2. CONTENTS
INTRODUCTION OF CENTRIFUGATION
PRINCIPLE OF SEDIMENTATION
INSTRUMENTATION OF CENTRIFUGATION
PREPARATIVE ULTRACENTRIFUGATION
ANALYTICAL ULTRACENTRIFUGATION
APPLICATIONS OF CENTRIFUGATION
3. CENTRIFUGATION
Definition:-It is the process by which particles are separated under the
applied centrifugal force which is greater than conventional gravitational
force.
In other words centrifugation
“overcomes sedimentation”
4. PRINCIPLE OF SEDIMENTATION
A particle whether it is a precipitate a macromolecule or a cell organelle is
subjected to a centrifugal force when it is rotated at a high rate of speed.
the rate of sedimentation is dependent upon the applied centrifugal field(G), that
is determined by the radial distance, r, of the particle from the axis of rotation (in
cm) and the square of the angular velocity(ω) of the rotor (in radians per second):
G=ω2 r
G depends on the radical distance of the particle from the rotation axis and the square
of the angular velocity.
5. 2π rev min-1
60
• rev: revolution per minute (r.p.m.)
Now:
4π (rev min-1)2 r
3600
G=
ω=
6. FACTORS AFFECTING THE SEDIMENTATION
the more dense a biological structure is, the faster it sediments in a
centrifugal field.
the more massive a biological particle is, the faster it moves in a centrifugal
field.
the denser the biological buffer system is, the slower the particle will move
in a centrifugal field.
the greater the frictional coefficient is, the slower a particle will move;
the greater the centrifugal force is, the faster the particle sediments;
the sedimentation rate of a given particle will be zero when the density of
the particle and the surrounding medium are equal.
8. Instrumentation for centrifugation
• The basic centrifuge consists of two components one is Electric motor with drive
shaft to spin the sample and a Rotor to hold tubes or other containers of the sample.
• A wide variety of centrifuges are available ranging from a low speed centrifuge used
for routine pelleting of relatively heavy particles. There are three types of centrifuges
• 1.Low –speed centrifuges .
It is also called as clinical centrifuge.
The common centrifuge has a maximum speed in the range of 4000 to 5000 rpm .
These instruments usually operate at low temperature.
Two types of rotors fixed angle and swinging bucket rotors may be used in this
instruments.
Low speed centrifuges are especially useful for the rapid sedimentation of coarse
precipitates or red blood cells.
Conti.
9. Instrumentation for centrifugation
2. High Speed Centrifuges:
High speed centrifuges for more sophisticated biochemical applications.
Higher speeds and temperature control of the rotor chambers are
essential.
Rotor chambers in most instruments are maintained at or near 40 C.
Three types of rotors are available for high speed centrifugation.
a. Fixed Angel rotor.
b. Swinging –bucket rotors
c. vertical rotor
High speed centrifuges are used to:-
sediment Cell debris after cell homogenization.
Ammonium sulfate precipitates of proteins and cellular organelles such
as chloroplasts, mitochondria and nuclei.
10.
11. Instrumentation for centrifugation
• 3. Ultracentrifuges:
The most complex of the centrifuges are the ultracentrifuges because of the
high speed attainable intense heat is generated in the rotor so the spin
chamber must be refrigerated and placed under high vacuum to reduce
friction.
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.
Ultracentrifuges can be used both for preparative work as well as for
analytical.
12.
13. Preparative ultracentrifugation
It aims to isolate and purify specific particles such as sub. cellular
organelles.
It is mainly of two types:
Differential centrifugation
Density gradient centrifugation
Molecules separate according to their size, density and centrifugal force.
14. Differential centrifugation
Differential centrifugation, also termed pelleting.
It fractionates particles according to their weight.
The suspension of isolated organelles is spun for different combination of
speed and time.
The lowest speed and shortest time separates out the heaviest organelles
and high speed and longer time separating the lightest organelles.
15.
16. Density gradient centrifugation
Isolated organelles are separated on the basis of their density.
Solutions of increasing density, such as sucrose solutions, are layered into a
test tube with the most concentrated solution at the bottom of the tube.
The suspension of isolated organelles is pipetted on the top of the most
dilute solution.
As the tubes are spun, the organelles collect in the layer which corresponds
to their own density.
17. Analytical ultracentrifugation
Analytical ultracentrifugation is the study of the behavior of
macromolecules in solution under the influence of a strong gravitational
force.
It is a centrifuge with one or several optical detection systems, which allow
the observation of the fractionation process while the sample is centrifuged.
The commercially used analytical centrifuge is Beckman-Coulter analytical
ultracentrifugation
18. Applications of centrifugation
It is used to concentrate samples(preparatory cent.)
Used to separate solute from solvent.
Used to isolate cell organelles.
Blood cell separation.
Used in washing machine.
Centrifuges are used to separate cream (remove fat) from milk.
