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CENTRIFUGATION

 Centrifugation is one of the most important separation technique.


 It exploits the inherent varied sedimenting property of substances
for their isolation by the application of centrifugation field.
 The resullting solution has 2 components namely the SEDIMENT
(Pellet), and SUPERNATENT.

C E N T R I F U G A T I O N - BASIC PRINCIPLES

 This method is based on the principle of sedimentation.

 When a particle sediments, it must displace some of the solution in


which it is suspended, resulting in an upthrust on the particle
equal to the weight of the liquid displaced.

 If a particle is assumed to be a sphere of known volume and


density, then the net force(f) is experienced when the centrifugal
force at an angular velocity of ω radians/sec is given by:

S = Volume x Density x ω2r


Or,
F = 4/3 Π rp3 (ρp – ρm) ω2r ------------------ 1

Where,
4/3 Π rp3 = Volume of sphere of radius ‘r’.
ρp = Density of the particle.
ρm = Density of the suspended medium.
ν = Distance of the particle from the center of rotation.
ω = Angular velocity of rotor.

FRICTIONAL COEFFICIENT

 Particles however generate friction as they migrate through the


solution.
 If the particle is spherical and is moving at a known velocity, then
its frictional force appearing opposing motion is given by Stoke’s
law.

f0 = 6Πηrpv ------------------------------ 2
Where:
f0 = Frictional coefficient of spherical particles.
η = Coefficient of viscosity, and v = Velocity of the sedimenting particle.
 A particle of known volume and density are present in a medium of
constant density will therefore be accelerated in a centrifugal field,
until the net force on the particle equals the force resisting its
motion through the medium
f = f0
or,
4/3 Π rp3 (ρp – ρm) ω2r = 6Πηrpv ---------------------- 3

 In practice, the balancing of this force occurs quickly, with the


result that the particles sediment at a constant ratio. Its rate of
sedimentation (v) is given by:

v = dx/dt = 2/9 rp2 (ρp – ρm) ω2r -------------------- 4


η

 The non-spherical molecules as in the case of rod like molecules,


such as DNA and proteins like myosin experience considerable
frictional resistance.
 Frictional coefficient (f) of the molecules can be increased as much
as 10 times of the frictional coefficient of the sphere (f0).
 This results in the particles sedimenting at a lower rate and thus,
equation 4 can be modified as:
v = dr/dt = 2/9 rp2 (ρp – ρm) ω2r --------------------
5

f/f0

 From equation 5, it is clear that the sedimentation rate is of a


particle is dependant on the following factors:
 It is directly proportional to the size of the particle,
since the equation involves the square of the particle
radius. It is apparent that the size of the particle has
the greatest influence upon sedimentation.
 It is directly proportional to the difference in density
between the particle & the medium. It will be zero,
when the density of the particle and the medium are
equal.
 It will decrease, when the viscosity of the medium
increases (i.e., ω2r) v = 1/η.
 The sedimentation rate (or) velocity decreases as the frictional
coefficient ratio (f/f0) increases the ratio f/f0 is approximately 1 for a
spherical molecule.
 The rate of sedimentation depends on the applied centrifugal field (G)
ω2r being directed radially outwards which is determined by the
square root of angular velocity of the rotor and radial distance of the
particle from the axis of rotation.
 According to the equation, G= ω2r, since one revolution of the rotor is
equal to 2Π radians, its angular velocity in rad/sec can be readily
expressed in terms of revolutions per minute. The common way of
expressing rotor speed is:

ω = 2Π r.p.m / 60 (angular velocity (ω))

The centrifugal field (G) in terms of r.p.m is then:

G = [ 4 Π2 (r.p.m)2r / 3600 ] G = ω2r

It is generally expressed as multiples of earth’s gravitiational field, the


ratio of the weight of the molecule in centrifugal field to the weight of the
same particle when acted upon by gravity alone. It is referred to as
RELATIVE CENTRIFUGAL FIELD (RCF).

R.C.F = [ 4 Π2 (r.p.m)2r / 3600 x 980(g) ] = (ω2r/g)

RELATION BETWEEN R.C.F AND g


The RCF is given by the equation:

Or, r.p.m = R.C.F x 3600g

4Π2 x r
It can be shortened as : r.p.m. = 9.549 RCF x g
r
By plotting the values of Π and g,

r.p.m = 298.93 RCF / r

Where, r = Radial distance from the axis of the rotor

Sedimentation Rate

 It is the sedimentation velocity of a particle in a centrifugal field


and is denoted as v.
 It is related to particle size, & its radial distance from the axis of
the rotor and the applied centrifugal field by the following
equation:
v = dv/dt = 2/9 rp2 (ρp – ρm) ω2r
η

Sedimentation coefficient (S)

 It is the sedimentation velocity (v) of a particle / unit centrifugal


field and is denoted as ‘S’.

