Centrifuge, Principle, Rotors, Types of centrifuge, Application

1. Centrifugation
Course: B.Sc Biotechnology
Subject: Bio Analytical Technique
Unit: II

2. Centrifugation
• Biological centrifugation is a process that uses centrifugal force to
separate and purify mixtures of biological particles in a liquid medium.
• It is a key technique for isolating and analysing cells, subcellular fractions,
supramolecular complexes and isolated macromolecules such as proteins
or nucleic acids.
• The development of the first analytical ultracentrifuge by Svedberg in the
late 1920s and the technical refinement of the preparative centrifugation
technique by Claude and colleagues in the 1940s positioned
centrifugation technology at the centre of biological and biomedical
research for many decades.
• Today, centrifugation techniques represent a critical tool for modern
biochemistry and are employed in almost all invasive subcellular studies.
• While analytical centrifugation is mainly concerned with the study of
purified macromolecules or isolated supramolecular assemblies,
preparative centrifugation methodology is devoted to the actual
separation of tissues, cells, subcellular structures, membrane vesicles and
other particles of biochemical interest.

3. General Steps in Biochemical
Separation

4. Principles of centrifugation
• A centrifuge is a device for separating particles from a solution according
to their size, shape, density, viscosity of the medium and rotor speed.
• In a solution, particles whose density is higher than that of the solvent
sink (sediment), and particles that are lighter than it float to the top.
• The greater the difference in density, the faster they move. If there is no
difference in density (isopyknic conditions), the particles stay steady.
• To take advantage of even tiny differences in
density to separate various particles in a solution,
gravity can be replaced with the much more
powerful “centrifugal force” provided by a
centrifuge.
1.

5. Centrifugation
• A centrifuge is used to separate particles or
macromolecules:
-Cells
-Sub-cellular components
-Proteins
-Nucleic acids
• Basis of separation:
-Size
-Shape
-Density
• Methodology:
-Utilizes density difference between the particles/macromolecules
and the medium in which these are dispersed
-Dispersed systems are subjected to artificially induced gravitational
fields

6. BASIC PRINCIPLES OF SEDIMENTATION
• the effect of sedimentation due to the influence of the
Earth’s gravitational field (g¼981 cm s–2) versus the
increased rate of sedimentation in a centrifugal field
(g>981 cm s–2) is apparent.
• To give a simple but illustrative example, crude sand
particles added to a bucket of water travel slowly to
the bottom of the bucket by gravitation, but sediment
much faster when the bucket is swung around in a
circle.
• Similarly, biological structures exhibit a drastic increase
in sedimentation when they undergo acceleration in a
centrifugal field.

7. When designing a centrifugation protocol,
it is important to keep in mind that :
• 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. • Biological particles moving through a viscous medium experience a
frictional drag, whereby the frictional force acts in the opposite
direction to sedimentation and equals the velocity of the particle
multiplied by the frictional coefficient.
• The frictional coefficient depends on the size and shape of the
biological particle.
• As the sample moves towards the bottom of a centrifuge tube in
swing-out or fixed-angle rotors, its velocity will increase due to the
increase in radial distance.
• At the same time the particles also encounter a frictional drag that
is proportional to their velocity.
• The frictional force of a particle moving through a viscous fluid is
the product of its velocity and its frictional coefficient, and acts in
the opposite direction to sedimentation.

9. • Essentially, the rate of sedimentation is dependent
upon the applied centrifugal field (cm s2), G,
• G=rω2 depends on the radical distance of the
particle from the rotation axis and the square of the
angular velocity.
• 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)
• The average angular velocity of a rigid body that
rotates about a fixed axis is defined as the ratio of the
angular displacement in a given time interval.

10. • One radian, usually abbreviated as 1 rad,
represents the angle subtended at the centre
of a circle by an arc with a length equal to the
radius of the circle. Since 360o equals 2
radians, one revolution of the rotor can be
expressed as 2 rad.
• Accordingly, the angular velocity in rads per
second of the rotor can be expressed in terms
of rotor speed s as:

11. CALCULATION OF CENTRIFUGAL FIELD
and therefore the centrifugal field can be expressed as:

12. CALCULATION OF ANGULAR VELOCITY
The centrifugal field is generally expressed in multiples of the gravitational field,
g (981cms–2). The relative centrifugal field (g), RCF, which is the ratio of the centrifugal
acceleration at a specified radius and the speed to the standard acceleration of gravity,
can be calculated from the following equation:

13. • Although the relative centrifugal force can easily be calculated, centrifugation
manuals usually contain a nomograph for the convenient conversion between
relative centrifugal force and speed of the centrifuge at different radii of the
centrifugation spindle to a point along the centrifuge tube.

14. Types of centrifuges
• Centrifugation techniques take a central position in modern
biochemical, cellular and molecular biological studies.
• Depending on the particular application, centrifuges differ in their
overall design and size.
• However, a common feature in all centrifuges is the central motor
that spins a rotor containing the samples to be separated.
• Particles of biochemical interest are usually suspended in a liquid
buffer system contained in specific tubes or separation chambers
that are located in specialised rotors.
• Thebiological medium is chosen for the specific centrifugal
application and may differ considerably between preparative and
analytical approaches.
• The optimum pH value, salt concentration, stabilising cofactors and
protective ingredients such as protease inhibitors have to be
carefully evaluated in order to preserve biological function.

