Centrifugation is a process which involves the use of the centrifugal force for the sedimentation of heterogeneous mixtures to separate the two miscible substances ,and also to analyze the hydrodynamic properties of macro molecule with a centrifuge , used in industry and in laboratory setting.

1. By
Dr. Rachana Choudhary
Asstt. Prof. Department of Microbiology
SHRI SHANKRACHARYA MAHAVIDYALAY JUNWANI, BHILAI
CENTRIFUGATION

2. SYNOPSIS
 INTRODUCTION
 CENTRIFUGE
 HISTORY
BASIC PRINCIPLE OF CENTRIFUGATION /SEDIMENTATION
TYPES OF ROTOR
TECHNIQUE OF CENTRIFUGATION
SEDIMENTATION COEFFICENT
FACTOR AFFECTING SEDIMENTATION COEFFICIENT.
PRECAUTION
APPLICATION OF CENTRIFUGATION
REFERENCE

3. INTRODUCTION
Centrifugation is a process which involves the use of the
centrifugal force for the sedimentation of heterogeneous mixtures
to separate the two miscible substances ,and also to analyze the
hydrodynamic properties of macromolecule with a centrifuge ,
used in industry and in laboratory setting.
 More dense components of the mixture move away from the axis
of the centrifuge while less dense components of the mixture
move towards the axis.
 The theoretical basis of this technique is the effect of gravity on
particles (including macromolecules) in suspension. Two
particles of different masses will settle in a tube at different rates
in response to gravity.

4. Device:
Centrifuge is a piece of equipment, generally driven by a motor, that
puts an object in rotation around a fixed axis , applying force
perpendicular to the axis.
It is device for separating particles from a solution according to
there size shape , density and viscosity of the medium.
 Centrifuge uses centrifugal force to
separate phases of different densities.
 The centrifugal force is proportional to
the rotation rate of the rotor.
Basic components of centrifuge:
i. A metal rotor with holes (it accommodates vessel of liquid)
ii. A motor or alternative means of spinning- the rotor at a selected
speed.

5. HISTORY
• English military engineer Benjamin Robins (1707-1751) invented
a whirling arm apparatus to determine drag.
• In 1864, Antonin Prandtl proposed the idea of a dairy
centrifuge to separate cream from milk.
• The potential of the centrifuge in the laboratory setting was first
exploited by Friedrich Miescher in 1869 ,Miescher used a crude
centrifuge system to isolate a cell organelle.
• In 1879,the first continuous centrifugal separator was
demonstrated by Gustaf de lava & development made the
commercialization of the centrifuge.
• In 1923 the Svedberg and Nicols employed it for increase the
gravitational force.

6. BASIC PRINCIPLE OF CENTRIFUGATION
A particle, whether it is precipitate a macromolecule or a cell organelle is
subjected to a centrifugal force when is rotated at high rate of speed. The
centrifugal force F is denoted by equation:
F= m2r
F = intensity of the centrifugal force.
m= effective mass of the sedimenting particle.
= angular velocity of rotation.
r= distance of the migrating particles from
the central axis of rotation.
A more common measurement of F in the terms of the earths gravitation
force, g is the relative centrifugal force , RCF its defined by
RCF=(1.11910ˉ5)(rpm2(r)
This equation relates RCF to revolutions per minute of the sample. Equation
dictates that the RCF on a sample will vary with r, the sedimenting particles
from the axis of rotation.

7. TYPES OF ROTORS
• Basically there are four types of otors
• Fixed Angle Rotors
• Vertical Tube Rotors
• Swinging Bucket Rotors
• Zonal Rotors

8. FIXED ANGLE ROTORS
Consist of metal with 6 to 12 holes
within their body .
 The hole angle between( 14˚ & 40˚)
to vertical axis.
 Particles travels short distance and
strike with the wall.

