different technique of centrifugation.

1. Topic: Centrifugation
Presented by-
Vl-a
M.Sc
Department of biotechnology
Central Mizoram university

2. Centrifugation
• Its Principle
Types of Centrifuges &
Its Uses &
Characteristics
• Applications of
Centrifugations

3. Centrifugation is a procedure that involves the use of
centrifugal force for the sedimentation of mixture with a
centrifuge used in industry and in laboratory settings. More
dense components of the mixture move away from the axis of
the centrifuge while less dense components of the mixture
move towards the axis

4. A particle whether it is a precipitate a macromolecule or a cell organelle is
subjected to a centrifugal force when it is rotated at a high rate of speed. The
centrifugal force F is denoted by equation
F=mω2 r
Where
F= intensity of the centrifugal force
m= effective mass of the sedimenting particle
ω= angular velocity of rotation
r= distance of the migrating particles from the central axis of
rotation
A more common measurement of F in terms of the earths gravitation force , g, is
relative centrifugal force, RCF its defined by
RCF = (1.119 x 10-5 (rpm)2 (r)
This equation relates RCF to revolutions per minute of the sample . Equation dictates
that the RCF on a sample will vary with r, the distance of the sedimenting particles
from the axis of rotation . The RCF value is reported as “ a number times gravity ,g .”

5. The basic centrifuge consists of two components:-
1. one is Electric motor with drive shaft to spin the sample.
2. Rotor to hold tubes or other containers of the sample.
There are three types of centrifuges
1.Low –speed centrifuges
Its also called as clinical centrifuge.
 has 4000 to 5000 rpm with RCF value approx. up to 3000Xg, operate at
low temperature with no means of temperature control of the samples.
 Two types of rotors fixed angle and swinging bucket may be used in this
instruments.
 Rotors used – fixed angle and swing bucket.
 Centrifuge tube containing 12 – 50 mL of sample are commonly used.
 useful for rapid sedimentation of coarse precipitates or red blood cells

6.  The sample is centrifuged until the particles are packed into
a pellet at the bottom of the tube. The upper liquid portion,
the supernatant, is then separated by decantation.

7. 2. High Speed Centrifuges:
 operator can control speeds and temperature of the rotor .
 Rotor chambers are usually maintained at or near 40C.
Three types of rotors are available for high speed centrifugation.
a. Fixed Angel rotor
b. Swinging –bucket rotors
c. vertical rotor
Rotors are made from carbon-fiber composition.
 12,000- 15,000 rpm and centrifugal field of 11,000-12,000 x g.
 High speed centrifuges are used to sediment
>Cell debris after cell homogenization.
>Ammonium sulfate precipitates of proteins
> cellular organelles such as chloroplasts, mitochondria and nuclei.

8. Fixed Angel rotor. Swinging –bucket rotors vertical rotor

9. 3.Ultracentrifuges:
 due to its high speeds, intense heat is generated in the rotor, so the
spin chamber must be refrigerated and placed under high vacuum
to reduce friction.
 rotor chamber is covered with protective steel armor plate.
 can hold six tubes of 0.2-2.2 mL samples.
 Spins sample up to 60,000 rpm and generate up to 600,000 x g.
 Ultracentrifuges can be used both for preparative work as well as for
analytical
Preparative models its primarily used for separations and purification
of samples for further analysis and analytical models which are
designed for performing physical measurements on the sample by
equipped with optical system to monitor directly the sedimentation of
the sample during sedimentation.

11. Types of Centrifuges & applications
Types of centrifuge
Characteristic Low Speed High Speed Ultracentrifuge
Range of Speed (rpm) 1-6000 1000-25,000 20-80,000
Maximum RCF (g) 6000 50,000 6,00,000
Refrigeration some Yes Yes
Applications
Pelleting of cells Yes Yes Yes
Pelleting of nuclei Yes Yes Yes
Pelleting of organelles No Yes Yes
Pelleting of ribosomes No No Yes
Pelleting of Macromolecules No No Yes

12. There are two techniques on which the application of
centrifuge is based on:-
1.Preparative Techniques
 quite straightforward, consisting of placing the sample
in a tube in the rotor and spinning the sample for a fixed
period.
 sample is removed and the two phases ,pellet and
supernatant may be separated by careful decantation.
 separates particles ranging in size from coarse
precipitates to cellular organelles .

14. 2.Analytical technique:
In this mostly measurements are taken to determine
molecular weight, density and purity of the sample
All analytical techniques require use of an ultracentrifuge and
can be classified as :-
1)DIFFERENTIAL CENTRIFUGATION
2)DENSITY GRADIENT CENTRIFUGATION

15. 1)DIFFERENTIAL CENTRIFUGATION
 In this tissue is first homogenized to break the cell membranes and mix up
the cell contents.
 The homogenate is then subjected to repeated centrifugations. Each time
removing the pellet and increasing the centrifugal force.
 Finally purification may be done through equilibrium sedimentation and
the desired layer extracted for further analysis .
 Separation is based on size and density, with larger and denser particles
pelleting at lower centrifugal force. Although the technique is similar to
preparative differential centrifugation but the goal of an experiment is to
measure the sedimentation coefficient of the particle.

17. 2)DENSITY GRADIENT CENTRIFUGATION
 Samples are centrifuged in a fluid medium that gradually
increase in density from top to bottom.
 It permits separation of multicomponent mixture of
macromolecules and the measurement of sedimentation
coefficient.
 Density gradient centrifugation permits the separation of
multicomponent mixtures of macromolecules and the
measurement of sedimentation coefficient .
 Two method are used – zonal centrifugation and isopycnic
centrifugation

18.  also known as band or gradient centrifugation
 it relies on the concept of sedimentation coefficient (i.e.
movement of sediment through liquid medium)
 in this technique a density gradient is created in a test tube
with sucrose (up to 60% conc.) and high density at the
bottom.
 the sample of protein is placed on the top of the gradient and
then centrifuged.
 the proteins sediment according to their sedimentation
coefficient and the fractions are collected by creating a hole
at the bottom of tube.
 Can be used in separation and isolation of macromolecules
and to the determination of s.

20.  also called as density gradient centrifugation.
 Density gradient is formed during cntrifugation.
 the solution of biological sample and cesium salt is uniformly
distributed in a centrifuge tube and rotated in an ultra
centrifuge.
 under the influence of centrifugal force the cesium salts
redistributes to form a density gradient from top to bottom.
 the sample molecules move to the region where their density
equals to the density of gradient.

22. Always :-
 carefully read operating manual of the
centrifuge.
 checks the rotor chamber for cleanliness and for
damage.
 Always balance centrifuge tubes before
centrifugation.
 Keep an accurate record of centrifuge and rotor
used.