Centrifugation
is the technique of separating components where the centrifugal force/
acceleration causes the denser molecules to move towards the periphery while
the less dense particles move to the center.
— The process of centrifugation relies
on the perpendicular force created when a sample is rotated about a fixed
point.
— The rate of centrifugation is
dependent on the size and density of the particles present in the solution.
— A centrifuge is a device used to
separate components of a mixture on the basis of their size, density, the
viscosity of the medium, and the rotor speed.
— The centrifuge is commonly used in
laboratories for the separation of biological molecules from a crude extract.
— In a centrifuge, the sample is kept
in a rotor that is rotated about a fixed point (axis), resulting in strong
force perpendicular to the axis.
— There are different types of
centrifuge used for the separation of different molecules, but they all work on
the principle of sedimentation.
— Rotors in centrifuges are the motor
devices that house the tubes with the samples. Centrifuge rotors are designed
to generate rotation speed that can bring about the separation of components in
a sample. There are three main types of rotors used in a centrifuge, which are:
1. Fixed angle rotors
2. Swinging bucket rotors/ Horizontal rotors
3. Vertical rotors
1.Fixed angle rotors
— These rotors hold the sample tubes
at an angle of 45° in relation to the axis of the rotor.
— In this type of rotor, the particles
strike the opposite side of the tube where the particles finally slide down and
are collected at the bottom.
— These are faster than other types of
rotors as the pathlength of the tubes increases.
— However, as the direction of the
force is different from the position of the tube, some particles might remain
at the sides of the tubes.
2.
Swinging bucket rotors/ Horizontal rotors
— Swinging bucket rotors hold the
tubes at an angle of 90° as the rotor swings as the process is started.
— In this rotor, the tubes are
suspended in the racks that allow the tubes to be moved enough to acquire the
horizontal position.
— In this type of rotors, the
particles are present along the direction or the path of the force that allows
the particles to be moved away from the rotor towards the bottom of the tubes.
— Because the tubes remain horizontal,
the supernatant remains as a flat surface allowing the deposited particles to
be separated from the supernatant.
3.
Vertical rotors
— Vertical rotors provide the shortest
pathlength, fastest run time, and the highest resolution of all the rotors.
— In vertical rotors, the tubes are
vertical during the operation of the centrifuge.
— The yield of the rotor is not as
ideal as the position of the tube doesn’t align with the direction of the
centrifugal force.
— As a result, instead of settling
down, particles tend o spread towards the outer wall of the tubes.
— These are commonly used in isopycnic
and density gradient centrifugation.
TYPES OF
CENTRIFUGATION
— There are two main types of
centrifugation.
— They are Differential centrifugation
and Density gradient centrifugation.
Differential centrifugation
— Differential centrifugation is a
type of centrifugation process in which components are separately settled down
a centrifuge tube by applying a series of increasing centrifugal force.
Principle of Differential centrifugation
— Differential centrifugation is based
upon the differences in the sedimentation rate of biological particles of
different size and density.
— As the increasing centrifugal force
is applied, initial sedimentation of the larger molecules takes place.
— Further particles settle down
depending upon the speed and time of individual centrifugation steps and the
density and relative size of the particles.
— The largest class of particles forms
a pellet on the bottom of the centrifuge tube, leaving smaller-sized structures
within the supernatant.
— Thus, larger molecules sediment
quickly and at lower centrifugal forces whereas the smaller molecules take
longer time and higher forces.
— In the case of particles that are
less dense than the medium, the particles will float instead of settling.
Steps of Differential centrifugation
— The sample solution is homogenized
in the medium containing buffer.
— The sample is then placed in the
centrifuge tube, which is operated at a particular centrifugal force for a
specific time at a particular temperature.
— By the end of this operation, a
pellet will be formed at the bottom of the tube, which is separated from the
supernatant.
— The supernatant is added to a new
centrifuge tube where it is centrifuged at another speed for a particular time and
particular temperature.
— Again, the supernatant is separated
from the pellets formed.
— These steps are continued until all
particles are separated from each other.
— The particles can then be identified
by testing for indicators that are unique to the specific particles.
Uses of Differential centrifugation
— Differential centrifugation is
commonly used for the separation of cell organelles and membranes found in the
cell.
— It can also be used for
low-resolution separation of the nucleus.
— As this technique separates
particles based on their sizes, this can be used for the purification of
extracts containing larger-sized impurities.
Density gradient centrifugation
— Density gradient centrifugation is
the separation of molecules where the separation is based on the density of the
molecules as they pass through a density gradient under a centrifugal force.
Principle of Density gradient centrifugation
— Density gradient centrifugation is
based on the principle that molecules settle down under a centrifugal force
until they reach a medium with the density the same as theirs.
