Cell Fractionation Centrifugation botany

1. CELL FRACTIONATION:
CENTRIFUGATION
Sanjay Kumar Behera
Head Of Department Of Botany
Remuna Degree College

2. Cell fractionation
• It is a technique used in biological analysis to study the structure, chemical
composition and functions of cellular constituents.
• In this process the cells are ruptured and the cellular fractions are
separated by a process called centrifugation.
• Cell fractionation is done mainly in three steps:
• Extraction and homogenization, Centrifugation and Separation.
• Cells or tissues are first suspended in a solution of appropriate pH and
isotonic salt or sucrose solution.
• Then the suspended cells are disrupted by a process called
homogenization by applying physical forces through grinding, or through
osmotic shocks or by some sound vibrations (ultrasonic vibrations).

3. • These shearing forces ensure that all of the cells in the sample get broken
and the cellular fractions have come out.
• The liquid now contains the suspension of cell organelles and other
constituents and is called as a homogenate.
• The isotonic solution preserves the cellular organelles.
• The separation of the various fractions of the homogenate is done by a
process called centrifugation by use of different types of centrifuge
machines depending on the purpose.
• In the following sections we will discuss the centrifugation and separation
process in details.
Cell fractionation

4. Centrifugation
• Centrifugation is a process of separation of the cell homogenate into
fractions by spinning them at very high speeds.
• This centrifugation is done by different types of machines called
centrifuges (which, however, work on the same basic principles of
separation of particles.)
• The first analytical centrifuge was developed by Svedberg in late 1923 and
refinements and improvements were done in 1940s by Clande and his
team.
• Since then the centrifugation has become a key technique for isolating and
analyzing cells sub-cellular fractions, supra-molecular complexes and
isolated macro-molecules such as proteins or nucleic acids.
• Today centrifugation process represents a critical tool for modem
biochemistry and is employed in all most all invasive sub-cellular studies.

5. Basic Principles of Centrifugation
• It is a common observation that when a muddy water is kept undisturbed
for sometime, the soil particles settle down leaving a colloidal or turbid
water above it.
• This settlement of soil particles is done due to gravity and is called
sedimentation (settlement under force). But this takes a longer time.
• Only the particles having high weight or density settle and particles with
lower density do not settle down making the upper water turbid.
• In other words, these particles require more force on them for Settlement
or quick settlement. So, if gravitational force can be increased or is
replaced by some other higher force then these particles can settle down.
• In 1923, Svedberg and Nicholas for the first time put forth that if
centrifugal force is applied on the particles by spinning the suspension at a
high speed, then the particles will speed up their sedimentation.

6. • The centrifugal force due to spinning creates a similar effect like the
gravitational force on the particles in suspension but it is generated due to
rotation and is directed outwardly from the centre of rotation along the
radius.
• Centrifugation process is based on this principle of sedimentation.
• In a centrifuge machine the suspension is rotated at high speed around a
central axis which creates a centrifugal force on the particles in the
suspension.
• The particles move outwards in radial direction. However, two forces
counteract this centrifugal force acting on the particles- buoyant force and
the frictional force (generated by the particles as they migrate through the
solution).
• If the centrifugal force exceeds these two opposite forces, then the
particle sediment at a constant rate.
Basic Principles of Centrifugation

7. • So, the sedimentation depends on the applied centrifugal field (G), which
is given by the expression:
G = rω2
• Where, 'ω' (omega) is the angular velocity in radians / sec. and 'r' is the
radial distance (in cm) of the particle from the axis of rotation.
Angular velocity ω = radians / sec
Since the revolution is 360°, it is 2π radians.
So the revolution / minute (rpm) = ω × 60/2π
Or ‘ω’= 2π × rpm/60
• Since 'r' is fixed for a centrifuge, and ‘π’ has a fixed value, G can only be
increased by increasing the rpm; so that sedimentation particles can be
done quickly.
Basic Principles of Centrifugation

8. • Therefore Sedimentation depends on
Applied centrifugal field
Density of the particle.
Density or viscosity of the suspension medium.
• The G is generally expressed in multiples of the gravitational field, i.e. 'g'
(980 cms-1)
• The sedimentation rate or velocity (v) of the biological particle is
expressed as its sedimentation coefficient (S).
• Sedimentation rate is the rate at which particles of a given size and shape
travel to the bottom of the tube under centrifugal force.
S = v/rω2
• The sedimentation coefficients of biological macromolecules are relatively
small and are usually expressed as Svedberg's unit (S). One 'S' is equal to
10-13 s.
Basic Principles of Centrifugation

9. Applications of Centrifuge
• Centrifuge machine and centrifugation process has become the basic
requirement in all laboratories and research institutions, medical labs etc.
due to its various applications.
• Centrifugation process is done:
• To separate two miscible substances
• To analyze the hydrodynamic properties of macromolecules
• To purify mammalian cells
• For fractionation of sub-cellular organelles, fractionation of membrane
vesicles.
• To remove fat from milk to produce skimmed milk
• For separation of particles from an air-flow using cyclonic separation

10. • For clarification and stabilization of wine
• For separation of urine components and blood components in forensic
and research laboratories
• For separation of proteins using purification techniques
• For separation of protein bound or antibody bound ligand from free ligand
in immunological assay
• For removal of cellular debris from blood to separate cell free plasma or
serum.
Applications of Centrifuge

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