Centrifugation

Centrifugationis a process that involves the use of the centrifugal force for the separation of mixtures, used in industry and in laboratory settings. Inchemistryandbiology, 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 remainingsolutionis 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 acentrifuge, and the vessel that spins the samples is called arotor. 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 1923Theodor Svedbergand 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 acolloid.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, Svedbergs attention shifted from gold to proteins.

By 1900, it was generally accepted that proteins were composed of amino acids; however, whether proteins were colloids ormacromoleculeswas still under debate.

One protein being investigated at the time washemoglobin. 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 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 biologists 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.Azonal 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 centrifuges manufacturer.Fiberlite Rotors: Advanced Carbon Fiber Centrifuge Rotors

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Increased productivity with higher G-forcesTypes of Rotor CentrifugesSwing-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.

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:Theultracentrifugeis acentrifugeoptimized for spinning a rotor at very high speeds, capable of generating acceleration as high as2000000g(approx.19600 km/s).There are two kinds of ultracentrifuges, the preparative and the analytical ultracentrifuge. Both classes of instruments find important uses inmolecular biology, biochemistry, andpolymerscience.

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 rotorsandcontinuous flowsystems are capable of handing bulk and larger sample volumes, respectively, in a laboratory-scale instrument.

Isotope separationIsotope separationis the process of concentrating specificisotopesof achemical elementby removing other isotopes. The use of thenuclidesproduced is various. The largest variety is used in research (e.g. inchemistrywhere atoms of "marker" nuclide are used to figure out reaction mechanisms). By tonnage, separatingnatural uraniumintoenriched uraniumanddepleted uraniumis 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 orgrade. While different chemical elements can be purified throughchemical processes, isotopes of the same element have nearly identical chemical properties, which makes this type of separation impractical, except for separation ofdeuterium.

Separation techniquesThere are three types of isotope separation techniques: