CENTRIFUGATION - SUJITHA

1. CENTRIFUGATION
BY,
S.SUJIDHA
2ND YEAR
BIOTECHNOLOGY.

2. DEFINITION:
• Biological centrifugation is a process that uses centrifugal force to separate
and purify mixture of biological particles in a liquid medium.
• It is a key technique for isolating and analysing the cells, subcellular fractions,
supra molecule complexes and isolated macro molecules such as proteins and
nucleic acids.

3. DISCOVERY:
• In 1864, Antonin Prandtl proposed the idea of a diary centrifuge to separate cream from milk.
• In 1923, theodor svedberg & his student H.Rinde had sucessfully analysed large grained sols in terms of
their gravitational sedimentation.

4. BASIC PRINCIPLE:
The basic physics on which the centrifuge works is gravity & generation of the centrifugal force to sedimant
different fractions.
• Rate of sedimentation depends on: applied centrifugal field (g) being directed radially outwards.
• G depends on
Angular velocity [ω in radians/sec]
Radical distance [r in CMS] particle from axis of rotation.
G=ω²r

5. TYPES OF ROTORS:
1. Fixed angle rotors.
2. Vertical tube rotors.
3. Swinging bucket rotors.

6. 1.FIXED ANGLE ROTORS:
• Tubes are held at angle of 14-40°c to the vertical.
• Particles moves radially outwards, travel a short distance.
• Useful for differential centrifugation.
• Reorientation of the tube occurs during acceleration &
declaration of the rotor.

7. 2.VERTICAL ANGLE ROTORS:
• Held vertical parallel to rotor axis.
• Partical moves short distance.
• Time of separation is shorter.
DISADVANTAGE: Pellet may fall back into solution at the end of
the centrifugation.

8. 3.SWINGING BUCKET ROTORS:
• Sing out to horizontal position when rotor accelerates.
• Longer distance of travel may allow better separation, such
as in density gradient centrifugation.
• Easier to withdraw supernatant without disturbing the pellet.
• Normally used for density gradient centrifugation.

9. TYPES OF CENTRIFUGE:

10. 1.DESKTOP CENTRIFUGE:
• Very simple and small.
• Maximum speed of 3000 rpm.
• Donot have any temperature regulatory system.
• Used normally to collect rapidly sediment substance such as blood cells, yeast cells, or
bulky precipitates of chemical reactions.

11. 2.HIGH SPEED CENTRIFUGES:
• Maximum speed of 25,000 rpm, providing 90,000 g centrifugal force.
• Equipped with refrigeration to remove heat generated.
• Temperature maintained at 0-4°c by means of thermocouple.
• Used to collect micro organism, cell debris, cells, large cellular organelles,precipitates
of chemical reactions.

12. 3.ULTRA CENTRIFUGE:
• Operate at speed at 75,000 rpm, providing the centrifugal force to 500,000 g.
• Rotor chamber is sealed and evacuated by pump to attain vaccum.
• Refrigeration system [temp: 0-4°c].
• Rotor chamber is always enclosed in a heavy Armor plate.
There are two types in ultra centrifugation:
Preparative
Analytical.

13. 3.1 .PREPARATIVE CENTRIFUGATION:
• Centrifugation for isolation and purification of components is known as preparatory centrifugation.
• It is concerned with the actual isolation of biological material for subsequent biochemical investigation.
Dividing into 2 main techniques depend on suspension medium in which separation occurs:
a. Homogenous medium – differential centrifugation.
b. Densityy gradient – density gradient centrifugation.

14. 3.1.A. DIFFERENTIAL CENTRIFUGATION:
• Separation is acheived based in the size of the particles in differential centrifugation.
• Commonly used in simple pelleting and obtaining the partially pure separation of subcellular organelles and
macro molecules.
• Used for study of sub cellular organells, tissues or cells.
• During centrifugation, larger particles sediment faster than the smaller ones.
• DRAWBACK: it is poor yielding fact that preparation obtained is never pure.

15. DIFFERENTIAL CENTRIFUGATION:

16. 3.1.B. DENSITY GRADIENT CENTRIFUGATION:
• It is the preferred method to purify subcellular organells and macromolecule.
• Density gradient can be generated by placing layer after layer of gradient media such as sucrose in tube, with heaviest
layer at the bottom and lightest at the top in either.
Classified into 2 categories:
- Rate zonal [ size ] separation.
- Isopycnic [ density ] separation.

17. - RATE ZONAL CENTRIFUGATION:
• It is a gradient centrifugation.
• Take advantage of particle size and mass instead of particle density for
sedimentation.
EX: for common application include separation of cellular organells such as endosomes
or proteins ( such as antibody ).

18. - ISOPYCNIC CENTRIFUGATION:
• Particle of a particular density will sink during centrifugation until a position is
reached where the density of the surrounding solution is exactly the same as the
density of the particle.
• Once quasi- equilibrium is reached, the lenght of centrifugation does not have any
influence on the migration of particle.
EX: separation of nucleic acids in CS CL [ cesium chloride] gradient.

19. 3.2 . ANALYTICAL CENTRIFUGATION:
• Speed – 70,000 rpm. RCD – 5 lakh g.
• Motor, rotor, chamber that is refrigerated and evacuated and optical system.
• Optical system has light absorption system, schleiren system and reyleigh inferometric system.
2 cells – analytical cell and counterpoise cell.

21. APPLICATIONS:
1. Remove cellular elements from blood to mprovide cell free plasma or serum for analysis
2. Remove chemically precipitates protein from an analytical specimen.
3. Separate protein bound from free ligand in immuno chemical and other assay.
4. Separation of sub-cellular organelles: DNA, RNA.
5. Exact solutes in biological fluids from aqueous to organic solvents.
6. Separates lipids components.