— In this case, a medium with a
density gradient is employed, which either has to decrease density or
increasing density.
— Molecules in a sample move through
the medium as the sample is rotated creating a centrifugal force.
— The more dense molecules begin to
move towards the bottom as they move through the density gradient.
— The molecules then become suspended
at a point in which the density of the particles equals the surrounding medium.
— In this way, molecules with
different densities are separated at different layers which can then be
recovered by various processes.
Steps of Density gradient centrifugation
— A density gradient of a medium is
created by gently laying the lower concentration over the higher concentrations
in a centrifuge tube.
— The sample is then placed over the
gradient, and the tubes are placed in an ultracentrifuge.
— The particles travel through the
gradient until they reach a point at which their density matches the density of
the surrounding medium.
— The fractions are removed and
separated, obtaining the particles as isolated units.
Uses of Density gradient centrifugation
— Density gradient centrifugation can
be applied for the purification of large volumes of biomolecules.
— It can even be used for the
purification of different viruses which aids their further studies.
— This technique can be used both as a
separation technique and the technique for the determination of densities of
various particles.
— Density gradient centrifugation is
of two types.
— They are Isopycnic centrifugation
and Rate-zonal density gradient centrifugation
Isopycnic centrifugation
— Isopycnic centrifugation is a type
of centrifugation where the particles in a sample are separated on the basis of
their densities as centrifugal force is applied to the sample.
Principle of Isopycnic centrifugation
— Isopycnic centrifugation is also
termed the equilibrium centrifugation as the separation of particles takes
place solely on the basis of their densities and not on their sizes.
— The particles move towards the
bottom, and the movement is based on the size of the particles. And, the flow
ceases once the density of the particle becomes equal to the density of the
surrounding medium.
— The density in the gradient
increases as we move down the tube towards the bottom. As a result, the
particles with higher densities settle down at the bottom, followed by less
dense particles that form bands above the denser particles.
— It is considered as a true
equilibrium as this depends directly on the buoyant densities and not the sizes
of the particles.
Steps of Isopycnic centrifugation
— A gradient prepared with an
increasing density towards the bottom of the tube is prepared. A pre-performed
gradient can also be used.
— The solution of the biological
sample and salt is uniformly distributed in the centrifuge tube and placed
inside the centrifuge.
— Once the centrifuge is operated, a
density gradient of the salt is formed in the tube.
— The particles move down the tube and
settle down as they reach the region with their respective densities.
— The particles are then separated and
identified using different other processes.
Uses of Isopycnic centrifugation
— Isopycnic centrifugation can be
applied for the purification of large volumes of biomolecules.
— This technique can be used as a
technique for the determination of densities of various particles.
Rate-zonal density gradient centrifugation/
Moving Zone Centrifugation
— Rate-zonal density gradient
centrifugation is a type of centrifugation that separates particles on the
basis of their shape as size and works on the same principle of density
gradient centrifugation but works in a different way. It is also called the moving
zone centrifugation.
Principle of Rate-zonal density gradient
centrifugation
— Rate zonal centrifugation
fractionates particles by both size and shape.
— The procedure is to layer a sample
in a restricted zone on top of a pre-poured density gradient. The density
gradient is then centrifuged.
— All particles migrate into the
density gradient because the density gradient has only densities much lower
than the densities of the particles being centrifuged.
— The particles are fractionated
primarily by size and shape. The larger a particle is, the more rapidly it
sediments.
— The more spherically symmetrical a
particle is, the more rapidly it sediments.
— The particles sediment through the
gradient at a rate that is a function of their sedimentation coefficient.
— Unlike differential centrifugation
where the sample is distributed throughout the medium, in rate-zonal
centrifugation, the sample is initially present only on top of
the gradient as a narrow band.
Steps of Rate-zonal density gradient
centrifugation
— A density gradient is prepared in a
centrifuge tube before applying the sample.
— The same is then layered on the top
of the gradient in the form of a band.
— During centrifugation, fast-moving
particles (larger in size and circular in shape) move ahead of slower particles
so that different particles are separated as various bands on different parts
of the gradient.
— The particles are separated on the
basis of their sedimentation coefficients, and they are obtained from the
bottom of the tube through a perforation.
Uses of Rate-zonal density gradient
centrifugation
— Rate-zonal differential
centrifugation has been used for the separation of viruses as they have
components that are of different size and density that are unique to each
virus.
— This method has been employed for
the fractionation of RNA on sucrose gradients.
— Besides, rate-zonal differential
centrifugation has also been used for the separation, purification and
fractionation of DNA molecules from both viruses and bacteria.
— The fractionation of polysomes and
ribosome subunits has been one of the earliest applications of this method.