Principle, types, uses

1. CENTRIFUGATION
Dr. Sneha Paul
Executive-Medical team
Spi-Global
Principle, types and uses

2. Instrumentation
Electric motor- to
spin the sample
Rotor- to hold the
sample

3. 1659:
• Dutch mathematician and scientist
• Christiaan Huygens-centrifugal force
1864
• German master brewer
• Antonin Prandtl-dairy centrifuge
1869
• Swiss physician and biologist
• Friedrich Miescher-used centrifugal force to gain what he called “nuclein” (now known as DNA)
from cells, and was therefore the first researcher who isolated nucleic acids
1877
• Gustav De Laval - added turbines to prandtl design
• Invented the first continuous cream separator, which revolutionized the dairy industry
1925
• colloid chemist
• Theodor Svedberg-first ultracentrifuge, Nobel prize
1930
• Belgian medical doctor and cell biologist
• Albert Claude discovered the process of cell fractionation in which centrifugation is an essential
step
1930
• Jesse Beams, an American physicist at the University of Virginia, worked on high vacuum
ultracentrifuges to achieve even higher speeds than Svedberg’s ultracentrifuge.
1942
• Albert Claude and his colleague James S. Potter published an innovative paper called “Isolation
of chromatin threads from the resting nucleus of leukemic cells” in 1942
1946/
47
• Edward Pickles (a student of Jesse Beams) founded a company called Spinco that specialized in
the design and manufacturing of ultracentrifuges. The company introduced the first commercial
preparative ultracentrifuge in 1949, the Model L
1950
• American biochemist Myron K. Brakke used density gradient centrifugation as a separation
technique to purify potato yellow dwarf virus.
1964
• Eppendorf presented the Centrifuge 3200, the first centrifuge to form an integral part of
the Eppendorf Microliter System
1980s
.
• Hettich went on showing novelties such as the first robotically operated centrifuge, dramatically
improving work efficiency in high-throughput screening and medical diagnostic laboratories
HISTORY

4. Centrifuge
force
PRINCIPLE
 A centrifuge is a device used to separate components of a mixture on
the basis of their size, density, the viscosity of the medium, and the
rotor speed.


5. PRINCIPLE
The sedimentation principle is where the acceleration of the rotor causes a centripetal force to
act upon the rotor and centrifuge tubes. This causes the denser substances in the tubes to be
forced outward in a radial direction. This also causes the lighter particles to be displaced and
move towards the centre. Many particulates can become ‘stuck’ at the bottom of centrifuge
tubes, especially when using a laboratory centrifuge. These particles are commonly known as
pellets, and the clarified solution is coined the supernatant.

6. PRINCIPLE

7. PRINCIPLE
Sedimentation Coefficient:
Measure of the rate of sedimentation of a molecule or particle; it is equal to the
velocity per unit centrifugal field (acceleration), and is measured in Svedberg units.
Fc- centripetal force
Fb: Buoyant force
Ff-Force of friction

8.  Relative centrifugal force is the measure of the strength of rotors
of different types and sizes.
 This is the force exerted on the contents of the rotor as a result of
the rotation.
 RCF is the perpendicular force acting on the sample that is
always relative to the gravity of the earth.
 The formula to calculate the relative centrifugal force (RCF) can
be written as:
 RCF (g Force)= 1.118 × 10-5 × r × (RPM)2
 where r is the radius of the rotor (in centimeters), and RPM is
the speed of the rotor in rotation per minute
RELATIVE CENTRIFUGAL FORCE (RCF)

9. PRINCIPLE
RBC
WBC
Plasma
Platelets
Filtration
sedimentation
Centrifugation
process

10. To separate two miscible substances
To analyze the hydrodynamic properties of macromolecules
Purification of mammalian cells
Fractionation of subcellular organelles (including
membranes/membrane fractions) Fractionation of membrane
vesicles
Separating chalk powder from water
Removing fat from milk to produce skimmed milk
Separating particles from an air-flow using cyclonic separation
The clarification and stabilization of wine
Separation of urine components and blood components in
forensic and research laboratories
Aids in the separation of proteins using purification techniques
USE’S/ APPLICATIONS…..

11. TYPES OF CENTRIFUGE ROTORS
Fixed Angle rotor
• These rotors hold the sample tubes at an angle of 45° in relation to the axis
of the rotor.
• In this type of rotor, the particles strike the opposite side of the tube where
the particles finally slide down and are collected at the bottom.
• These are faster than other types of rotors as the pathlength of the tubes
increases.

12. TYPES OF CENTRIFUGE ROTORS
Swinging bucket rotor
• Swinging bucket rotors hold the tubes at an angle of 90° as the rotor
swings as the process is started.
• In this rotor, the tubes are suspended in the racks that allow the tubes to
be moved enough to acquire the horizontal position.
• In this type of rotors, the particles are present along the direction or the
path of the force that allows the particles to be moved away from the rotor
towards the bottom of the tubes.

