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41K views12 pagesImportant aspects of Centrifugation; Basic principles, classification and applications.

Jul 08, 2007

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Important aspects of Centrifugation; Basic principles, classification and applications.

Attribution Non-Commercial (BY-NC)

41K views

1616 upvotes00 downvotes

Important aspects of Centrifugation; Basic principles, classification and applications.

Attribution Non-Commercial (BY-NC)

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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

which it is suspended, resulting in an upthrust on the particle

equal to the weight of the liquid displaced.

density, then the net force(f) is experienced when the centrifugal

force at an angular velocity of ω radians/sec is given by:

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

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

result that the particles sediment at a constant ratio. Its rate of

sedimentation (v) is given by:

η

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

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:

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).

The RCF is given by the equation:

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

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

field and is denoted as ‘S’.

Integration of the above equation between the limits r1, r2 and t1,

t2, we have:

S= ln t2/r1

ω2(t2’ - t1’)

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

• PREPARATIVE CENTRIFUGATION

• ANALYTICAL CENTRIFUGATION

PREPARATIVE CENTRIFUGATION

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.

η (f/f0)

ρp = Density of the particle

ρm = Density of the medium

ω2r = Applied centrifugal field

η = Viscosity of the medium

f/f0 = Frictional coefficient.

various fractions of substances, preparative centrifugation can be

classified into:

DIFFERENTIAL CENTRIFUGATION

DIFFERENTIAL CENTRIFUGATION

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:

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

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

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

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

This involves the separation of fractions on the basis of their

molecular size or molecular traces or sedimentation rate.

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.

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 irrespective of the time.

This is because the sedimented particles keep floating on a

cushion of the medium having greater density than its own.

– 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

centrifugation procedure.

This can be done in 2 ways:

– Careful sucking

– Puncturing the bottom of the tube.

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.

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