TECHNIQUES IN BIOCHEMISTRY
BIOCHEM-505 4 (2+2)
Block 1: Unit 4
CENTRIFUGATION
(HYDRODYNAMICS METHOD FOR SEPARATION
OF BIOMOLECULES)
Dr. ALKA KATIYAR
Teaching Associate
DEPARTMENT OF AGRICULTURAL BIOCHEMISTRY
C.S. AZAD UNIVERSITY OF AGRICULTURE & TECHNOLOGY, KANPUR
Block 1: Unit 4
CENTRIFUGATION
(HYDRODYNAMICS METHOD FOR SEPARATION
OF BIOMOLECULES)
LECTURE NO- 5 to 6
Objective-
Introduction
Principle
Instrumentation
Application
DEFINE: CENTRIFUGATION
Centrifugation is a technique of separating substances
which involves the application of centrifugal force.
The particles are separated from a solution according to
their size, shape, density, the viscosity of the medium and
rotor speed.
PRINCIPLE OF CENTRIFUGATION
In a solution, particles whose density is higher than that of
the solvent sink (sediment), and particles that are lighter
than it floats to the top.
The greater the difference in density, the faster they move. If
there is no difference in density (isopycnic conditions), the
particles stay steady.
To take advantage of even tiny differences in density to
separate various particles in a solution, gravity can be
replaced with the much more powerful “centrifugal force”
provided by a centrifuge.
A centrifuge is a piece of equipment that puts an object in
rotation around a fixed axis (spins it in a circle), applying a
potentially strong force perpendicular to the axis of spin
(outward).
The centrifuge works using the sedimentation
principle, where the centripetal acceleration
causes denser substances and particles to move
outward in the radial direction.
At the same time, objects that are less dense are
displaced and move to the center.
In a laboratory centrifuge that uses sample
tubes, the radial acceleration causes denser
particles to settle to the bottom of the tube,
while low- density substances rise to the top.
TYPES OF CENTRIFUGE
1. LOW-SPEED CENTRIFUGE
2. HIGH-SPEED CENTRIFUGES
3. ULTRACENTRIFUGES
LOW-SPEED CENTRIFUGE
1) Most laboratories have a standard low-speed centrifuge used for
routine sedimentation of heavy particles
2) The low-speed centrifuge has a maximum speed of 4000-
5000rpm
3) These instruments usually operate at room temperatures with no
means of temperature control.
4) Two types of rotors are used in it- Fixed angle and
Swinging bucket.
5) It is used for sedimentation of red blood cells until the particles
are tightly packed into a pellet and supernatant is separated by
decantation.
HIGH-SPEED CENTRIFUGES
High-speed centrifuges are used in more sophisticated
biochemical applications, higher speeds and
temperature control of the rotor chamber are essential.
The high-speed centrifuge has a maximum speed of
15,000 – 20,000 RPM
The operator of this instrument can carefully control
speed and temperature which is required for sensitive
biological samples.
Three types of rotors are available for high-speed
centrifugation-
1. Fixed angle
2. Swinging bucket
3. Vertical rotors
ULTRACENTRIFUGES
It is the most sophisticated instrument.
Ultracentrifuge has a maximum speed of 65,000 RPM
(100,000’s x g).
Intense heat is generated due to high speed thus the
spinning chambers must be refrigerated and kept at a
high vacuum.
It is used for both preparative work and analytical
work
TYPES OF CENTRIFUGATION
1. Differential Pelleting (differential
centrifugation)
2. Density Gradient Centrifugation
3. Rate-Zonal Density-Gradient Centrifugation
4. Isopynic Centrifugation
DIFFERENTIAL PELLETING (DIFFERENTIAL
CENTRIFUGATION)
It is the most common type of centrifugation
employed.
Tissue such as the liver is homogenized at 32 degrees
in a sucrose solution that contains buffer.
The homogenate is then placed in a centrifuge and
spun at constant centrifugal force at a constant
temperature.
After some time a sediment forms at the bottom of a
centrifuge called pellet and an overlying solution
called supernatant.
The overlying solution is then placed in another
centrifuge tube which is then rotated at higher
speeds in progressing steps.
DENSITY GRADIENT CENTRIFUGATION
This type of centrifugation is mainly used to
purify viruses, ribosomes, membranes, etc.
A sucrose density gradient is created by gently
overlaying lower concentrations of sucrose on
higher concentrations in centrifuge tubes
The particles of interest are placed on top of the
gradient and centrifuge in ultracentrifuges.
The particles travel through the gradient until
they reach a point at which their density matches
the density of surrounding sucrose.
The fraction is removed and analyzed.
RATE-ZONAL DENSITY-GRADIENT
CENTRIFUGATION
Zonal centrifugation is also known as band or gradient
centrifugation
It relies on the concept of sedimentation coefficient (i.e.
movement of sediment through the liquid medium)
In this technique, a density gradient is created in a test
tube with sucrose and high density at the bottom.
The sample of protein is placed on the top of the
gradient and then centrifuged.
With centrifugation, faster-sedimenting particles in
sample move ahead of slower ones i.e. sample separated
as zones in the gradient.
The protein sediment according to their sedimentation
coefficient and the fractions are collected by creating a
hole at the bottom of the tube.
ISOPYNIC CENTRIFUGATION
The sample is loaded into the tube with the gradient-forming
solution (on top of or below pre-formed gradient, or mixed in
with self-forming gradient)
The solution of the biological sample and cesium salt is
uniformly distributed in a centrifuge tube and rotated in an
ultracentrifuge.
Under the influence of centrifugal force, the cesium salts
redistribute to form a density gradient from top to bottom.
Particles move to point where their buoyant density equals
that part of gradient and form bands. This is to say the
sample molecules move to the region where their density
equals the density of gradient.
It is a “true” equilibrium procedure since depends on bouyant
densities, not velocities
Eg: CsCl, NaI gradients for macromolecules and nucleotides
– “self-forming” gradients under centrifugal force.
APPLICATIONS OF CENTRIFUGATION
1. To separate two miscible substances
2. To analyze the hydrodynamic properties of macromolecules
3. Purification of mammalian cells
4. Fractionation of subcellular organelles (including
membranes/membrane fractions) Fractionation of membrane
vesicles
5. Separating chalk powder from water
6. Removing fat from milk to produce skimmed milk
7. Separating particles from an air-flow using cyclonic separation
8. The clarification and stabilization of wine
9. Separation of urine components and blood components in
forensic and research laboratories
10. Aids in the separation of proteins using purification
techniques such as salting out, e.g. ammonium sulfate
precipitation.