13. TYPES OF CENTRIFUGE ROTORS
Vertical rotor
• Vertical rotors provide the shortest pathlength, fastest run time, and the highest resolution of
all the rotors.
• the tubes are vertical during centrifugation
• The yield of the rotor is not as ideal as the position of the tube doesn’t align with the direction
of the centrifugal force.
• As a result, instead of settling down, particles tend spread towards the outer wall of the
tubes

14. 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
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.
TYPES OF CENTRIFUGE

15. HIGH-SPEED CENTRIFUGES:
High-speed centrifuges are used in more sophisticated
biochemical applications, higher speeds and temperature control
of the rotor chamber are essential.
The high-speed centrifuge has a maximum speed of 15,000 –
20,000 RPM
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
TYPES OF CENTRIFUGE

16. ULTRACENTRIFUGES:
It is the most sophisticated instrument.
Ultracentrifuge has a maximum speed of 150,000 RPM (100,000’s
x g).
Intense heat is generated due to high speed thus the spinning
chambers must be refrigerated and kept at a high vacuum.
Ultracentrifuges can separate molecules in large batches and in a
continuous flow system.
In addition to separation, ultracentrifuges can also be used for
the determination of properties of macromolecules like the size,
shape, and density.
It is used for both preparative work and analytical work.
TYPES OF CENTRIFUGE

17. ULTRACENTRIFUGES:
Preparative ultracentrifuges sediment and separate
biological/organic components such as DNA, RNA, lipoproteins,
membranes, organelles and viruses.
Analytical ultracentrifuges detect samples in real time. They can
detect the equilibrium and velocity sedimentation, the shape of
molecules and the mass of molecules.
TYPES OF CENTRIFUGE

18. Analytical Centrifugation:
Analytical centrifugation is a separation method where the
particles in a sample are separated on the basis of their density
and the centrifugal force they experience.
Analytical ultracentrifugation (AUC) is a versatile and robust
method for the quantitative analysis of macromolecules in
solution.
The sedimentation coefficient can be used to characterize
changes in the size and shape of macromolecules with changing
experimental conditions.
Three optical systems are available for the analytical
ultracentrifuge (absorbance, interference, and fluorescence) that
permit precise and selective observation of sedimentation in real-
time.
TYPES OF CENTRIFUGATION

19. Density gradient centrifugation:
 Density gradient centrifugation is based on the principle that
molecules settle down under a centrifugal force until they reach a
medium with the density the same as theirs.
 This type of centrifugation is mainly used to purify viruses,
ribosomes, membranes, etc.
 A sucrose density gradient is created by gently overlaying lower
concentrations of sucrose on higher concentrations in centrifuge tubes
 The particles of interest are placed on top of the gradient and
centrifuge in ultracentrifuges.
 The particles travel through the gradient until
they reach a point at which their density matches
the density of surrounding sucrose.
 The fraction is removed and analyzed.
TYPES OF CENTRIFUGATION

20. Uses:
Density gradient centrifugation can be applied for the
purification of large volumes of biomolecules.
It can even be used for the purification of different viruses which
aids their further studies.
This technique can be used both as a separation technique and
the technique for the determination of densities of various
particles.
Types:
1. Rate-Zonal Density-Gradient centrifugation
2. Isopynic Density-Gradient Centrifugation
TYPES OF CENTRIFUGATION

21. Rate-Zonal Density-Gradient centrifugation:
 Zonal centrifugation is also known as
band or gradient centrifugation
 It relies on the concept of sedimentation
coefficient (i.e. movement of sediment
through the liquid medium)
 In this technique, a density gradient is
created in a test tube with sucrose and
high density at the bottom.
 The sample of protein is placed on the
top of the gradient and then centrifuged.
 With centrifugation, faster-sedimenting
particles in sample move ahead of slower
ones i.e. sample separated as zones in
the gradient.
 The protein sediment according to their
sedimentation coefficient and the
fractions are collected by creating a hole
at the bottom of the tube.
TYPES OF CENTRIFUGATION

22. Isopynic Density-Gradient Centrifugation:
 The sample is loaded into the tube with the
gradient-forming solution (on top of or
below pre-formed gradient, or mixed in with
self-forming gradient)
 The solution of the biological sample and
cesium salt is uniformly distributed in a
centrifuge tube and rotated in an
ultracentrifuge.
 Under the influence of centrifugal force, the
cesium salts redistribute to form a density
gradient from top to bottom.
 Particles move to point where their buoyant
density equals that part of gradient and
form bands. This is to say the sample
molecules move to the region where their
density equals the density of gradient.
 It is a “true” equilibrium procedure since
depends on bouyant densities, not velocities
 Eg: CsCl, NaI gradients for macromolecules
and nucleotides – “self-forming” gradients
under centrifugal force.
TYPES OF CENTRIFUGATION

23. Differential centrifugation:
 It is the most common type of centrifugation employed.
 Tissue such as the liver is homogenized at 32 degrees in a sucrose
solution that contains buffer.
 The homogenate is then placed in a centrifuge and spun at
constant centrifugal force at a constant temperature.
 After some time a sediment forms at the bottom of a centrifuge
called pellet and an overlying solution called supernatant.
 The overlying solution is then placed in another centrifuge tube
which is then rotated at higher speeds in progressing steps.
TYPES OF CENTRIFUGATION

24. TYPES OF CENTRIFUGATION
Subcellular organelles separation by cell fraction
Smaller
Larger

25. Differential velocity (Moving Boundary) centrifugation:
Differential velocity centrifugation is a type of centrifugation
process in which components are separately settled down a
centrifuge tube by applying a series of increasing velocities.
Equilibrium density gradient centrifugation: Equilibrium density
gradient centrifugation is a modified and specialized form of
density gradient centrifugation.
Sucrose gradient centrifugation: Sucrose gradient centrifugation
is a type of density gradient centrifugation where the density
gradient is formed of sucrose by changing the concentration of
sucrose.
TYPES OF CENTRIFUGATION

26. OTHER CENTRIFUGES
Vacuum centrifuge/ Concentrators Refrigerated centrifuges
Microcentrifuge Hematocrit centrifuge
Gas centrifuge
Continuous flow centrifuge
Benchtop centrifuge

27. Type of sample
Vessel selection
Type of centrifuge
Type of rotor
Determination of desired relative centrifugal force
Defined temperature during centrifugation
HOW TO SELECT THE RIGHT
CENTRIFUGE FOR YOUR APPLICATION