15. The most obvious differences between
centrifuges are:
• The maximum speed at which biological specimens
are subjected to increased sedimentation;
• The presence or absence of a vacuum;
• The potential for refrigeration or general
manipulation of the temperature during a
centrifugation run; and
• The maximum volume of samples and capacity for
individual centrifugation tubes.

16. Many different types of centrifuges
are commercially available including:
• large-capacity low-speed preparative centrifuges;
• refrigerated high-speed preparative centrifuges;
• analytical ultracentrifuges;
• preparative ultracentrifuges;
• large-scale clinical centrifuges; and
• small-scale laboratory microfuges.

17. Types of rotors
2.

18. 3.

19. Care and maintenance of centrifuges
• Corrosion and degradation due to biological buffer systems used within rotors or
contamination of the interior or exterior of the centrifuge via spillage may
seriously affect the lifetime of this equipment.
• Another important point is the proper balancing of centrifuge tubes. This is not
only important with respect to safety, but it might be also cause vibration-induced
damage to the rotor itself and the drive shaft of the centrifuge.
• Thus, proper handling and care, as well as regular maintenance of both centrifuges
and rotors is an important part of keeping this biochemical method available in the
laboratory.
• Corrosion may be triggered by longterm exposure of rotors to alkaline solutions,
acidic buffers, aggressive detergents or salt. Thus, rotors should be thoroughly
washed with distilled or deionised water after every run.
• For overnight storage, rotors should be first left upside down to drain excess liquid
and then positioned in a safe and dry place.
• To avoid damage to the hinge pins of swinging-bucket rotors, they should be dried
with tissue paper following removal of biological buffers and washing with water.
• Regular maintenance of rotors and centrifuges by engineers is important for
ensuring the safe operation of a centralised centrifugation facility.
• Accurate record-keeping of run times and centrifugal speeds is important, since
cyclic acceleration and deacceleration of rotors may lead to metal fatigue.

20. Mechanical stress
• Always ensure that loads are evenly balanced before a run.
• Always observe the manufacturers maximum speed and sample
density ratings.
• Always observe speed reductions when running high density
solutions, plastic adapters, or stainless steel tubes.
Corrosion
• Many rotors are made from either titanium or aluminum alloy,
chosen for their advantageous mechanical properties. While
titanium alloys are quite corrosion-resistant, aluminum alloys are
not.
• When corrosion occurs, the metal is weakened and less able to
bear the stress from the centrifugal force exerted during operation.
• The combination of stress and corrosion causes the rotor to fail
more quickly and at lower stress levels than an uncorroded rotor

21. PREPARATIVE CENTRIFUGATION
• Preparative ultracentrifuges are available with a wide variety of rotors suitable for
a great range of experiments. Most rotors are designed to hold tubes that contain
the samples. Swinging bucket rotors allow the tubes to hang on hinges so the
tubes reorient to the horizontal as the rotor initially accelerates. Fixed angle rotors
are made of a single block of metal and hold the tubes in cavities bored at a
predetermined angle. Zonal rotors are designed to contain a large volume of
sample in a single central cavity rather than in tubes. Some zonal rotors are
capable of dynamic loading and unloading of samples while the rotor is spinning at
high speed.
• Preparative rotors are used in biology for pelleting of fine particulate fractions,
such as cellular organelles (mitochondria, microsomes, ribosomes) and viruses.
They can also be used for gradient separations, in which the tubes are filled from
top to bottom with an increasing concentration of a dense substance in solution.
Sucrose gradients are typically used for separation of cellular organelles. Gradients
of caesium salts are used for separation of nucleic acids. After the sample has spun
at high speed for sufficient time to produce the separation, the rotor is allowed to
come to a smooth stop and the gradient is gently pumped out of each tube to
isolate the separated components.

22. Two types of ultracentrifuges
developed:
Analytical
· Uses small sample size (less than 1 ml)
· Built in optical system to analyze
progress of molecules during
centrifugation
· Uses relatively pure sample
· Used to precisely determine
sedimentation coefficient and MW of
molecules
· Beckman Model E is an example of
centrifuge used for these purposes.
Preparative
· Larger sample size can be used
· No optical read-out – collect fractions
and analyze them after the run
· Less pure sample can be used
· Can be used to estimate sedimentation
coefficient and MW
· Generally used to separate organelles
and molecules. Most centrifugation
work done using preparative
ultracentrifuge
· Several models available, including L5-
65 and L5-75 used for preparative
purposes.

23. 1.Differential centrifugation
Based on the differences in the sedimentation rate of the biological particles of
different size, shape and density
Preparative Centrifugation Types
4.

24. Moving Boundary (differential
velocity) Centrifugation
1) The entire tube is filled with sample and centrifuged
2) Through centrifugation, one obtains a separation of two particles but any particle in
the mixture may end up in the supernatant or in the pellet or it may be distributed in
both fractions, depending upon its size, shape, density, and conditions of
centrifugation
3) Repeat sedimentation at different speed
Application: low resolution separation such as preparation of nucleus
5.

