1. Introduction

- centrifuge: device for separating particles from a solution

according to their size, shape, density, viscosity of the medium

and rotor speed

- effect of gravity

- centrifugal force

- pellet and supernatant

- used to separate particles or macromolecules.

II. Types of Centrifugation

a) Preparative :

1. Differential Centrifugation

2. Density gradient Centrifugation

2a. Rate-Zonal

2b. Isopycnic

b) Analytical

a) Preparative Centrifugation

- used to separate organelles and molecules

- can handle larger liquid volumes

- no optical read-out

- Separation methods used in preparative ultracentrifugation:

1. Differential Centrifugation- pelleting,

2. Density Gradient Centrifugation.

1.Differential Centrifugation

- based on the size of the particles

- used for simple pelleting, for the separation of sub

cellular organelles and macromolecules

- first, sample must be homogenised

- ultra centrifugation

- sedimentation depends on mass, shape and partial

specific volume of a macromolecule, as well as

solvent density, rotor size, rate of rotation.

-Usually uses a fixed angle rotor.

2.Density Gradient Centrifugation

- method to purify subcellular organelles and

macromolecules.

- density gradients generated by placing layer after layer of

gradient media

- Density gradient centrifugation classified into two:

2a. Rate-Zonal separation (size)

2b. Isopycnic Separation (Density)

2a.Rate- Zonal Centrifugation

- use of continuous density gradient of solvent such as

sucrose.

- density increases towards the bottom of the tube

- sample layered on the top

- molecules form discrete bands after centrifugation

- separation based on size of the molecules

- Swinging bucket rotors.

2b. Isopycnic Separation

- based on the density of the molecules

- Mix gradient material with the sample molecule (CsCl)

- molecules move to the position where their density is same

as the gradient material (isopycnic position)

- inorder to generate a gradient, we select a CsCl

concentration that will give a range of densities that includes

the range of molecules that have to be separated.

- used for the separation of DNA

- Swinging bucket or fixed angle rotor.

b) Analytical Centrifugation

- uses small size samples

- built-in optical system

- uses relatively pure sample.

III. Theory of Centrifugation

- Centrifugation:any object moving in a circle at a steady

angular velocity is subject to an outward directed force, F.

The magnitude of this force depends on the angular velocity

in radians, w, and the radius of rotation, r, in centimeters.

F= w 2r

F= gravitational force and also referred to as the relative

centrifugal force, RCF

Where the earth's gravitational field (g=980 cm/s-2).

RCF= w 2r /980

- Sedimentation of a molecule influenced by

> properties of the molecules (Size, shape, density)

> properties of the solvent, or the gradient material(density,

viscosity, temperature)

> interactions between the solute molecules and the solvent

gradient molecule

-As the rotor spins, centrifugal force is applied to each

molecule in the sample:

Centrifugal force= Mω2r

M=mass(molecular weight),

ω=angular velocity(radius/sec),

r= distance from the axis of the rotation

- For a spherical molecule, f= 6πηrm

η = viscosity of the medium

rm= radius of the molecule

Sedimentation coefficient:

S= v/ω2r

where, v =velocity

v can be given as (dr/dt)

S= (dr/dt)/ω2r

IV. Types of Centrifuges

- Desktop clinical centrifuges: below 3000rpm

- High speed centrifuges : 20,000 to 25,000rpm

- The Ultracentrifuge :75,000rpm

a. Drive and speed control

b. Temperature control

c. Vacuum System

d. Rotors.

V. Types of Rotors

- Swinging bucket rotors

- Fixed angle

- Vertical

Centrifugation

Centrifugation is a process that involves the use of the centrifugal force for the sedimentation of mixtures with a centrifuge, used in industry and in laboratory settings. More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture migrate towards the axis. Chemists and biologists may increase the effective gravitational force on a test tube so as to more rapidly and completely cause the precipitate ("pellet") to gather on the bottom of the tube. The remaining solution is properly called the "supernate" or "supernatant liquid". The supernatant liquid is then either quickly decantedfrom the tube without disturbing the precipitate, or withdrawn with a Pasteur pipette.[citation needed]

The rate of centrifugation is specified by the angular velocity measured in revolutions per minute (RPM), or acceleration expressed as g. The conversion factor between RPM and g depends on the radius of the sample in the centrifuge rotor. The particles' settling velocity in centrifugation is a function of their size and shape, centrifugal acceleration, the volume fraction of solids present, the densitydifference between the particle and the liquid, and the viscosity.

In the chemical and food industries, special centrifuges can process a continuous streamof particle-laden liquid.

Centrifugation is the most common method used for uranium enrichment, relying on the slight mass difference between atoms of U238 and U235 in uranium hexafluoride gas.[citation needed]

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