Principle of centrifugation

CENTRIFUGE

Centrifugation is a technique used for the separation of particles using a centrifugal field. The particles are suspended in liquid medium and placed in a centrifuge tube.  The tube is then placed in a rotor and spun at a definitive speed. Rotation of the rotor about a central axis generates a centrifugal force upon the particles in the suspension.

Two forces counteract the centrifugal force acting on the suspended particles:

· Buoyant force: This is the force with which the particles must displace the liquid media into which they sediment.

· Frictional force: This is the force generated by the particles as they migrate through the solution.

Particles move away from the axis of rotation in a centrifugal field only when the centrifugal force exceeds the counteracting buoyant and frictional forces resulting in sedimentation of the particles at a constant rate.

Particles which differ in density, size or shape sediment at different rates. The rate of sedimentation depends upon:

1.       The applied centrifugal field

2.       Density and radius of the particle.

3.       Density and viscosity of the suspending medium.

Angular velocity = w radians / second;

since one revolution = 360^o = 2p radians,

(r =  radial distance of the particle from the axis of rotation)

As the centrifugal field acting on the particle is much greater than the Earth's gravitational field, CF is generally expressed relative to the Earth's gravitational field as multiples of g, the acceleration due to gravity (g= 980 cm/s²)

This expression relates relative centrifugal field (RCF) to the speed of the centrifuge (rpm) and and the radius of the rotor (r). For example, if a rotor with an average radius of 7 cm revolves at a speed of 20,000 rpm, a centrifugal field of 31,300 g is created.

The sedimentation rate of velocity (v) of a particle can be expressed in terms of its sedimentation rate per unit centrifugal field. This is termed as sedimentation coefficient (s). The sedimentation rate is proportional to w² r, the centrifugal field,

Sedimentation velocity depends upon the mass of the particle, its density, shape and also on the density and viscosity of the medium in which the particle is suspended.

So, In summary, Centrifugation is the process of using centrifugal force to separate the lighter portion of solution, mixture or suspension from the heavier portions. In laboratory centrifuge is used to:

· Remove cellular debris from blood to separate cell free plasma or serum

· Concentrate cellular elements and other components for microscopic analysis or chemical analysis.

· Separate protein bound or antibody bound ligand from free ligand in immunological assay.

· Extract solutes from aqueous or organic solvents.

· Separate lipid components like chylomicrons from other components of plasma.

Types of centrifuges

Horizontal head or swinging bucket centrifuges: 

This type of centrifuge allow the tubes placed in the cups of the rotor to assume a horizontal plane when the rotor is in motion and a vertical position when it is at rest. During centrifugation particles travel uniformly and constantly along the tube while the tube is at right angle to the shaft of centrifuge; thus the sediment is distributed uniformly against the bottom of the tube and remains there when rotor stops, with liquid above it. This liquid can be decanted off and both liquid and sediment can be separated for analysis. The spinning rotor offers considerable resistance to rotation and generates heat due to air friction.

Fixed-angle or Angle-head centrifuge: 

Here tubes are held in a fixed position at angles from 25⁰ to 40⁰to the vertical axis of rotation. Upon centrifugation particles are driven outward horizontally but strike the side of the tube so that the sediment packs against the side and bottom of the tube with the surface of sediment parallel to the shaft of the centrifuge. As rotor slows down or stops, gravity causes the sediment to slide down the tube, usually a poorly packed pellet is formed.

Ultracentrifuge

It is a very high speed centrifuge that has fixed head rotors. It is mainly used in separation of lipoproteins. Since the separation is long process there is generation of heat and thus are provided with internal cooling system.

Axial centrifuge

An axial centrifuge is based on a centrifugal concept that allows tubes of blood to be spun in a vertical orientation as opposed to horizontal orientation used in traditional centrifuges.

In centrifugation, relative centrifugal force (RCF) is the force required to separate two phases, this force also called relative centrifugal field. Units are expressed as number of times greater than gravity (e.g., 500xg). By accelerating the g speedy sedimentation can be achieved.

RCF is calculated as follows:

Where 1.18 x 10^-5 are an empirical factor

r = radium in cm from the center of rotation to the bottom of the tube in rotor cavity or bucket during centrifugation

RPM= speed of rotation of rotor in revolutions per minute.  

Time required to sediment particles depends on the rotor speed, radium of the rotor, and effective path length travelled by sedimented particles, that is, the depth of liquid in the tube. 

The length of time for centrifugation can be calculated so that running with an alternate rotor of a different size is equivalent to running with original rotor

Time (alternate rotor) = [time x RCF (original rotor)]/[RCF (alternate rotor)]

A Svedberg unit (S/Sv) is a non-SI unit for sedimentation rate. The sedimentation rate is the rate at which particles of a given size and shape travel to the bottom of the tube under centrifugal force. The Svedberg is technically a measure of time, and is defined as exactly 10^-13 seconds (100 fs). The Svedberg unit (S) offers a measure of particle size based on its rate of travel in a tube subjected to high g-force. Svedberg units are successful in classifying ribosomes as 50S and 80S in eukaryotes.  A substance with a sedimentation coefficient of 26S (26x10^-13s) will travel at 26 microns per second (26x10^-6 m/s) under the influence of an acceleration of a million gravities (10⁷ m/s²).

TYPES OF CENTRIFUGES

	Low speed	High speed	Ultracentrifuge
1. Speed range(rpm)	2000-10000	18000-30000	40000-100000
2. Refrigeration	Some	Yes	Must
3. Vacuum system	None	Some	Must
4. Application for pelleting
Cells	Yes	Yes	Yes
Nuclei	Yes	Yes	Yes
Membranous Organelles	Yes	Yes
Membrane	-	Some	Yes
Ribosome or Polysome	-	-	Yes
Macromolecules	-	-	Yes

The low speed centrifuge is commonly used in the clinical laboratory to separate serum or plasma from whole blood and also in deproteinisation of physiological fluids.

PRECAUTIONS DURING CENTRIFUGATION

1. It is important that the tubes/ centrifuge cups in the rotor head be balanced before centrifugation. This will permit maximum RCF and minimize breakage of tubes, wear on the motor and bearings and loss of sample.

2. Tubes should be properly capped and the lid of the centrifuge closed during centrifugation. This will prevent the release of infectious material inside the centrifuge by aerosol formation. If breakage occurs resulting in the spillage of potentially infectious material the centrifuge bowl will be contaminated. Spillage of the sample can lead to corrosion of the centrifuge. Therefore in case of any spillage, the centrifuge should be properly decontaminated and cleaned.