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.
centrifugation techniques: Two main
types of centrifugation techniques are in
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.
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
and their uses: The
instrument used for this technique is known as centrifuge.Four major types of centrifuges are generally used.
capacity refrigerated centrifuges
speed refrigerated centrifuges
(i) preparative (ii) analytical
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.
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.
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
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
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.