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These techniques are based up on the behaviour of particles in an applied centrifugal field such as density, shape or size of the molecules being separated. The particles are normally suspended in a specific liquid medium held in tubes or bottles which are located in rotor. The rotor is positioned centrally on the drive shaft of the centrifuge. Particles which differ in density, shape and size can be separated since they sediment at different rates in the centrifugal field. Each particle sediments at a rate which is proportional to the applied centrifugal field. The rate of sedimentation can be expressed as rpm (revolutions per minute) or as g (gravitational force).
The rate of sedimentation is dependent up on the applied centrifugal field (G) which is determined by the square of the angular velocity of the rotor (ω) and the radial distance (r) of the particle from the axis of rotation according to the equation G= ω2r. The sedimentation rate or velocity (v) of a particle can also be expressed in terms of its sedimentation per unit centrifugal field, known as sedimentation co-efficient (s).
v = s ω2 r.
Types of centrifugation techniques: Two main types of centrifugation techniques are in general use.
· Preparative centrifugation techniques: This technique is applied for the actual separation, isolation and purification of whole cells, plasma membrane, ribosomes, chromatin, nucleic acids, lipoproteins, viruses and many sub-cellular organelles. Large amount of materials may be involved for the bulk isolation.
· Analytical centrifugation techniques: This technique is applied to study the characteristic features of pure macromolecules or particles. It requires only a small amounts of materials and utilizes specially designed rotors and detector system to continuously monitor the process of sedimentation of the material in the centrifugal field.
Centrifuges and their uses: The instrument used for this technique is known as centrifuge.Four major types of centrifuges are generally used. They are
· small bench centrifuges
· large capacity refrigerated centrifuges
· high speed refrigerated centrifuges
· ultracentrifuge (i) preparative (ii) analytical
a. Small bench centrifuges: These are the simplest and less expensive instruments.They are used to isolate erythrocytes from blood and other separations which require low centrifugal force. These centrifuges generally have a maximum speed of 4000-6000 rpm (revolutions per minute).The speed can also be expressed as g / min.
b. Large capacity refrigerated centrifuges: These centrifuges have a maximum speed of 6000 rpm/ min . Compounds to be separated can be taken in bulk.The instrment is provided with refrigeration facility . By this method, biological materials can be isolated without any loss in their biological properties. Erythrocytes, coarse or bulky precipitates, yeast cells, nuclei and chloroplasts can be isolated by using this centrifuge.
c. High speed refrigerated centrifuges: These instruments have maximum speed of 25000 rpm/min. They are used to collect microorganisms, cellular debris, large cellular organells and precipated proteins
d. (i) Preparative ultracentrifuges: A maximum speed of 80000 rpm / min can be attained by this centrifuge. The rotor chamber is refrigerated , sealed and evacuvated to minimize excessive rotor temperature. These centrifuges are used for the separation of lipoprotein fractions and for deproteinisation of physiological fluids for aminoacid analysis
(ii) Analytical ultracentrifuges: These instruments are capable of operating at about 70000rpm/min. The rotor is present inside an evacuated , refrigerated chamber. An optical system is attached to observe the materials getting sedimented and to determine concentration distributions within, at any time during centrifugation.This technique finds applications for the separation and isolation of hormones, enzymes , ribosomal units, viruses and subcellular organells from animal and plant tissue homogenates.
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