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sample is placed in an ultracentrifuge and spun in low speed - nuclei settle out, forming a pellet The supernatant (suspension containing remaining organelles) is spun at a higher speed - chloroplasts settle out The supernatant is spun at a higher speed still - mitochondria and lysosomes settle out The supernatant is spun at an even higher speed - ribosomes, membranes settle out The ribosomes, membranes and Golgi complexes can be separated by another technique called density gradient centrifugation. Ultracentrifugations Main articles: Differential centrifugation, Isopycnic centrifugation, and ultracentrifugation Ultracentrifugation makes use of high centrifugal force for studying properties of biological particles. Compared to microcentrifuges or high-speed centrifuges, ultracentrifuges can isolate much smaller particles, including ribosomes, proteins, and viruses. Ultracentrifuges can also be used in the study of membrane fractionation. This occurs because ultracentrifuges can reach maximum angular velocities in excess of 70,000 rpm. Additionally, while microcentrifuges and supercentrifuges separate particles in batches (limited volumes of samples must be handled manually in test tubes or bottles), ultracentrifuges can separate molecules in batch or continuous flow systems.
In addition to purification, analytical ultracentrifugation (AUC) can be used for determination of the properties of macromolecules such as shape, mass, composition, and conformation. Samples are centrifuged with a high-density solution such as sucrose, caesium chloride, or iodixanol. The high-density solution may be at a uniform concentration throughout the test tube ("cushion") or a varying concentration ("gradient"). Molecular properties can be modeled through sedimentation velocity analysis or sedimentation equilibrium analysis. During the run, the particle or molecules will migrate through the test tube at different speeds depending on their physical properties and the properties of the solution, and eventually form a pellet at the bottom of the tube, or bands at various heights. Density Gradient Centrifugation Density gradient centrifugation is considered one of the more efficient methods of separating suspended particles. Density gradient centrifugation can be used both as a separation technique and as a method of measuring the densities of particles or molecules in a mixture.[7] A tube, after being centrifuged by this method, has particles in order of density based on height. The object or particle of interest will reside in the position within the tube corresponding to its density.[8]



Linderstorm-Lang, in 1937, discovered that density gradient tubes could be used for density measurements. He discovered this when working with potato yellow-dwarf virus.[7] This method was also used in Meselson and Stahl's famous experiment in which they proved that DNA replication is semi-conservative by using different isotopes of nitrogen. They used density gradient centrifugation to determine which isotope or isotopes of nitrogen were present in the DNA after cycles of replication.[8] Nevertheless, some non-ideal sedimentations are still possible when using this method. The first potential issue is the unwanted aggregation of particles, but this can occur in any centrifugation. The second possibility occurs when droplets of solution that contain particles sediment. This is more likely to occur when working with a solution that has a layer of suspension floating on a dense liquid, which in fact have little to no density gradient.[7] Differential Centrifugation Differential Centrifugation is a type of centrifugation in which one selectively spins down components of a mixture by a series of increasing centrifugation forces. This method is commonly used to separate organelles and membranes found in cells. Organelles generally differ from each other in density in size, making the use of differential centrifugation, and centrifugation in general, possible. The organelles can then be identified by testing for indicators that are unique to the specific organelles.[9] Other applications 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 separation of proteins using purification techniques such as salting out, e.g. ammonium sulfate precipitation.[10] History By 1923 Theodor Svedberg and his student H. Rinde had successfully analyzed large-grained sols in terms of their gravitational sedimentation.[11] Sols consist of a substance evenly distributed in another substance, also known as a colloid.[12] However, smaller grained sols, such as those containing gold, could not be analyzed.[11] To investigate this problem Svedberg developed an analytical centrifuge, equipped with a photographic absorption system, which would exert a much greater centrifugal effect.[11] In addition, he developed the theory necessary to measure molecular weight.[12] During this time, Svedberg's attention shifted from gold to proteins.[11] By 1900, it had been generally accepted that proteins were composed of amino acids; however, whether proteins were colloids or macromolecules was still under debate.