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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