C E N T R I F U G A T I O N

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Centrifugation

is one of the most important separation technique.

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It exploits the inherent varied sedimenting property of substancesfor their isolation by the application of centrifugation field.

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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

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This method is based on the principle of sedimentation.

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When a particle sediments, it must displace some of the solution in which it is suspended, resulting in an upthrust on the particleequal to the weight of the liquid displaced.

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If a particle is assumed to be a sphere of known volume anddensity, then the net force(f) is experienced when the centrifugalforce at an angular velocity of ω radians/sec is given by:S = Volume x Density x ω2rOr,

F = 4/3 Π r

(ρ

– ρ

) ω

------------------ 1Where,

4/3 Π r

= Volume of sphere of radius ‘r’.

= Density of the particle.

= Density of the suspended medium.

= Distance of the particle from the center of rotation.

= Angular velocity of rotor.

FRICTIONAL COEFFICIENT

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Particles however generate friction as they migrate through thesolution.

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If the particle is spherical and is moving at a known velocity, thenits frictional force appearing opposing motion is given by

Stoke’s law.

= 6Πηr

------------------------------ 2Where:f

= Frictional coefficient of spherical particles.

η = Coefficient of viscosity, and v = Velocity of the sedimenting particle.

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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 itsmotion through the medium

f = f

or,

4/3 Π r

(ρ

– ρ

) ω

r = 6Πηr

---------------------- 3

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In practice, the balancing of this force occurs quickly, with theresult that the particles sediment at a constant ratio. Its rate of sedimentation (v) is given by:

v = dx/dt = 2/9 rp

(ρ

– ρ

) ω

-------------------- 4

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The non-spherical molecules as in the case of rod like molecules,such as DNA and proteins like myosin experience considerablefrictional resistance.

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Frictional coefficient (f) of the molecules can be increased as muchas 10 times of the frictional coefficient of the sphere (f0).

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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

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From equation 5, it is clear that the sedimentation rate is of aparticle is dependant on the following factors:

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It is directly proportional to the size of the particle,since the equation involves the square of the particleradius. It is apparent that the size of the particle hasthe greatest influence upon sedimentation.

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It is directly proportional to the difference in densitybetween the particle & the medium. It will be

zero

, when the density of the particle and the medium areequal.

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It will decrease, when the viscosity of the mediumincreases (i.e., ω2r) v = 1/η.

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The sedimentation rate (or) velocity decreases as the frictionalcoefficient ratio (f/f0) increases the ratio f/f0 is approximately 1 for aspherical molecule.

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The rate of sedimentation depends on the applied centrifugal field (G)ω2r being directed radially outwards which is determined by thesquare root of angular velocity of the rotor and radial distance of theparticle from the axis of rotation.

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According to the equation, G= ω2r, since one revolution of the rotor isequal to 2Π radians, its angular velocity in rad/sec can be readilyexpressed 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 Π

(r.p.m)

r / 3600 ] G = ω

It is generally expressed as multiples of earth’s gravitiational field, theratio of the weight of the molecule in centrifugal field to the weight of thesame particle when acted upon by gravity alone. It is referred to as

RELATIVE CENTRIFUGAL FIELD (RCF).

R.C.F =

[ 4 Π

(r.p.m)

r / 3600 x 980(g) ] = (ω

r/g)

RELATION BETWEEN R.C.F AND g

The RCF is given by the equation:

Or, r.p.m = R.C.F x 3600g4Π

x r

It can be shortened as :

r.p.m. = 9.549 RCF x gr