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C E N T R I F U G A T I O N - BASIC PRINCIPLES
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
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
f/f0
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,
Sedimentation Rate
Integration of the above equation between the limits r1, r2 and t1,
t2, we have:
S= ln t2/r1
ω2(t2’ - t1’)
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
• PREPARATIVE CENTRIFUGATION
• ANALYTICAL CENTRIFUGATION
PREPARATIVE CENTRIFUGATION
DIFFERENTIAL CENTRIFUGATION
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
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.
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)
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