[13] 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 dalton (Da) but it was uncertain whether this value was a multiple of one or four (dependent upon the number of iron atoms present).[14] Through a series of experiments utilizing the sedimentation equilibrium technique, two important observations were made: hemoglobin has a molecular weight of 68,000 Da, suggesting that there are four iron atoms present rather than one, and that no matter where the hemoglobin was isolated from, it had exactly the same molecular weight.[11][12] How something of such a large molecular mass could be consistently found, regardless of where it was sampled from in the body, was unprecedented and favored the idea that proteins are macromolecules rather than colloids.[13] In order to investigate this phenomenon, a centrifuge with even higher speeds was needed, and thus the ultracentrifuge was created to apply the theory of sedimentation-diffusion.[11] The same molecular mass was determined, and the presence of a spreading boundary suggested that it was a single compact particle.[11] Further application of centrifugation showed that under different conditions the large homogeneous particles could be broken down into discrete subunits.[11] The development of centrifugation was a great advance in experimental protein science A centrifuge is a piece of equipment that puts an object in rotation around a fixed axis (spins it in a circle), applying a force perpendicular to the axis of spin (outward) that can be very strong. The centrifuge works using the sedimentation principle, where the centrifugal acceleration causes denser substances and particles to move outward in the radial direction. At the same time, objects that are less dense are displaced and move to the center. In a laboratory centrifuge that uses sample tubes, the radial acceleration causes denser particles to settle to the bottom of the tube, while low-density substances rise to the top.[1] There are three types of centrifuge designed for different applications. Industrial scale centrifuges are commonly used in manufacturing and waste processing to sediment suspended solids, or to separate immiscible liquids. An example is the cream separator found in dairies. Very high speed centrifuges and ultracentrifuges able to provide very high accelerations can separate fine particles down to the nano-scale, and molecules of different masses. Large centrifuges are used to simulate high gravity or acceleration environments (for example, high-G training for test pilots). Medium-sized centrifuges are used in washing machines and at some swimming pools to draw water out of fabrics. Gas centrifuges are used for isotope separation, such as to enrich nuclear fuel for fissile isotopes. Contents 1 History 2 Types 3 Uses 3.1 Laboratory separations 3.2 Isotope separation 3.3 Aeronautics and astronautics 3.4 Industrial centrifugal separator 3.5 Geotechnical centrifuge modeling 3.6 Synthesis of materials 3.7 Commercial applications 4 Mathematical description 5 See also 6 References and notes 7 Further reading 8 External links History English military engineer Benjamin Robins (17071751) 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.[2] Types Whole blood is often separated, using a centrifuge, into components for storage and transportation A centrifuge machine can be described as a machine with a rapidly rotating container that applies centrifugal force to its contents. There are multiple types of centrifuge, which can be classified by intended use or by rotor design: Types by rotor design:[3][4][5][6] Fixed-angle centrifuges are designed to hold the sample containers at a constant angle relative to the central axis. Swinging head (or swinging bucket) centrifuges, in contrast to fixed-angle centrifuges, have a hinge where the sample containers are attached to the central rotor. This allows all of the samples to swing outwards as the centrifuge is spun. Continuous tubular centrifuges do not have individual sample vessels and are used for high volume applications. A peeler centrifuge made by Krauss-Maffei An inverting filter centrifuge made my Heinkel A decanter centrifuge made by Sharples Types by intended use: Laboratory centrifuges, are general-purpose instruments of several types with distinct, but overlapping, capabilities. These include clinical centrifuges, superspeed centrifuges and preparative ultracentrifuges. Analytical ultracentrifuges are designed to perform sedimentation analysis of macromolecules using the principles devised by Theodor Svedberg. Haematocrit centrifuges are used to measure the volume percentage of red blood cells in whole blood. Gas centrifuges, including Zippe-type centrifuges, for isotopic separations in the gas phase. Industrial centrifuges may otherwise be classified according to the type of separation of the high density fraction from the low density one. Generally, there are two types of centrifuges: the filtration and sedimentation centrifuges. For the filtration or the so-called screen centrifuge the drum is perforated and is inserted with a filter, for example a filter cloth, wire mesh or lot screen. The suspension flows through the filter and the drum with the perforated wall from the inside to the outside. In this way the solid material is restrained


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