DENSITY GRADIENT

CENTRIFUGATION
 CENTRIFUGATION

• It is a technique involving centrifugal force for the active

separation of particles based on their size, shape,
density, viscosity of medium, and rotor speed.

• The particles are suspended in a solvent medium and

placed in a centrifuge tube.

• This tube is then placed in a rotor and spun at a defined

speed for a period of time.
Centrifugation contd.
 TYPES OF CENTRIFUGATION
Centrifugation is mainly of two types-

i. Differential Centrifugation

ii. Density-gradient centrifugation

 Differential Centrifugation

It is a common method in
biochemistry and cell-
biology.

It is used to separate
organelles and other sub-
cellular particles on the
basis of sedimentation
rate.
DENSITY GRADIENT ANALYSIS
 INTRODUCTION
• This technique was introduced and developed by N. G.
Anderson and M. K. Brakke in the era of 1940-1950.

• This technique came forward to increase the dissolution

in a mixture to assist in better centrifugation.

• In the centrifuge tube, we put successive layers of

solvents in decreasing density, with the solvent with
highest density being at the bottom of the tube.
 TYPES OF DENSITY GRADIENT
There are 2 ways to make the density gradient:

1. DISCONTINUOUS: The solvent mediums are poured

step by step, according to their densities.

2. CONTINUOUS: All the solvents of varying densities are

poured simultaneously and the solvents gets settled inlayers
on their own on centrifugation.
 TYPES OF DENSITY GRADIENT
CENTRIFUGATION
• Based on the way of sedimentation of particles, density
gradient centrifugation can be divided into:

1. RATE-ZONAL SEDIMENTATION

2. ISO-PYCNIC SEDIMENTATION
 RATE-ZONAL SEDIMENTATION
• Here, the particles fractionates on the basis of both size and shape.

• In this method, the density of the particles is always greater than the density
of the medium.

• After centrifugation, the partcles migrate to their respective density gradient

layer.

• The larger the particle, the more rapidly the particle sediments.

• This method takes more time than iso-pycnic centrifugation.

Rate-zonal centrifugation contd.
 ISO-PYCNIC SEDIMENTATION
• This method is also known as Buoyant or Equilibrium Separation.

• The particles are separated on the basis of their density.

• The method starts with an uniform mixture of sample and density gradient.

• Upon centrifugation, particles of specific density settles in the layer of same

density gradient media.

• Particles are separated based on their buoyancy too.

• It takes less time than Rate-zonal technique.

Iso-pycnic sedimentation contd.
 APPLICATIONS
1. Isolation of enzymes.
2. Purification of particles.
3. Purification and separation of:
• Viruses(pox virus), bacterias, etc.
• Proteins
4. Separation of:
• Biomolecules
• RNA & DNA Hybrids
• RNA sub-units
• Antibodies
 FORENSIC APPLICATIONS
• Comparison of soil samples.
• Separation and purification of microorganisms (bacteria)
from soil.
• Isolation of spermatozoa in vaginal contents.
• Comparison and separation of planktons from liver
tissues.
• Purification of sperm cells.
 CASE STUDY

On 16 March 1978, the prime minister of Italy, Mr. Aldo Moro, was
kidnapped on his way to the Parliament House.
In the morning of 8 May 1978, Mr. Moro's body was found in the
trunk of a car.
The forensic investigation conducted on the evidence found on
Mr. Moro's clothes and shoes found beach sand, bitumen,
vegetable materials, and polyester fragments,which were same
as that present in the car.
The study of the vegetable materials suggested that they had
been picked up at a time close to the killing.
Thermosetting polyester, of the type used in boat manufacturing,
was found under the fenders, in the tires, and inside the car, as
well as under Mr. Moro's shoes, supporting proximity of a beach.
Also, low quality ashphalt were found in the tires which isn't
generally found in beaches.
THANK YOU