S = v/ ω2r = [dv/dt]/ ω2r = [dv/dt] x (1/ ω2r)

 Integration of the above equation between the limits r1, r2 and t1,
t2, we have:
S= ln t2/r1

ω2(t2’ - t1’)

 S is determined by observing the particles at different positions


and at different times help in the calculation of the molecular
weight of the macromolecules and is related as follows:

M = RTS / D(1-v ρ)
S20W
► It is the standard sedimentation coefficient of a substance in water
at 200C.
► Since sedimentation rate studies may be preformed using a wide
variety of solvent, solute system.
► The measured value of sedimentation coefficient, which is affected
by temperature, solution viscosity, and density is often connected
to a value that could be obtained in a medium with viscosity and
density of water at 200C and expressed as the sedimentation
coefficient (or) S2Ow.
► It is given by the equation:
S2Ow = Sobserved = 1-v ρ2Ow nT n
1-v ρT n20 n0
Where:
Sobserved = Sedimentation coefficient observed in a
particular medium.
ρ2Ow = Density of water at 200C.
v = Partial specific volume
n/no = Derived velocity of the solvent to that of water.
nT/n20 = Relative velocity of water at the temperature ‘T’,
compared to that at 200C.
► In the above equation, the partial specific volume is assumed to be
unaffected.
Svedberg Unit
► It is the time dimensional constant of sedimentation coefficient.
The basic unit of which is taken is 10-13s. It is denoted as ‘S’
► For example, a molecule possessing a sedimentation coefficient of
5 x 10-13 secs, will have a value of 5S.
► This gives and idea about the size of the molecule.

CENTRIFUGATION – CLASSIFICATION

Centrifugation can be classified in 2 ways:

• PREPARATIVE CENTRIFUGATION
• ANALYTICAL CENTRIFUGATION

PREPARATIVE CENTRIFUGATION

• It is a separation technique, used for the preparation of cellular,


sub cellular and molecular fractions, in a uniform (or) density
gradient medium.
• It separates various substances into fractions on the basis of
sedimentation rate, which in turn depends on the size, shape,
density of the molecule and viscosity of the medium.

v = 2/9 rp2 (ρp – ρm) ω2r ------------------- 1


η (f/f0)

Where, rp= Radius of the particle


ρp = Density of the particle
ρm = Density of the medium
ω2r = Applied centrifugal field
η = Viscosity of the medium
f/f0 = Frictional coefficient.

 Depending on the method of approach adapted for preparation of


various fractions of substances, preparative centrifugation can be
classified into:

 DIFFERENTIAL CENTRIFUGATION

 DENSITY GRADIENT CENTRIFUGATION


DIFFERENTIAL CENTRIFUGATION

 It is a type of preparative centrifugation, in which the substance is


separated as fraction by applying increasing centrifugal field for
different time durations, in a uniform medium, keeping ρm and η
constant, and assuming f/f0 as 1.
 From eqn 1 it is clear that the sedimentation velocity will depend
on vp, ρp and ω2r as:

v α vp2, ρp, ω2r

Or, v = 2/9vp2 ρp ω2r ------------ 2

 Thus at different magnitudes of the centrifugal field, different


fractions having different fractions, having different size and
density are separated.
 From equation 2 the rate of sedimentation depends on size, than on
density of the particle.
 Moreover the density of the protein particles of different sizes and
density are made to sediment as pellet by applying varying
centrifugal force for varying time periods.
 This can be done by varying the speed of the centrifuge as:
Centrifugal field = ω2r = (2Πn)2r
= 4Π2(r.p.m)2r

Where, r=Distance from the rotor.

 Thus by making the centrifuge revolve at different speeds, different


magnitude of centrifugal field is created which makes the particles
of particular dimension to sediment at a particular time as pellet,
and the unsedimented particles remain in the supernatent.
 They can also be made to sediment by applying greater centrifugal
force by increasing the speed.
ISOLATION OF SUBCELLULAR ORGANELLES

 It is also known as cell fractionation.


 The procedure involves 2 main types
 HOMOGENIZATION
 DIFFERENTIAL CENTRIFUGATION
 Homogenization is the 1st step in cell fraction and involves
disorganization of tissues & breaking of cell wall/cell membrane,
leading to the release of the cellular contents.
 The tissue in this process gets converted into a homogentate.

