Centrifugation is a process used to separate or concentrate materials suspended in a liquid medium. It is a method to separate molecules based on their sedimentation rate under the centrifugal field. It involves the use of centrifugal force for the sedimentation of molecules.

1. CENTRIFUGATION:
Basic Principle &
Theory
By: Mayank Sagar

2. WHAT IS CENTRIFUGATION?
• Centrifugation is a process used to separate or concentrate materials suspended
in a liquid medium.
• It is a method to separate molecules based on their sedimentation rate under
centrifugal field.
• It involves the use of the centrifugal force for the sedimentation of molecules.
• It is also used to measure physical properties such as molecular weight, density
& shape of molecules.

3. TYPES OF
CENTRIFUGATION
Preparative Analytical
For separation of one type of material
from others
For measurement of physical properties
of macromolecules

4. PRINCIPLE OF CENTIFUGATION
• Particles suspended in a solution are pulled downward by Earth's gravitational
force , In a solution, particles whose mass or density is higher than that of the
solvent sink, and particles that are lighter than it float to the top.
• The greater the difference in mass or density, the faster they sink.
• This sedimentation movement is partially offset by the buoyant of the particle.
• Because the Earth’s gravitational field is weak , a solution containing particle of
very small masses usually remain suspended due to the random thermal motion.

5. • Gravitational force depends only on the mass
and not on charge, shape and chemical
composition.
• Forces much larger than Earth's gravitational
force are required to cause appreciable
sedimentation of such very small masses.
• Such forces can be obtained by subjecting
particles to centrifugation. Sedimentation of
these particles can be enhanced by applying
centrifugal forces.
• A centrifuge does the same thing. It increases
the sedimentation by generating centrifugal
forces as great as 1,000,000 times the force of
gravity.

6. CENTRIFUGAL FORCE
• A centrifugal force is a kind of force that is exerted on particle when they are
subjected to circular motion.
• Lets consider a solution being spun at centrifuge.
Where, m = Mass of particle
ω = Angular velocity in radians per second
r = Radius of a particle from the axis of rotation
ω2 r =Centrifugal acceleration

7. OTHER INTERACTING FORCES
• Other than, centrifugal force, there are some other interacting forces, namely
buoyant and frictional force.

8. Buoyant force
• A buoyant force is an exerted force that causes movement of particles upwards
and develops as a result of force applied to the particles by the fluid.
• The opposing buoyant force is smaller for a dense particle and can express as:
• Where ρ= Fluid density
g = Gravitational or centrifugal acceleration
V= Volume of fluid displaced
• So, Net force = centrifugal force – buoyant force

9. Frictional force
• A frictional force is a kind of force that restricts the movement of dispersed particles
in the solution. The frictional coefficient is smaller for a minute particle than the dense
particle.
• When particles move downward through the solution, the motion is also opposed by
the frictional force.
• The frictional force is equal to the product of the frictional coefficient and the
sedimentation velocity.
• It acts in the opposite direction to the net force:
Where f= Frictional coefficient
v= Sedimentation velocity

10. • In a steady-state, the frictional force is equal to the net force.
fv= mω2r – ω2rVρ
= mω2r – ω2rmv̅ρ
= mω2r (1– v̅ρ)
• The mass of 1mole of particles represented by,
M= mN (Where N is the Avogadro’s number)
fv= Mω2r (1– v̅ρ)
N

11. SEDIMENTATION COEFFICIENT
• The Sedimentation coefficient (S) is defined as sedimetation rate per unit of
centrifugal force.
• It is equals to the ratio of velocity to the gravitational acceleration.
S= v/ω2r = M/N.(1– v̅ρ)/f
• A sedimentation coefficient is measured by the units of a second.
1 S = 10-13second.
• This unit is named for The Svedberg, a pioneer in the field of centrifugation.

12. REALTIVE CENTRIFUGAL FORCE
• Also known as g- force.
• Relative centrifugal force is the force acting on samples during centrifugation.
• RCF is the ratio of the centrifugal acceleration at a specified radius and the speed to the
standard acceleration of gravity.
• It is expressed as multiples of the earth's gravitational field (g).
E.g, 500 g = 500 RCF
• It depends on the revolutions per minute (RPM) and
radius of the rotor, and is relative to the force of Earth’s gravity.
RCF = 11.2 × r (RPM/1000)2 or RCF = 1.12 × 10-5 (RPM)2
• RCF units are therefore dimensionless (denoting multiples of g).

