Centrifugation

When a solid-liquid suspension is rotated in a cylindrical container (bowl) the suspension is subject to a centrifugal force in the radial direction.

Centrifugation is a process by which solid particles separated from a liquid using centrifugal force as a driving force.

The centrifugal force can be many times greater than the force of gravity , allowing even very small particles or particle slightly denser than the fluid to settle.

The rate of centrifugation is specified by the angular velocity measured in revolution per minute (rpm), or acceleration expressed as g.

The conversion factor between rpm and g depend on the radius of the sample in the centrifugal rotor.

The particles’ setting velocity in centrifugation is a function of their size and shape, centrifugal acceleration ,the volume fraction of solid present, the density difference between the particle and liquid, and viscosity.

Centrifugation

Suspension In process

Induced gravitational field

Precipitate

Supernatant

Separation complete

Centrifuges are classified into two categories:• Laboratory centrifuges • Preparative centrifuges

Laboratory centrifuges

Rotor

Centrifuge tube

Supernatant Precipitate

Suspension

•Used for small-scale separation and particle free sample preparations

•Typical liquid volumes handled is about 1 – 5000 ml

•The material to be centrifuged is distributed in centrifuge tubes

•Tubes are attached rotor in a symmetric manner

•Two types of rotors: fixed rotors and swing out rotors

•Induced gravitational field move particles towards the bottom of the tubes

•Typical rotation speeds:1,000 – 15,000 rpm

•Induced gravitational field is measured in terms of the G value

•G value depends on the rotation speed as well as the manner in which the centrifuge tubes are held by the rotor

Laboratory centrifugation

g

rn

g

rG

22 2

•G value will depend on the location

•Highest: bottom of tube

•Lowest: top of tube

•Particle experience variable G values during their motion

•Average value is frequently used

•Typical G values: 1,000 – 20,000

Rotors

•Fixed angled rotors have smaller difference between rmax and rmin

•Time required for precipitation is less with the fixed angled rotor

•Distance travelled by particles is less with the angled rotor

•Fixed angled rotors are heavier and require much higher energy to operate

•Swing out rotors are preferred for centrifuging substances such as cells and coarse particles

•Precipitated macromolecules and finer particles are centrifuged using fixed angled rotors

Fixed angled rotor Swing-out rotor

Rmax - Rmin Rmax - Rmin

Preparative centrifugation

•Handle larger liquid volumes (i.e. 1 to several thousand litres)

•Range of designs

•Common feature: rotating chamber into which the suspension is fed from one end while the supernatant and precipitate is collected from the other end in a continuous or semi-continuous manner

•Most common type: tubular centrifuge

•Typical rotating speed: 500 - 2000 rpmSupernatant collection

Rotating tubular bowl

Developing precipitatelayer

Precipitate collection

Supernatant

Feed

Annular space

CENTRIFUGE

Ultracentrifugation:

Ultracentrifugation

Important technique for purifying proteins and particularly nucleic acids.

Density gradient Density gradient centrifugationcentrifugation

DENSITY GRADIENT CENTRIFUGATION

DNA separation