This instrument is based on centrifugal forces. Basically, it has containers rotated around the central axis with the help of electric motor. Cooling centrifuges, high speed centrifuges and ultracentrifuges are available with the different types of rotors i.e. angle head and swinging bucket types. In the angle type rotor, the sample kept at an angle of about 30° to the horizontal whereas in the latter, the sample while spinning is horizontal. The most common centrifuge is clinical centrifuge. In the case of ultracentrifuge, the spun rotors produce force under vacuum to reduce friction.
For the separation of the components of a mixture, the sample in solution is centrifuged. The sedimentation takes place in a solution present in a column. The density of the solution increases as we move down the column. The solution should be inert as a result of which gradient is formed. This sample mixture is now put in a column which allows the sample to form different bands according to their rate of sedimentation. The components with high sedimentation rate are at lower end of the column. The size and shape of the molecule also affect the sedimentation, as sedimentation coefficient is a function of a mass of the particle. The bands are formed at various positions which can be separated by puncturing the tube, bands are collected separately.
Density Gradient Centrifugation
The sample is mixed in a dense solution possessing low concentration and having fast diffusing property. The mixture is spun in centrifuge but before starting the process the sample is to be in a uniform mixture. After centrifugation, the solution forms a density gradient and the sample components occupy those positions in the density gradient which correspond to their density. The bands of sample formed are separated by puncturing the tube. This method is also called as isopycnic centrifugation.
In this type of centrifugation the components of a mixture are separated according to their size, shape and density. Generally bigger molecules reach the lower part of the column before smaller one get sedimented. These particles which have the same size get sedimented according to their density difference whereas those particles which have same density are separated by differential centrifugation techniques. In this method,a homogenous solution of mixture of components is taken and centrifuged for a fixed duration and the centrifugal field is also fixed. After a certain period of time, it is found that some components are sedimented and they form supernatant solution. The pellet part is taken out and again centrifuged for fixed duration at a particular centrifugal field. Because first centrifugation does not give a pure pellet, by repeated centrifugation of the pellet portion only a pure pellet can be obtained. The supernatant fluid is separated from which the particles having lowest sedimentation rate can be separated out. As the supernatant fluid left at last, has lost all large, medium, small sized particles and it contains only the smallest sized particle pellets, after repeated centrifugation contain only large sized particles and thus is now pure. In the end, bands of sample components are formed the large sized particles at the bottom of the tube and the small sized particles present at the top of the tube. Thus the different components of a mixture are separated.
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