Centrifugation is a technique that uses centrifugal force to separate mixtures based on density. It works by spinning samples at high speeds, which causes heavier components to sediment. There are several types including preparative centrifugation to separate/purify biological samples, analytical centrifugation to determine physical characteristics, differential centrifugation to separate cell components, density gradient centrifugation to separate mixtures based on density differences, and ultracentrifugation using very high speeds. Centrifugation has many applications in research, medicine, and industry.

1. CENTRIFUGATION
BCH 503
5th Semester

2. Introduction
• Centrifugation is one of the most important and widely applied
research cellular techniques in bio-chemistry and molecular
biology, pharmacy and in medicine
• Centrifugation is a process which involves the use of the
centrifugal force for the sedimentation of heterogeneous
mixtures with a centrifuge
• It is carried out by spinning a biological sample at high rate of
speed

3. Categories
Preparative Centrifugation
Separation & purification of Biological samples
( cells, organelles, macromolecules)
Analytical Centrifugation
Physical characteristics of biological sample such as size ,shape
and density

4. 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 idea was subsequently put into practice by his brother, Alexander Prandtl, who
made improvements to his brother's design, and exhibited a working butterfat
extraction machine in 1875.
• 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. This
process led to the discovery of an important new class of biological constituents,
later to be known as nucleic acids.
• The work of Miescher was quickly recognized and developed further by others.
• In 1879, the first continuous centrifugal separator was demonstrated by Gustaf de
Laval. This development made the commercialization of the centrifuge a possibility
for the first time.

5. Principal
• Utilizes density difference between the particles
and the medium in which these are dispersed
• Dispersed systems are subjected to artificially
induced gravitational fields.
• The centrifugal force causes the sedimentation
of heavier solid particles.

6. Continued
• A particle is subjected to centrifugal force when it is
rotated at high rate of speed
• The centrifugal force, F is defined by the equation
F= mω2r
Where,
F= intensity of the centrifugal force
m= effective mass of the sedimenting particle
ω= angular velocity of rotation
r= distance of the migrating particles from the
central axis of rotation

7. Continued
A more common measurement of F in terms of the earths
gravitation force , g, is relative centrifugal force, RCF its defined
by
RCF = (1.119 x 10-5 (rpm)2 (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 distance of the sedimenting particles from the axis of
rotation . The RCF value is reported as “ a number times gravity
,g .”

8. Instrumentation
Consist of two components
Electric motor
(Spin sample)
Rotor
(hold tubes and containers of sample)

9. Types of Centrifuge
• Low speed centrifuge
• High speed centrifuge
• Ultra centrifuge

10. Low Speed Centrifuge
• Most laboratories have a standard low-speed centrifuge used
for routine sedimentation of heavy particles.
• The low speed centrifuge has a maximum speed of 4000-
5000rpm.
• These instruments usually operate at room temperatures with
no means of temperature control.
• Two types of rotors are used in it, fixed angle and swinging
bucket.
• It is used for sedimentation of red blood cells until the
particles are tightly packed into a pellet and supernatant is
separated by decantation.

12. High Speed Centrifuge
• High speed centrifuges are used in more sophisticated
biochemical applications, higher speeds and temperature
control of the rotor chamber are essential
• The operator of this instrument can carefully control speed and
temperature which is required for sensitive biological samples
• Three types of rotors are available for high speed
centrifugation-fixed angle, swinging bucket, vertical rotors

14. Types of rotors
A centrifuge rotor is the rotating unit of the centrifuge,
which has fixed holes drilled at an angle. Test tubes are
placed inside these holes and the rotor spins to aid in
the separation of the materials
Types
Swing-bucket Rotor
Fixed-angle Rotor
Vertical rotor

15. Swing-bucket Rotor
• A swing-bucket rotor usually supports samples
ranging in volume from 36 mL to 2.2 mL
• Swing-buckets can support two types of separations: rate-zonal and
Isopycnic
• Swing-buckets are preferred for rate-zonal separations, because the
distance between the outside of the meniscus and the outside of the bottom
of the tube is long enough for separation to occur

16. Fixed-angle rotors
• Fixed-angle rotors are usually used for pelleting applications
to either pellet particles from a suspension and remove the
excess debris, or to collect the pellet
• Rotor cavities range from 0.2 mL to 1 mL
• The most important aspect in deciding to use a fixed-angle
rotor is the K factor
• The K factor indicates how efficient the rotor can pellet at
maximum speed
• The lower the K factor, the higher the pelleting efficiency

17. Vertical rotors
• Vertical rotors are highly specialized
• They are typically used to band DNA in cesium
chloride
• Vertical rotors have very low K factors, which is
useful if the particle must only move a short distance
until it pellets
• Run time on vertical rotors is short

18. Ultra centrifuge
• It is the most sophisticated instrument
• Intense heat is generated due to high speed thus the spinning
chambers must be refrigerated and kept at high vacuum
• It is used for both preparative work and analytical work

20. Types of Centrifugation Techniques
• Density Gradient Centrifugation
• Differential Centrifugation
• Ultra Centrifugation

21. Density Gradient Centrifugation
• It allow separation of many or all components in
a mixture and allows for measurement to be
made
• There are two forms of Density gradient
centrifugation
Rate zonal centrifugation
Isopycnic or sedimentation equilibrium
centrifugation

22. 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.

23. Continued
The particles will begin sedimenting in separate zones according
to their size shape and density

24. Isopycnic or sedimentation equilibrium
centrifugation
• In this type of centrifugation , 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 only to the position in the
centrifuge tube at which the gradient density is equal
to its own density

25. Continued
• In Isopycnic centrifugation separation of particles occurs into
zones on the basis of their density differences, independent of
time

26. Differential Centrifugation
• Differential centrifugation is a common procedure in
microbiology and 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 and mix up the cell contents
• The homogenate is then subjected to repeated centrifugations,
each time removing the pellet and increasing the centrifugal
force

28. Ultra Centrifugation
• An important tool in biochemical research is the centrifuge,
which through rapid spinning imposes high centrifugal forces
on suspended particles, or even molecules in solution, and
causes separations of such matter on the
basisULTRACENTRIFUGATION of differences in weight
• Example: Red cells may be separated from plasma of blood,
nuclei from mitochondria in cell homogenates, and one protein
from another in complex mixtures

29. Applications
Pharmaceutical Industry
 Production of bulk drug
Biopharmaceutical analysis of drugs
Determination of molecular weight of colloids
Evaluation of Suspension and Emulsions
Production of biological products

30. Continued
Application in water treatment
 Separation of solid substance from highly concentrated
suspension
Separation of oily suspension
Separation of oily concentrated sludge
Separation of Heavy products

31. Advantages
• Centrifuges have a clean appearance and have little to no odor
problems
• Not only is the device easy to install and fast at starting up and
shutting down, but also only requires a small area for operation
• They can be selected for different applications
• The device is simple to operate
• Centrifuge has more process flexibility and higher levels of
performance

32. Disadvantages
• The machine can be very noisy and can cause vibration
• The device has a high-energy consumption due to high G-
forces
• High initial capital costs

33. Precautions
• A centrifuge user should strictly observe the
following precautions
• Manufacturer’s manual should be strictly followed
• Rotor should be stored in proper containers
• Attention should be given to imbalance detectors
• Rotor speed should not exceed the assigned speed
• Lid of the rotor chamber should remain locked
during operation
• To avoid the rotor failure, manufactures instructions
regarding rotor care and use should always be
followed