25. • Cellular and subcellular fractionation techniques are indispensable
methods used in biochemical research. Although the proper separation of
many subcellular structures is absolutely dependent on preparative
ultracentrifugation, the isolation of large cellular structures, the nuclear
fraction, mitochondria, chloroplasts or large protein precipitates can be
achieved by conventional high-speed refrigerated centrifugation.
• Differential centrifugation is based upon the differences in the
sedimentation rate of biological particles of different size and density.
• Crude tissue homogenates containing organelles, membrane vesicles and
other structural fragments are divided into different fractions by the
stepwise increase of the applied centrifugal field.
• Following the initial sedimentation of the largest particles of a
homogenate (such as cellular debris) by centrifugation, various biological
structures or aggregates are separated into pellet and supernatant
fractions, depending upon the speed and time of individual centrifugation
steps and the density and relative size of the particles.
• To increase the yield of membrane structures and protein aggregates
released, cellular debris pellets are often rehomogenised several times
and then recentrifuged. This is especially important in the case of rigid
biological structures
• With respect to the separation of organelles and membrane vesicles,
crude differential centrifugation techniques can be conveniently
employed to isolate intact mitochondria and microsomes.

26. 6.

27. 2. Density-gradient centrifugation
• Important technique for purifying proteins and
particularly nucleic acids.
 Two different types of density gradien
centrifugation, for two different purposes are:
1. Zonal (or Rate Zonal) Centrifugation
(Sucrose density gradient centrifugation)
2. Iso-density (Isopycnic) Centrifugation
(Caesium chloride density gradient centrifugation)

28. Moving Zone Centrifugation
1. Preparation of gradient sucrose density for centrifugation medium
Density1 < Density2 < Density 3 < Density4 < DensityAnalyte
2.Sample is applied in a thin zone at the top of the centrifuge tube on a density
gradient
7.

29. 3. Under centrifugal force, the particles will begin sedimenting through the gradient in
separate zones according to their size shape and density
Insufficient time--------- Incomplete separation
Overtime--------------------co precipitation of all analytes
8.

30. Iso-density (Isopyncic) Centrifugation
1. Preparation of gradient sucrose density for centrifugation medium
The gradient density has to cover the range of different densities of analytes
9.

31. 10.

32. 11.

33. Subcellular fractionation
• A typical flow chart outlining a subcellular
fractionation is Depending on the amount of
starting material, which would usually range
between 1 and 500 g in the case of skeletal
muscle preparations, a particular type of rotor
and size of centrifuge tubes is chosen for
individual stages of the isolation procedure. The
repeated centrifugation at progressively higher
speeds and longer centrifugation periods will
divide the muscle homogenate into distinct
fractions.

34. 12.

35. Step 1- Cell homogenization
13.

36. 14.

37. 15.

38. Step 2-Cell Fractionation by
Centrifugation.
• Repeated centrifugation at progressively
higher speeds will fractionate homogenates of
cells into their components.
• In general, the smaller the subcellular
component, the greater is the centrifugal
force required to sediment it.

39. Step 3- Density Gradient
Centrifugation
16.

40. 17.

41. Analytical Ultracentrifugation
• An analytical ultracentrifuge spins a rotor at an accurately
controlled speed and temperature. The concentration
distribution of the sample is determined at known times using
absorbance measurements. It can determine:
 Continuously monitor the sedimentation process
• Purity of macromole
• Relative molecular mass of solute (within 5% SD)
• Change in relative molecular mass of supermolecular
complexes
• Conformational change of protein structure
• Ligand-binding study

42. 18.

43. Sedimentation Velocity Method
Sedimentation velocity experiments
are performed at high speed to
overcome the effect of diffusion. For a
sedimentation velocity experiment, an
initially uniform solution is placed in a
cell and a sufficiently high angular
velocity is applied to cause rapid
sedimentation of solute towards the
cell bottom. As a result, there is a
depletion of solute near the meniscus,
causing a characteristic spectrum as
shown in the following figure. A sharp
boundary occurs between the depleted
region and the sedimenting solute (the
plateau)
19.

44. Book and Web References
• http://en.wikipedia.org/wiki/Centrifugation
• http://www.sigmaaldrich.com/content/dam/sigma-
aldrich/docs/Sigma-Aldrich/Brochure/1/biofiles_v6_n5.pdf
• http://www.phys.sinica.edu.tw/TIGP-
NANO/Course/2007_Spring/Class%20Notes/AC_Chapter
%203%20Centrifugation%200321.pdf
• Book name : Wilson and Walker

45. Image References
• 1.,5.
to11.,13.,14.,15.,17.http://www.phys.sinica.edu.tw/TIGP-
NANO/Course/2007_Spring/Class%20Notes/AC_Chapter
%203%20Centrifugation%200321.pdf
• 2.,3.,4.,12.,16.,18. Book Name: Principles and Techniques of
Biochemistry and Molecular Biology by Wilson n Walker.