centrifugation

1. Centrifugation
Centrifugation is a process that involves the use of the centrifugal force for the separation of
mixtures, used in industry and in laboratory settings. In chemistry and biology, centrifugation increases
the effective gravitational force on a mixture in a test tube, to rapidly and completely bring the precipitate
("pellet") to the bottom of the tube. The remaining solution is called the "supernate," "supernatant," or
supernatant liquid. The supernatant liquid is then separated from the precipitate by decantation or
withdrawal with a Pasteur pipette.
The equipment used for centrifugation is called a centrifuge, and the vessel that spins the
samples is called a rotor. Generally, a motor causes the rotor to spin around a fixed axis, applying a force
perpendicular to the axis. The centrifuge works using the sedimentation principle, where the centripetal
acceleration is used to separate substances of greater and lesser density.
There are many different kinds of centrifuges, including those for very specialized purposes. In
the chemical and food industries, special centrifuges can process a continuous stream of particle-laden
liquid.
English military engineer Laval (1707-1751) invented a whirling arm apparatus to determine
drag, and Antonin Prandl invented the first centrifuge in order to separate cream from milk to make it
easier to churn butter.
History
By 1923 Theodor Svedberg and his student H. Rinde had successfully analyzed large-grained sols
in terms of their gravitational sedimentation. Sols consist of a substance evenly distributed in another
substance, also known as a colloid. However, smaller grained sols, such as those containing gold, could
not be analyzed. To investigate this problem Svedberg developed an analytical centrifuge, equipped with
a photographic absorption system, which would exert a much greater centrifugal effect. In addition, he
developed the theory necessary to measure molecular weight. During this time, Svedberg’s attention
shifted from gold to proteins.
By 1900, it was generally accepted that proteins were composed of amino acids; however,
whether proteins were colloids or macromolecules was still under debate.
One protein being investigated at the time was hemoglobin. It was determined to have 712
carbon, 1,130 hydrogen, 243 oxygen, two sulfur atoms, and at least one iron atom. This gave hemoglobin
a resulting weight of approximately 16,000 Da but it was uncertain whether this value was a multiple of
one or four (dependent upon the number of iron atoms present).
Differential Centrifugation
If you had sufficient time and a vibration-free environment, you could patiently wait and the
force of gravity would bring most suspended particles to the bottom of a centrifuge tube. The smallest

2. particles would probably stay in suspension due to brownian motion, and most macromolecules would be
uniformly distributed because they would be in solution rather than suspension. I don't know about you,
but I don't have the kind of patience needed in order to rely solely on gravity for separation of solid from
liquid components. Besides, for practical purposes the pellet you obtained would be way too easily
disrupted for effective separation of solid material from supernatant. Gravity would not be a terribly
effective way of separating suspended materials based on size or other characteristics.
Density gradient centrifugation using tubes is the most widely employed technique for separating
cells and cell organelles and for isolating cellular macromolecules. However, although it is one of the cell
biologist’s most valuable tools, it is not without disadvantages, as the amount of material that can be
fractionated in a single tube is so small.
When large quantities of sample must be fractionated (to isolate sparse organelles such as
lysosomes or peroxisomes), a very large number of tubes and gradients is needed. Much larger quantities
of sample may be fractionated using zonal rotors.
A zonal rotor consists of a large cylindrical chamber subdivided into a number of sector-shaped
compartments by vertical septa (or vanes) that radiate from the axial core to the rotor wall. The entire
chamber is used during centrifugation and is loaded with a single density gradient, each sector-shaped
compartment serving as a large centrifuge tube.
The large chamber capacity of these rotors (typically 1 and 2 liters) eliminates the need for
multiple runs and multiple density gradients.
Centrifuges can be divided into types based on their rotor design: fixed angle, swinging bucket
and continuous flow. Choice of centrifuge rotor and lid will depend on application. Fixed-angle
centrifuges hold the sample containers at a constant angle relative to the central axis and are used
primarily for differential centrifugation. Swinging head (or swinging bucket) centrifuges, have a hinge
where the sample containers are attached to the central rotor and are used primarily for gradient work.
Continuous flow centrifuges don't have individual sample vessels and are used for large volume batch
separations. Look for rotors that are easily attached, exchangeable and allow access for cleaning. Be sure
to check if your intended rotor is compatible with your centrifuge’s manufacturer.
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Types of Rotor Centrifuges
Swing-Bucket Rotors
• 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.