9. VERTICAL TUBE ROTORS:-
 Have holes within their body lie parallel to the rotor shaft and
not at an angle.
As the rotor accelerates and centrifugal field is applied, the
solution within the tube reorients through 90˚ (makes solution to
the axis of rotation).
 As the rotor decelerates the
 solution orients back to the
 original position.

10. Swinging bucket rotors
These rotor have bucket that swing out to a horizontal position
when the rotor accelerates.
The solution in the tube reorients to lie perpendicular to the axis
of rotation and parallel to the applied centrifugal field.
 As the rotor decelerate the solution orient back to the original
position.

11. Zonal rotors
The zonal rotor is more or less sphere which has its interior
subdivided into four equal quadrants (sector shaped) by means
of a four –veined core.
The zonal rotor is rotated while
it is still empty i.e. without gradient.
When the speed has been brought
up to 3000- 4000rpm, the gradient
is loaded into the rotor.

12. TECHNIQUES OF CENTRIFUGATION
 Preparative centrifugation
i. Differential centrifugation
ii. Density gradient centrifugation.
 Rate –zonal centrifugation.
 Isopycnic centrifugation.
 Analytical centrifugation.
 Ultracentrifugation.

13. Preparative centrifugation :-
 Preparative centrifugation is con-
cerned with the actual isolation of
biological material for subsequent
biochemical investigation.
It can be divided into two main techniques
depending upon the medium of suspension
in which the separation is carried out in a
sus-pending medium which is homo-genous
are known as differential centrifugation.
• Those carried out in a suspending medium
having density gradient are known as
density gradient centrifugation.

14. Differential centrifugation:-
 Differential centrifugation is a
common procedure in microbiology
& cytology used to separate certain
organelles from whole cells for
further analysis of specific parts of
cells.
In the process, a tissue sample is
first homogenized to break the cell
membranes & mix up the cell
contents.
The homogenate is then subjected
to repeated centrifugations, each
time removing the pellet &
increasing the centrifugal force.

15. DENSITY GRADIENT CENTRIFUGATION
• Also known as equilibrium sedimentation.
• It employs medium which has gradients.
• Separation under centrifugal force is depend upon buoyant
densities of the particles.
• The gradients, apart from exerting their separating effect,
eliminate mixing of separated components due to convection &
mechanical vibrations, hence gives much better separation than
Types of differential centrifugation.
 Rate-zonal centrifugation.
 Isopycnic centrifugation .

16. RATE –ZONAL CENTRIFUGATION
 In Rate zonal centrifugation the solution have a density
gradient . The sample has a density i.e. greater than all the layers
in the solution.
 The sample is applied in a thin zone at the top of the centrifuge
tube on a density gradient . Under centrifugal force, the particles
will begin sedimenting
through the gradient.
 The particles will begin sedimen-
ting in separate zones according
to their size shape and density.

17. ISOPYCNIC CENTRIFUGATION
 In, isopycnic separation, also called equilibrium separation
 The solution contains a greater range of densities.
 The density gradient contains the whole range of densities of
the particles in the sample.
 Each particle will sediment on to the position in the centrifuge
tube at which the gradient
densities is equal to its own density.
 In Isopycnic centrifugation
separation of particles occurs into
zones on the basis of their density
differences, independent of time.

18. DIFFERENCE BETWEENRATE-ZONAL & ISOPYCNIC
CENTRIFUGATION.
Rate zonal isopycnic
synonym Sedimentation
velocity.
Sedimentation
equilibrium.
gradient Shallow, gradient
continuous.
steep, continuous or
discontinuous gradient.
centrifugation Incomplete
sedimentation.
Complete
sedimentation.
seperations RNA-DNA hybrids. DNA, plasma etc.

