Final Centrifugation

Centrifugation
In a suspension of particles, particle sedimentation rate depends on the nature of the particles, nature of
the medium in which the particles are suspended and the force applied to the particles.
One important factor affecting the sedimentation of particles is viscosity.

The rate at which a macromolecule sediments is characterized by its sedimentation coefficient.
This has been defined by Svedberg and Pederson as the sedimentation velocity per unit of centrifugal
field, commonly referred to as its sedimentation coefficient, S.
The rate of sedimentation is dependent on the applied centrifugal field (G) being directed radially
outwards, determined by the square of the angular velocity of the rotor (ω, in radians s–1) and the radial
distance (r, in cms) 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 radians s–1, can
be expressed in terms of revolutions per minute (revmin–1).
Centrifugal field (G) is then:
It is generally expressed as a multiple of the earth’s

gravitational field (g = 981/cm2), and is referred to
as the relative centrifugal field (RCF)
TYPES OF CENTRIFUGES
• Low-speed Centrifuge
• High-speed Refrigerated Centrifuge
• Ultracentrifuge
Preparative ultracentrifuge
Analytical ultracentrifuge
TYPES OF CENTRIFUGES
Low-speed Centrifuge :
It is used routinely for the initial processing of biological samples,
Have maximum speed 4000 to 6000 revmin–1 and
Have maximum relative centrifugal fields of 3000 to 7000 g.
Applications include rapid sedimentation of blood samples, and of synaptosomes.
High-speed Refrigerated Centrifuge

It is used to collect microorganisms, cellular debris, large cellular organelles and
proteins precipitated by ammonium sulphate.
Maximum speed 25,000 revmin-1 and
Relative centrifugal field of about 60,000 g.
The rotor can be of fixed angle or swinging bucket type.
A synaptosome is an isolated synaptic terminal from a neuron. Synaptosomes are
obtained by mild homogenization of nervous tissue under isotonic conditions and
subsequent fractionation using differential and density gradient centrifugation.
Ultracentrifuge : subdivided into: analytical and preparative ultracentrifuge
Preparative ultracentrifuge
Can spin rotors to a maximum speed of 80,000 rpm
Can produce a relative centrifugal field of about 6,00,000 g.
The rotor chamber is refrigerated, sealed and evacuated to minimize excessive rotor temperature.
Centrifuge tubes must be accurately balanced within 0.1 g of each other.
Applications include study of macromolecule/ligand binding kinetics, steroid hormone receptor assays,
separation of the major lipoprotein fractions from plasma, and deproteinization of physiological fluids for
amino acid analysis.
Analytical ultracentrifuge
Can spin rotors to a maximum speed of 70,000 rpm
Can produce a relative centrifugal field of about 5,00,000 g, and consist of motor.
The rotor contained in a protective armored chamber that is refrigerated and evacuated.
An optical system enable observation of sedimenting material during centrifugation to determine
concentration distribution in the sample at any time during centrifugation.
The optical system measures the difference in refractive index between the reference solvent and the
solution by the displacement of interference fringes caused by slits placed behind the two liquid columns .
TYPES OF ROTORS
Preparative centrifuge rotors are four main types: swing-out (swing bucket), fixed angle,
vertical and zonal.
• Swing-Out Rotors
• Fixed Angle Rotors
• Vertical Rotors
• Zonal Rotors
TYPES OF ROTORS
Preparative centrifuge rotors are four main types: swing-out (swing bucket), fixed angle, vertical and
zonal.
Swing-Out Rotors
In these rotors, the sample solutions in tubes are in individual buckets, which move out perpendicular
to the axis of rotation, as the rotor rotates. The centrifugal force is exerted along the axis of the tube
in these rotors. Since the centrifugal force is axial, some particles are sedimented against the wall of
the tubes.
Fixed Angle Rotors

In these rotors, the tubes are at fixed angle, varying from 14° to 20°. Rotors with shallow angles are
more efficient in pelleting because the sedimentation path length is shorter. Moreover, reorientation
of the solution in the tubes enhances the loading capacity of isopycnic gradients.
Vertical Rotors
The tubes are held in a vertical position. The diameter of the tube and their design enables them to
generate very high centrifugal forces. Vertical rotors are not suitable for pelleting, but can be used for
isopycnic centrifugation.
Zonal Rotors
Zonal rotors are of two types: batch type and continuous flow. Continuous flow type rotors are less frequently
used. Zonal rotors can be used for separation of human blood cells, fractionation of membranes from a rat liver,
nuclear pellet, fractionation of tissue cultures cell, post-nuclear supernatant, and harvesting of virus from tissue
culture fluid.
TYPES OF CENTRIFUGAL SEPARATION
Differential Pelleting
Differential centrifugation separates particles according to size and density. However, the centrifugal
force to pellet the larger particles from the top of the solution is also often sufficient to pellet the smaller
particles near the bottom of the tube. Hence, a pure preparation of the smallest particles cannot be
obtained in a single step.
Rate-zonal Centrifugation
The sample is layered as a narrow zone on the top of a density gradient and thereby, minimizes the
convection currents in the liquid column during centrifugation. This is ideal for the particles of defined
size (e.g. proteins, ribosomes and RNA). However, particles of the same type are often heterogeneous.
In such cases, particles can be separated by some other means, such as density.
Isopycnic Centrifugation
Particles are separated on the basis of density. Prolonged centrifugation does not affect the
separation as long as the density gradient remains stable
https://www.youtube.com/watch?v=ncr-9iMEzwU
Separation Techniques; Centrifugation
Experiment: Separation of plasma and blood cells by centrifugation.
•Requirements: Blood (EDTA), centrifuge tube, centrifuge machine
•Procedure: Take blood in two centrifuge tubes / vacutainers in equal quantity and place
in centrifuge machine opposite to each other. Centrifuge at 4000 rpm for 15 minutes.
After centrifugation, separate plasma from cellular part.
Note: There are two factors to consider.
• First ensure that the sample tubes are at an equal volume.

• Second, make sure that the tubes are balanced within the centrifuge by placing tubes directly
opposite of each other.

Final Centrifugation

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Final Centrifugation

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Centrifugation

One important factor affecting the sedimentation of particles is viscosity.

According to the equation, G = ω2r

Centrifugal field (G) is then:

It is generally expressed as a multiple of the earth’s

• High-speed Refrigerated Centrifuge

High-speed Refrigerated Centrifuge

• Fixed Angle Rotors

Fixed Angle Rotors

Experiment: Separation of plasma and blood cells by centrifugation.

•Requirements: Blood (EDTA), centrifuge tube, centrifuge machine

Note: There are two factors to consider.

• First ensure that the sample tubes are at an equal volume.

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