Ultra centrifugation

Bhawna Jukariya
M.Sc. III sem
(LSMGPGC Pithoragarh)
 Overview
• Centrifugation
• Princile
• Ultra centrifugation
• Types
• Applicaation
 Centrifugation
• A centrifugation is a device for separating particles from a solution
according to their size, shape, density, viscosity, of the medium and rotor
speed. In biology, the particles are usually cells, sub-cellular organelles,
viruses, large molecules such as proteins and nucleic acid.
 Principle of centrifugation
• A particle whether it is precipitate, a macromolecule or cell organelle when rotated at
high speed is subjected to a centrifugal force.
• Centrifugal force is defined as F = mwr
• Where F = intensity of centrifugal force
m = effective mass of sedimenting particle
w = angular velocity of rotation
r = distance of migration particles from central axis of rotation
Centrifugation
 Centrifugation
• Sedimentation of particles under gravity would take a larger amount of time, and
that is why an additional force is applied to aid the sedimentation process.
• Larger molecules move faster, whereas smaller molecules move slower.
• Denser molecules move outward the periphery of the tubes whereas the less dense
molecules are rotated toward the center of the tube.
• Once the process is complete, the larger and more dense particles settle down,
forming pellets at the bottom of the tube. In comparison, the smaller and less dense
particles remain either in the suspended in the supernatant or float on the surface.
 Ultra centrifugation
• Invented by Theodor Svedberg in 1923. (Nobel in1926)
• It is an important tool in biochemical research. It imposes high centrifugal force on
suspended particles and separates them on the basis of difference in molecular weight.
• Its rotational speed is up to 80000 rpm.
• It create a centrifugal force up to 600000g.
• Example –
1. RBC separation from plasma
2. Separation on mitochondria from nuclei
 Types of ultra centrifugation

Analytical ultra
centrifugation
Ultra
centrifugation Preparative
ultra
centrifugation
 Analytical ultra centrifugation
• The aim of analytical centrifugation is to study molecular interaction
between macromolecules or to analyze the properties of sedimenting
particles.
• The sample can be monitored in real time through a optical detection
system using UV light absorption or through interference of optical
refractive index sensitive system.
 Analytical centrifugation
• Sedimentation velocity experiment • Sediment equilibrium experiment
The objective of sedimentation Sedimentation equilibrium exp are
velocity exp to interperate the entire design to determine the final steady
time- state of the experiment.
Course of sedimentation
Determine the shape and mass of
dissolved macromolecules and their
sie distribution
 Function of analytical centrifugation
• It uses small sample size (less than 1mm).
• It built optical system to analyze the progress of separated molecules.
• It uses relatively pure sample.
• It is used to determine sedimentation coefficient and molecular weight of
the molecules.
 Preparative ultra centrifugation
• The aim of preparative centrifuge is to isolate and purify particles such as
sub-cellular organelles.
• These are available in wide variety of rotors.
• Most rotor are design to hold tubes that contain the samples E.g. swinging
bucket rotor and fixed angle rotors.
 Centrifuge rotors
• Fixed angle rotor • Swinging bucket rotor
Sedimenting particles have only Longer distance of travel may allow
short distance to travel before better separation, such as in density
pelleting. gradient centrifugation.
Shorter run time. Earlier to withdraw supernatant
Most widely used rotor types. without disturbing pellet.
 Differential ultra centrifugation
It is common procedure in microbiology and cytology to separate organelles
from whole cell for further analysis of those components.
In this process a tissue sample is first homogenized is then subjected to
repeated centrifugating each time removing the pallet and increasing the
centrifugal force.
Differential ultra centrifugation
• Density gradient centrifugation
It allows separation of many or all components in a mixture and also allow
for their measurement.
It is divided into two categories-
1. Rate zonal
2. Isopycnic
 Rate zonal
• In the rate zonal the solution have density gradient and the sample has
density which is greater than all the layer in solution.
• The sample is applied in a thin zone at the top of the centrifuge tube.
• Under centrifugal force the particles will begin to separate according to
their zones (Density gradient).
• The particles will sediment in separate zones according to their shape and
size.
Rate zonal centrifugation
 Isopycnic
• Isopycnic means of the same density. In this technique molecules are
separated on equilibrium position.
• Each molecules floats or sink in equivalent position.
• Separation of particles occurs on their density differences, independent of
time.
Isopycnic centrifugation
 Functions of preparative centrifuge
• Large sample size can be used.
• No inbuilt optical system for read out.
• Less pour sample can be used.
• It can be used to determine sedimentation coefficient and molecular
weight of molecules.
• It is generally used to separate organelles and cell component.
 Commercial applicaations
• Centrifuges with a bath weight of up to 2,200 kg per charge are used in the
sugar industry to separate the sugar crystal from the mother liquor.
• Standalone centrifuges for drying (hand-washed) clothes – usually with
water outlet.
• Larger industries centrifuges are also used in the oil industry to remove
solids from the drilling fluid.
 Applications
• Separating biomolecules from samples on the basis of their mass, size.
• Separating particles from an air flow using cyclonic separation.
Thank you