19. ANALYTICAL ULTRACENTRIFUGATION
• The aim of analytical ultracentri-
fugation is use to study molecular
interactions between macro-
molecules or to analyze the pro-
perties of sedimenting particles
such as their apparent molecular
weight.
• Which through rapid spinning imposes high centrifugal forces on suspended
particles , or even molecules in solution, and cause separations of such matter
on the basis of difference in weight.
• Can run at about 120,000-150,000rpm.
• RCF as high as 625,000 g., Volume a single tube accomodated-0.2-5.5ml.
• Separate particles as less as 10micron.

20. ULTRACENTRIFUGATION
• Svedberg coined the term “ultracentrifugation”.
• It is the most sophisticated instrument.
• Operate at speed of 75,000 rpm providing the
centrifugal force of 500,000g.
• The ultracentrifuge is a centrifuge optimized for
spinning a rotor at very high speeds , capable of
generating acceleration as high 19600km/s2.
• Intense heat is generated due to high speed thus the
spinning chambers must be refrigerated and kept at
high vaccum .

22. SEDIMENTATION COEFFICIENT
 The velocity of a particle per unit centrifugal field is referred to as its
sedimentation coefficient.
• It is expressed in Svedberg unit, S.
• Mathematically expression s=v/2r.
where, v= velocity of sedementing particle
= angular velocity of rotation
r= distance of migrating particle from the central
axis of the rotation
• The term s is most often defined under standard conditions, 20˚C and
water as the medium, and it is denoted by s20,w.
• The s value is a physical characteristic used to classify biological
macromolecules and cell organelles.
• 1S = 1 x 10-3 second.Range 1 x 10-13 to 10,000 x 10-13 second.

23. THE FACTORS ON WHICH THESE WORKS ARE :
 More dense a biological structure, faster it sediments in centrifugal force.
 More massive biological particle, faster it moves in centrifugal field.
 Dense the buffer system , slower particle moves.
 Greater the friction coefficient, slower particle move.
 Greater the centrifugal force, faster particle sediments.
 Sedimentation rate of a given particle will be zero when density of particle
and the surrounding medium are equal.

24. FACTOR AFFECTING SEDIMENTATION
COEFFICIENT
• Molecular shape :
• Large, rigid and extend molecule face more friction then
spherical molecules.
• A rigid molecule face more friction affecting its sedimentation.
Double stranded DNA is rigid due to hydrogen bonds. If
temperature of double stranded DNA sample is increased the
hydrogen bonds break decreasing its rigidity&
making its flexible & increasing
the sedimentation coefficient.

25. • Molecular weight :
Sedimentation coefficient also depend on molecular weight of the
molecules.
The relationship between sedimentation coefficient s and
molecular weight. M of DNA is
S20.w= 2.8 +0.0083M0.497
For protein molecules
S  M2/3
It is easier to characterize a protein by finding its sedimentation
coefficient rather than its molecular weight.

26. Solute binding
Binding of small solute molecules to large macromolecules
affects their S value .
If binding of the solute increases molecular weight of the
macromolecule its sedimentation coefficient will increase.
If binding of the solute
changes the shape of
macromolecule, it will
also affect the
sedimentation
coefficient.

27. PRECAUTIONS
• Centrifuge tube must be of strong glass and
tube must not be too long.
• Tube on opposite side must be balanced well.
• Speed/velocity must be increased slowly.
• Instrument be kept clean.
• Disinfectant the instrument regularly.

28. APPLICATION
• In laboratory, centrifugation is used to ;
 Remove cellular elements from blood to provide cell
free plasma or serum for analysis.
 Remove chemically precipitated protein from an
analytical specimen.
 Separate protein bound from free ligand in
immunochemical and other assay.
 Separation of the subcellular organelle , RNA ,DNA.
 Separate lipid components.

29. REFERENCE
 Modern and experimental biochemistry – RODNEY BOYER.
 Principles and technique in biochemistry – WILSON &
WALKER
Biophysical chemistry principle and techniques – UPADHYAY
& UPADHYAY NATH.
Google Search
Slideshare

30. THANK YOU