3. 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.
Ultracentrifuge:
The ultracentrifuge is a centrifuge optimized for spinning a rotor at very high speeds, capable of
generating acceleration as high as 2000000 g (approx. 19600 km/s² ).There are two kinds of
ultracentrifuges, the preparative and the analytical ultracentrifuge. Both classes of instruments find
important uses in molecular biology, biochemistry, and polymer science.
A wide variety of laboratory-scale centrifuges are used in chemistry, biology, biochemistry and clinical
medicine for isolating and separating suspensions and immiscible liquids. They vary widely in speed,
capacity, temperature control, and other characteristics. Laboratory centrifuges often can accept an range
of different fixed-angle and swinging bucket rotors able to carry different numbers of centrifuge tubes and
rated for specific maximum speeds. Controls vary from simple electrical timers to programmable models
able to control acceleration and deceleration rates, running speeds, and temperature regimes.
Ultracentrifuges spin the rotors under vacuum, eliminating air resistance and enabling exact temperature
control. Zonal rotors and continuous flow systems are capable of handing bulk and larger sample
volumes, respectively, in a laboratory-scale instrument.
Isotope separation
Isotope separation is the process of concentrating specific isotopes of a chemical element by
removing other isotopes. The use of the nuclides produced is various. The largest variety is used in
research (e.g. in chemistry where atoms of "marker" nuclide are used to figure out reaction
mechanisms). By tonnage, separating natural uranium into enriched uranium and depleted
uranium is the largest application.
This process is a crucial one in the manufacture of uranium fuel for nuclear power stations, and is
also required for the creation of uranium based nuclear weapons. Plutonium-based weapons use
plutonium produced in a nuclear reactor, which must be operated in such a way as to produce
plutonium already of suitable isotopic mix or grade. While different chemical elements can be
purified through chemical processes, isotopes of the same element have nearly identical chemical
properties, which makes this type of separation impractical, except for separation of deuterium.
Separation techniques
There are three types of isotope separation techniques:
• Those based directly on the atomic weight of the isotope.
• Those based on the small differences in chemical reaction rates produced by different
atomic weights.

4. • Those based on properties not directly connected to atomic weight, such as nuclear
resonances.
The third type of separation is still experimental; practical separation techniques all depend in some
way on the atomic mass. It is therefore generally easier to separate isotopes with a larger relative
mass difference.
For example deuterium has twice the mass of ordinary (light) hydrogen and it is generally easier to
purify it than to separate uranium-235 from the more common uranium-238. On the other extreme,
separation of fissile plutonium-239 from the common impurity plutonium-240, while desirable in that
it would allow the creation of gun-type nuclear weapons from plutonium, is generally agreed to be
impractical.
All large-scale isotope separation schemes employ a number of similar stages which produce
successively higher concentrations of the desired isotope. Each stage enriches the product of the
previous step further before being sent to the next stage. Similarly, the tailings from each stage are
returned to the previous stage for further processing. This creates a sequential enriching system
called a cascade.
Sedimentation equilibrium in a solution or suspension of different particles, such as molecules,
exists when the rate of transport of each material in any one direction due to sedimentation equals
the rate of transport in the opposite direction due to diffusion. Sedimentation is due to an external
force, such as gravity (for very large particles) or centrifugal force in a centrifuge.
Modern applications use the analytical ultracentrifuge. The theoretical basis for the measurements is
developed from the Mason-Weaver equation.