HOMOGENIZATION

 The animal is sacrificed, and the tissue is excised & washed in ice
cold buffer or sucrose.
 The tissue is weighed & mixed into small pieces and transferred
into a rotor tube.
 To the tissue, appropriate amounts of buffer or sucrose are added
and are homogenized by a Teflon pestle.
 The pestle is allowed to revolve at a speed of 2000rpm such that
the tissues are ruptured and but the cell organelles are intact and
the homogenate obtained is subjected to differential centrifugation.
DIFFERENTIAL CENTRIFUGATION - Assay of purity
 The subcellular organelles are subjected to differential
centrifugation.
 Assay of purity of the separated components is carried out by:
 MICROSCOPIC EXAMINATION OF ISOLATED
FRACTION
 CHEMICAL ANALYSIS
1. DNA content
2.Enzyme activity (Widely used)
3. Immunological precipitation
4. Spectrophotometry

DENSITY GRADIENT CENTRIFUGATION

 It is a type of preparative centrifugation.


 It involves the separation of various fractions by sedimenting them
in a density gradient medium.
 Depending on the approach adopted it can be further classified as:
 RATE ZONAL CENTRIFUGATION
 ISOPYCNIC CENTRIFUGATION

RATE ZONAL CENTRIFUGATION


 This involves the separation of fractions on the basis of their
molecular size or molecular traces or sedimentation rate.

v = 2/9 rp2 (ρp – ρm) ω2r (ρp > ρm)

η (f/f0)
 Here, the substance to be separated is carefully layered over a
preformed layer of density gradient.
 This is done in a preformed density gradient. The highest density
in the gradient should not be greater than the densest particle to
be separated.
 The density gradient is so established that it gives a sharp reduced
separation of fraction in the form of bands.
 This separation is achieved by centrifugation for definite period of
time where the applied centrifugal field makes the particle to
sediment at different rates.
 This is due to the fact that different particles will exhibit different
sedimentation velocity, which mainly depends on their size.

RATE ZONAL CENTRIFUGATION – CHARACTERISTICS

 It separates the particles on the basis of their size dimensions and


sedimentation rate.
 The highest density in the gradient should not be greater than the
densest particle to be separated.
 It is time dependant.

ISOPYCNIC CENTRIFUGATION
 It is also known as equilibrium density centrifugation.
 It separates the fractions on the basis of their characteristic
buoyant density in a preformed density gradient.
 The maximum density of the gradient exceeds the density of the
densest particle in a time independent fashion.
v = 2/9 rp2 (ρp – ρm) ω2r (ρp < ρm)
η (f/f0)

 Sedimentation occurs at their isodensity, and no further


sedimentation occurs irrespective of the time.
 This is because the sedimented particles keep floating on a
cushion of the medium having greater density than its own.

FORMATION OF DENSITY GRADIENT

 The 2 main types of density gradient are:


– Continuous density formation
– Discontinuous density formation
 Continuous density formation is done by careful layering of the
gradient medium of different densities one over the other.
 This gradient is not very sharp, and it rather forms in a linear to
the bottom of the tube.
 It is formed by gradient formers.
 Discontinuous density formation , also known as step gradient
is characterized by marked variation by dissolving the gradient in
various proportions and layered one over the other.
 Layering is done by using a pipette, and the high density region is
at the bottom of the tube and the lowest at the top.

CHOICE OF GRADIENT
 An ideal gradient must possess the following characteristics:
– Permit the desired type of separation.
– Be stable in the solution.
– Be inert to biological material.
– Be non-toxic
– Must be inexpensive and easily available
– Have negligible osmotic pressure and cause minimum
change in pH, ionic strength, & viscosity.
– Should not absorb light of wavelength used for the
spectrophotometric assay.
– Should not interfere with other assaying procedures.
 No material however fulfill all the conditions strictly.
 The commonly used gradients in biochemical separations are:
 Sucrose (Not preferred due to increase in viscosity at
density
greater than 1.1-1.2 g/cm3 and it exerts very high
osmotic effect at low concentrations)
 Ficoll
 Malrizamide
 Percol
 Cscb
 Ludose

SAMPLE APPLICATION
 The mixture of the substance is carefully layered on top of the
gradient.
 This is done with the help of a pipette or a hypodermic syringe

FRACTION COLLECTION

 The various fractions are collected after the completion of the


centrifugation procedure.
 This can be done in 2 ways:
– Careful sucking
– Puncturing the bottom of the tube.

Density gradient centrifugation – Applications


 Rate zonal centrifugation is used for the separation of DNA, RNA,
enzymes, hormones, hybrids, ribosomal subunits, and other
subcellular fractions.
 Isopycinc centrifugation is used in the separation of subcellular
organelles, and isotopic studies involving macromolecules.