Various techniques of Centrifugation 11.pptx

Presented by:
Dr. Sandeep Kumar Shah
PG-1st year

 Centrifugation is the technique of separating
components where the centrifugal force/
acceleration causes the denser molecules to move
toward 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.

 Principle of Centrifugation
•In a solution, particles whose density is higher
than that of the solvent sink (sediment), and
particles that are lighter than it floats to the top.
•The greater the difference in density, the faster
they move. If there is no difference in density
(isopycnic conditions), the particles stay steady.
•To take advantage of even tiny differences in
density to separate various particles in a
solution, gravity can be replaced with the much
more powerful “centrifugal force” provided by
a centrifuge.

•A centrifuge is a piece of equipment that puts an object in
rotation around a fixed axis (spins it in a circle), applying a
potentially strong force perpendicular to the axis of spin
(outward).
•The centrifuge works using the sedimentation principle, where
the centripetal acceleration causes denser substances and particles
to move outward in the radial direction.
•At the same time, objects that are less dense are displaced and
move to the center.
•In a laboratory centrifuge that uses sample tubes, the radial
acceleration causes denser particles to settle to the bottom of the
tube, while low- density substances rise to the top

1. Analytical Centrifugation
•Analytical centrifugation is a separation method where the
particles in a sample are separated on the basis of their
density and the centrifugal force they experience.
•Analytical ultracentrifugation (AUC) is a versatile and
robust method for the quantitative analysis of
macromolecules in solution.
Principle of Analytical Centrifugation
•Analytical centrifugation is based on the principle that particles
that are denser than others settle down faster. Similarly, the larger
molecules move more quickly in the centrifugal force than the
smaller ones.
•Analytical ultracentrifugation for the determination of the
relative molecular mass of a macromolecule can be performed by
a sedimentation velocity approach or sedimentation equilibrium
methodology.

•The hydrodynamic properties of macromolecules are
described by their sedimentation coefficients. They can be
determined from the rate that a concentration boundary of
the particular biomolecules moves in the gravitational field.
•The sedimentation coefficient can be used to characterize
changes in the size and shape of macromolecules with
changing experimental conditions.
•Three optical systems are available for the analytical
ultracentrifuge (absorbance, interference, and fluorescence)
that permit precise and selective observation of
sedimentation in real-time.

Uses of Analytical Centrifugation
•Analytical centrifugation can be used for the determination of the
purity of macromolecules.
•It can also be used for the examination of changes in the molecular
mass of supramolecular complexes.
•Besides, it allows the determination of the relative molecular mass
of solutes in their native state.

2. 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 increase in 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.

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.
Examples of Density gradient centrifugation
•This method was used in the famous experiment, which proved
that DNA is semi-conservative by using different isotopes of
nitrogen.
•Another example is the use of this technique for the isolation of
the microsomal fraction from muscle homogenates and
subsequent separation of membrane vesicles with a differing
density.

Various techniques of Centrifugation 11.pptx

3. 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.

•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.

4. 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.
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.

5. 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.

•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.

6. Differential velocity (Moving Boundary)
centrifugation
•Differential velocity centrifugation is a type of centrifugation
process in which components are separately settled down a
centrifuge tube by applying a series of increasing velocities.
Principle of Differential velocity (Moving Boundary)
centrifugation
•Differential centrifugation is based upon the differences in the rate
of sedimentation of biological particles of different size and density.
•As the increasing speed of the rotors 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.
•The pellet is then removed, and the supernatant is further
centrifuged to obtain smaller particles.
•Thus, larger molecules sediment quickly and at lower velocities,
whereas the smaller molecules take longer time and higher velocities.
•In the case of particles that are less dense than the medium, the
particles will float instead of settling.

•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 velocity (Moving Boundary)
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 identification and comparison of particles of
different sizes.

7. Equilibrium density gradient centrifugation
•Equilibrium density gradient centrifugation is a modified and
specialized form of density gradient centrifugation.
Principle of Equilibrium density gradient
centrifugation
•Equilibrium density gradient centrifugation is based on the
principle that particles in a solution are separated on the basis of
their densities.
•In this case, the particles move through the density gradient and
stop in a region where the density of the medium is equal to the
density of the particle.

Uses of Equilibrium density gradient centrifugation
•Equilibrium density gradient 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.
•At this point, the centrifugal force acting on the particle is
equal to the buoyant force pushing the particles up. As a result,
the particles cease to move and can be separated into different
layers.
•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.

Examples of Equilibrium density gradient
centrifugation
•This has been used in experiments performed by Meelson and
Stahl to determine the densities of different DNA molecules based
on where they reached on the density gradient.

8. Sucrose gradient centrifugation
•Sucrose gradient centrifugation is a type of density gradient
centrifugation where the density gradient is formed of sucrose by
changing the concentration of sucrose.
Principle of Sucrose gradient centrifugation
•Sucrose 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 sucrose gradient is employed, which
either has a lower density at the top and higher density at the
bottom.
•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.

Uses of Sucrose gradient centrifugation
•Sucrose gradient centrifugation is a powerful technique for the
separation of macromolecules like DNA and RNA.
•This has also been used for the analysis of protein complexes and
to determine the density as well as the size of various other
macromolecules.

Applications of Centrifugation
•To separate two miscible substances.
•To analyze the hydrodynamic properties of macromolecules
Purification of mammalian cells.
•Fractionation of subcellular organelles (including
membranes/membrane fractions) Fractionation of membrane
vesicles.
•Separation of urine components and blood components in forensic
and research laboratories.
•Aids in the separation of proteins using purification techniques
such as salting out, e.g. ammonium sulfate precipitation.

References
•Wilson, K., Walker, J. (2018). Principles and Techniques of
Biochemistry and Molecular Biology. Eighth edition. Cambridge
University Press: New York.
•Serwer, BACTERIOPHAGES: SEPARATION OF, Editor(s): Ian
D. Wilson, Encyclopedia of Separation Science, Academic Press,
2000, Pages 2102-2109,
http://trishul.sci.gu.edu.au/courses/7204BPS/Centrifugation_Lectu
re_2008.pdf
http://www.biologydiscussion.com/biochemistry/centrifugation/ce
ntrifuge-introduction-types-uses-and-other-details-with-
diagram/12489
https://www.slideshare.net/poojakamble1609/principles-and-
applications-of-centrifugation
https://en.wikipedia.org/wiki/Centrifugation
https://www.slideshare.net/khadeejaikram56/centrifugation-
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Various techniques of Centrifugation 11.pptx

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