Sedimentation is the process of allowing particles in suspension to settle down out of the suspension under the effect of gravitational field. The particles that settle down from the suspension are called sediment like mud settles from muddy water.

1. Centrifugation
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GATE, SET
Patkar-Varde college
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2. Centrifugation
•Principle
•Types of rotors in centrifugation
•differential centrifugation
•density gradient centrifugation.
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3. Sedimentation is the process of allowing particles in suspension to settle down out of the
suspension under the effect of gravitational field. The particles that settle down from the
suspension are called sediment like mud settles from muddy water.
For sedimentation to occur, it is required for particles to be heavier than the solution. In
this process, the Brownian particles attain a certain velocity under the action of
gravitational field (external field), which is known as sedimentation or settling velocity.
For small particles, the sedimentation velocity in the earth’s gravitational field is very
small, and sedimentation can only be observed by artificially increasing the gravitational
field by means of centrifugation.
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4. Basic Principle of Sedimentation
The basic principle of sedimentation is that particles of different sizes will settle at
different rates in a liquid, with the largest particles settling first. This is due to the fact
that larger particles have more mass and therefore require more energy to move them
through a liquid than smaller particles.
Centrifugation can only be used when the dispersed material is denser than the medium.
In centrifugation process, the rate of sedimentation is dependent upon the applied
centrifugal force (g) being directed readily outwards, which is determined by the square
of the angular velocity of the rotor (ω in radians S-1) and the radians (r, in centimeters)
of the particle from the axis of the rotation. Therefore, according to the equation.
g=ω²r
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5. Types of rotors in centrifugation
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6. 1. Horizontal (also called swinging bucket)
2. Fixed angle (or angle head) rotors.
3. Vertical tube rotor
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8. Rotors used in a centrifuge are categorized mainly into fixed angle rotors, swinging
bucket rotors, and vertical rotors depending upon the type of centrifugation, speed of
centrifuge, and volume of sample.
Rotors used for slow speed centrifugation are made up of steel, brass.
Rotors are fabricated from range of materials, such as carbon fiber, aluminum, and
titanium for high-speed centrifugation to withstand larger stress.
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9. Fixed angle rotor
In these rotors, particles move radially outward under the influence of centrifugal field
and they travel very short distance before reaching the outer wall of the centrifuge
tube.
Swinging-bucket rotor
Swinging-bucket rotors are ideal for separating large-volume samples at low speeds.
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10. Vertical tube rotor
Vertical rotors are commonly used in ultracentrifugation for banding of DNA in cesium
chloride(CsCl2). Vertical rotors have shortest path length for the particles to travel,
finally causing sedimentation of particles across diameter of the tube.
Thus, sedimentation of particles in vertical tube rotors is quick in comparison to fixed
angle or swinging bucket rotors.
It is generally used for the isolation of plasmid DNA and calculation of sedimentation
co-efficient.
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11. There are two main methods of separating particles by centrifugation:
differential centrifugation and density gradient centrifugation.
Differential centrifugation capitalizes upon the differential rates of sedimentation of
particles. The larger, denser particles have a higher rate of sedimentation.
Density gradient centrifugation produces a cleaner separation of particles than
differential centrifugation by employing a density matrix for the particles to move
through.
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12. The differential centrifugation process involves multiple centrifugation steps of
incrementally increased centrifugal force.
The largest and densest particles with the greatest rate of sedimentation will comprise
the pellet during the initial low-force spin while the smaller, less dense particles
remain in the supernatant.
The pellet and supernatant can then be separated, and the supernatant can be
placed back into the centrifuge at a higher centrifugal force to pull out the next group
of particles. This process is repeated as many times as necessary to isolate each desired
group of particles.
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13. In biochemistry and cell biology, differential centrifugation (also known as differential
velocity centrifugation) is a common procedure used to separate organelles and other
sub-cellular particles based on their sedimentation rate.
In a typical case where differential centrifugation is used to analyze cell-biological
phenomena (e.g. organelle distribution), a tissue sample is first lysed to break the cell
membranes and release the organelles and cytosol.
A fluid containing the contents of lysed cells is called a lysate.
The lysate is then subjected to repeated centrifugations, where particles that sediment
sufficiently quickly at a given centrifugal force for a given time form a compact "pellet" at
the bottom of the centrifugation tube.
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14. After each centrifugation, the supernatant (non-pelleted solution) is removed from the tube
and re-centrifuged at an increased centrifugal force and time. Differential centrifugation is
suitable for crude separations on the basis of sedimentation rate, but more fine purifications
may be done on the basis of density through equilibrium density-gradient centrifugation.
Thus, the differential centrifugation method is the successive pelleting of particles from the
previous supernatant, using increasingly higher centrifugation forces.
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16. Density gradient centrifugation employs a tube packed with a material that forms a
gradient of increasing density and viscosity. Various types of media are used for density
gradient separation including polyhydric alcohols, polysaccharides, inorganic salts, and
silica.
The type of matrix used is chosen based on the target molecule.
The density gradient matrix allows for more stringent separation of particles with
less contamination. The particles move through the matrix at different sedimentation
rates and settle out into clean bands.
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17. Rate-zonal centrifugation
As the name signifies, different-sized molecules will occupy different zones in a
centrifuge tube after centrifugation. This separation of macromolecules at different zones
is stabilized by using Sucrose gradient. So, this technique is also known as Sucrose
Density Gradient Centrifugation. This is used for separating all types of particles and
organelles.
Particle separation depends mostly on particle mass. Zones, or bands, are generated, each
containing a particle fraction of a specific mass. However, care must be taken when
performing rate-zonal centrifugation.
Because the mass of the particles is higher than the density of the solvent, if they are
centrifuged for too long, all particles will eventually deposit in the bottom of the tube.
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19. Isopycnic (equilibrium) centrifugation
In isopycnic separation, particles are mixed with the gradient solution, and during
centrifugation, they will move until they reach the gradient phase which equals their density
(isopycnic or equilibrium point).
Because the density of the gradient medium is always higher than the density of particles,
these will never sediment. Continuous gradients may be used in isopycnic centrifugation,
however, discontinuous gradients in which particles form bands at the interface between the
density gradient layers are more suitable for the separation of some biological samples, like
the separation of lymphocytes from whole blood.
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21. Centrifugation techniques
1. Preparative centrifugation technique-
Concerned with the actual separation, isolation, purification (eg.WBC, subcellular
organells, plasma membrane, polysomes,ribosomes,chromatin, nucleic acid, mitochondria,
stc.)
I. Differential centrifugation
II. Density gradient centrifugation-
a. Rate zonal
b. Isopycnic
2. Analytical centrifugation technique- Concerned with the study of sedimentation
characteristics of biological macromolecules and molecular structure.
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22. Analytical centrifugation technique
Require small amount of material, utilize specially design rotors and detector system to
continuously monitor the process of separation of the material in the centrifugal field.
Used for the purity, relative molecular mass and shape of material.
Analytical Centrifuge is Capable of speed approching7000 rev min -1 . Spins a rotor at an
accurately controlled speed and temperature.
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23. Application
Purity of macromolecules
Relative molecular mass of solute
Change in relative molecular mass of supermolecular complexes
Conformational change of protein structure
Ligand-binding study
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