomplished either by measuring the degree of blackening of a photographic film or by the pen deflection of the recorder of the scanning system, which is fed into a computer.

The other forms of centrifugations are preparative and the objective is to isolate specific particles, which can be reused. There are many types of preparative centrifugation such as rate zonal, differential, and isopycnic centrifugation.

2) Ultracentrifugation vs Low-speed Centrifugation:

Another system of classification is the rate or speed at which the centrifuge is turning. Ultracentrifugation is carried out at speed faster than 30,000 rpm. High-speed centrifugation is at speeds between 10,000 and 30,000 rpm. Low-speed centrifugation is at speeds below 10,000 rpm (mostly between 3,000 to 9,000 rpm).

3) Moving Boundary vs Zone Centrifugation:

A third method of defining centrifugation is by the way the samples are applied to the centrifuge tube. In moving boundary or differential centrifugation, the entire tube is filled with the sample and centrifuged. Through centrifugation, one obtains a separation of the mixture into two parts-a supernatant and a pellet. But any particle in the mixture may end up in the supernatant or in the pellet or it may be distributed in both fractions, depending upon its size, shape, density, and conditions of centrifugation. The pellet is a mixture of all of the sedimented components, and it is contaminated with whatever unsedimented particles were in the bottom of the tube initially. The only component that is purified is the slowest sedimenting one, but its yield is often very low. The two fractions are recovered by decanting the supernatant solution from the pellet. The supernatant can be recentrifuged at higher speeds to obtain further purification with the formation of a new pellet and supernatant.

Tags: Bio Technology, Bio Genetics , Biochemical Techniques

Related Posts by Categories