13. FACTORS AFFECTING CENTRIFUGATION
Density of
both sample
and solution
Temperature
Viscosity
Distance of
particle
displacement
Rotation
speed

14. FIXED- ANGLE ROTOR
cross-sectional diagram of a fixed-angle rotor
cross-sectional diagram of a centrifuge tube
positioned in a fixed-angle rotor

15. VERTICAL- TUBE ROTOR
cross-sectional diagram of a vertical
tube rotor
cross-sectional diagram of a centrifuge tube
positioned in a vertical tube rotor

16. SWINGING- BUCKET ROTOR
cross-sectional diagram of a
swinging-bucket rotor
cross-sectional diagram of a centrifuge tube positioned in
a swinging-bucket rotor

17. FIXED-ANGLE ROTOR VERTICAL TUBE ROTOR SWINGING BUCKET
ROTOR
Tubes are held at angle of 14°
to 40° to the vertical
Held vertical parallel to rotor
axis (7° – 10°)
Sing out to horizontal
position when rotor
accelerate
Particles moves radially
outwards, travel a short
distance.
Particles move short distance Longer distance of travel, may
allow better separation
Useful for differential
centrifugation
Used in density gradient
centrifugation
Used in density gradient
centrifugation
Comparatively low resolution Intermediate Best resolution
Take more separation time Very less time More time than fixed angle .
Pellet is formed at outermost
points of the tube
along outer wall of the tube
across diameter
At the bottom of the tube
RCF is intermediate Highest lower

18. CENTRIFUGES
• Centrifuge is primarily used to separate biological components based upon
differential sedimentation properties.
• Centrifugation is a technique for the separation of the components of an analyte based
on differences in the rate of migration under the influence of a centrifugal field.
• All centrifuges basically consist of a motor which spins a rotor containing the
experimental sample.
• The most obvious differences between centrifuges are:
1. speed at which biological specimens are subjected to increased sedimentation.
2. presence or absence of a vacuum
3. potential for refrigeration or general manipulation of the temperature during a
centrifugation run
4. maximum volume of samples and capacity for individual centrifugation tubes

19. TYPES OF CENTRIFUGES
• Based on the speeds at which the samples are centrifuged and the volumes of
samples following are the types of centrifuge:
• Microfuge
 so called because they centrifuge small volume samples in Eppendorf tubes
 0.5 to 1.5 cm 3 volumes.
 centrifugal fields of approximately 10000×g
sediment biological samples in minutes
 can also be used to concentrate protein samples

20. • Table/bench (/large preparative) top centrifuge :
 vary in design and are mainly used to collect small amounts of biological material,
such as blood cells.
 To prevent denaturation of sensitive protein samples,
refrigerated centrifuges should be employed.
maximum centrifugal fields of 3000×g to 7000×g
 used for the spinning of various types of containers
like (depending on the range of available adapters),
Volume of 5 to 250 cm3 plastic tubes, 96-well ELISA
simple and relatively inexpensive
central place in many high-throughput biochemical assays, where the quick and
efficient separation of coarse precipitates or whole cells is of importance.

21. • High-speed refrigerated centrifuges
 for the sedimentation of protein precipitates, large intact organelles, cellular debris
derived from tissue homogenisation and microorganisms.
centrifugal fields of approximately 100 000×g.
can be employed to differentially separate nuclei,
mitochondria or chloroplasts , bulky protein aggregates.
 example separation of myosin and actin macromolecules
filaments aggregates from muscle fibres lysate.
 Can be used to harvest yeast cells or bacteria from large
volumes of culture media, in a continuous flow mode with zonal rotors.
 but not sufficient to sediment smaller microsomal vesicles or ribosomes.

22. • Ultracentrifugation
 can be operated at relative centrifugal fields of up to 900 000×g -1 000 000 g
 the rotor chamber is sealed, evacuated and refrigerated to minimize
excessive rotor temperatures generated by frictional resistance
 Sophisticated instrument though very expensive
 Consists of specialised optical system enabling the sedimenting
material to be observed throughout the duration of a centrifuge run.
 detailed biochemical analysis of subcellular structures
and isolated biomolecules can be studied.
 concentration distributions can be recorded at any time during ultracentrifugation
 information about the purity/heterogeneity, sedimentation coefficient distribution, average
molar mass and molar mass distributions, and ligand interaction information can be obtained.

23. SAFETY ASPECTS OF CENTIFUGES
• Always close lid of centrifuge during operation.
• Always balance centrifuge.
• Do not open the lid while the rotor is moving
• Disinfect weekly and after all spills or breakages.
• Check tubes for cracks because they can break in centrifuge during operation.
• The work surface must be level and firm. Do not use the centrifuge on an
uneven or slanted work surface.

24. ROTOR BALANCE
• The mass of a properly loaded rotor will be evenly distributed on the centrifuge
drive hub, causing the rotor to turn smoothly with the drive
• An improperly loaded rotor will be unbalanced; consistent running of
unbalanced rotors will reduce ultracentrifuge drive life.
• To balance the rotor load , fill all opposing tubes to the same level with liquid of
same density.
• Weight of opposing tubes must be distributed equally.
• Place tubes in the rotor symmetrically.

25. REFERENCES
• Principles and techniques of Biochemistry and Molecular biology(7th ed.) –
Wilson and Walker
• Biophysical chemistry : principles and techniques- Avinash Upadhyay, Kakoli
Upadhyay.
• The cell: a molecular approach (4th ed.)- Geoffrey M.Cooper , Robert E. Hausman
• http://www.biologydiscussion.com/biochemistry/centrifugation/centrifuge-
introduction-types-uses-and-other-details-with-diagram/12489
• https://en.wikipedia.org/wiki/Centrifugation.
• Image courtesy- google images.