Sunday, November 24, 2013

Centrifugation

Centrifugation
Centrifugation is the separation and sedimentation of sub- cellular frac­tions of the homogenate according to their size, mass, density and specific gravity. The instrument used for this is called centrifuge . It consists of a pivoted cylinder, called rotor, in which the samples are placed. The rotor rotates or spins around a central axis at very high speeds. This produces gravitational fields, which are higher than earth's gravity. The high rotational speed of the rotor produces a centrifugal force, directed away from the cen­tre. It may be as much as 100,000 times greater than gravitational force. The magnitude of the centrifugal force is expressed in terms of earth's gravita­tional force, namely 'g' (g = 980 cms/sec2). The relative centrifugal force (RCF) is directly proportional to the number of revolutions per minute (rpm-ω), radius of rotation (r) and the gravitational force
 Centrifugal force causes the sample particles to move away from the cen­tre and also to settle in different layers according their sedimentation ratio. Heavier particles move away first and get deposited on the outer wall of the tube. Lighter particles follow them in regular sequence in the order of de­creasing mass.
The intensity of the centrifugal force in relation to the sedimentation of particles can be represented by the equation.
The rate of sedimentation of particles in centrifugation is called sedimen­tation coefficient. The unit of its measurement is called Svedberg unit(S). The suspension, left above the sediment after centrifugations, is called superna­tant. It may be subjected to repeated centrifugation, with progressively in­creasing speed. As a result, different particles settle down differently at different times, at different rates and at different sites. This may be called differential centrifugation.
In classical fractionation and centrifugation cell components separate into four successive fractions in the order nuclear fraction, mitochondrial fraction, microsomal fraction and soluble fraction. For the separation of DNA and RNAs an improved method of centrifugation has been deviced. It is called density gradient centrifugation.
Kinds of centrifuges
Three major kinds of centrifuges can be recognized, namely low-speed centrifuges, high-speed centrifuges, and ultracentrifuges. Low-speed centri­fuges are the most ordinary types of centrifuges, used for the routine sedimentation of heavy particles. Their rotor has a maximum speed up to 4,000 or 5,000 rpm, with RCF values up to 3000g. Low-speed centrifuges are used at room temperature, and they have no means for temperature control of the samples. High-speed centrifuges are advanced types of centrifuges, with higher speed and temperature control of the rotor chamber. They are especially important in the centrifugation of temperature-sensitive biological samples. Their rotor revolves at an average speed of 20,000 rpm, with an RCF value up to 50000g.
Ultracentrifuges are the most sophisticated and refrigerated types of cen­trifuges. In them the rotor rotates at extremely high speeds (75,000 rpm), producing a gravitational pull of above 50,000g. Ultracentrifuges are used for the separation of viruses and extremely minute sub-cellular components and also for the determination of molecular weights. They are used for pre­parative work and analytical measurements. So, there are two kinds of them, namely preparative Ultracentrifuges and analytical Ultracentrifuges. Analyti­cal Ultracentrifuges are provided with window and optical system. So, sedi­mentation of particles can be observed and their sedimentation rates can be measured during the run.
Methods of centrifugation
There are two main kinds of centrifugation, namely differential centrifuga­tion and density -gradient centrifugation.
(i) Differential centrifugation
Differential centrifugation is the successive centrifugation of the homogenate at progressively increasing rotor speeds, leading to the succes­sive separation of cell components. It involves the sedimentation of particles in a medium of homogeneous density.
(ii) Density-gradient centrifugation
Density-gradient centrifugation is the technique used for the separation of multi component mixtures of macromolecules and also for the measure­ment of sedimentation coefficients (S values). In this case, the suspending liquid and the multi component sample exhibit density difference. Density will be highest at the bottom of the centrifuge tube, and lowest at the top. In between these two extremes there will be a gradient (graded series) of den­sity- different zones. This may be called density gradient. The multi compo­nent sample is layered at the top of the gradient. Ultracentrifugation at a constant speed results in the sedimentation of the sample components according to their density. When a fraction reaches a liquid zone, whose density is the same as that of the fraction, the fraction stays there without any further sedimentation. In this manner different fractions of the sample sepa­rate into distinct density- different zones or bands.

Density-gradient centrifugation is widely used in the separation and puri­fication of biological macromolecules and also for the determination of S values. 

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