Contents 1 Mathematical formula 2 Centrifugation in biological research 2.1 Microcentrifuges 2.2 High-speed centrifuges 2.3 Fractionation process 2.4 Ultracentrifugations 2.5 Density Gradient Centrifugation 2.6 Differential Centrifugation 3 Other applications 4 History 5 See also 6 Sources 7 References


Mathematical formula[edit] The general formula for calculating the revolutions per minute (RPM) of a centrifuge is R P M = g r {\displaystyle RPM={\sqrt {g \over r}}} , where g represents the respective force of the centrifuge and r the radius from the center of the rotor to a point in the sample.[4] However, depending on the centrifuge model used, the respective angle of the rotor and the radius may vary, thus the formula gets modified. For example, the Sorvall #SS-34 rotor has a maximum radius of 10.8 cm, so the formula becomes R P M = 299 g r {\displaystyle RPM=299{\sqrt {g \over r}}} , which can further simplify to R P M = 91 g {\displaystyle RPM=91{\sqrt {g}}} .[5] the applied centrifugation field is square angular velocity in radians per sec .the radial distance o the particle from the axis; The most common formula used for calculculating Relative Centrifugal Force (x g) is: RCF (x g) = 1.118 x Radius (mm) x (rpm/1000)² Many clinical separations have historically been carried out at 3000 rpm. This is a somewhat arbitrary approach as the RCF applied is dependent upon the radius in a linear fashion - so a 10% larger radius means that a 10% higher RCF is applied at the same speed. To discover the RCF that you have been applying at 3000 rpm, the above formula can be simplified to: [Short cut for 3000 rpm, with only a 0.62% error:] RCF (x g) = 10 x Radius (mm)


Centrifugation in biological research[edit] Microcentrifuges[edit] Microcentrifuges are used to process small volumes of biological molecules, cells, or nuclei. Microcentrifuge tubes generally hold 0.5 - 2.0 mL of liquid, and are spun at maximum angular speeds of 12,000–13,000 rpm. Microcentrifuges are small enough to fit on a table-top and have rotors that can quickly change speeds. They may or may not have a refrigeration function. High-speed centrifuges[edit] High-speed or superspeed centrifuges can handle larger sample volumes, from a few tens of millilitres to several litres. Additionally, larger centrifuges can also reach higher angular velocities (around 30,000 rpm). The rotors may come with different adapters to hold various sizes of test tubes, bottles, or microtiter plates. Fractionation process[edit] General method of fractionation: Cell sample is stored in a suspension which is: Buffered - neutral pH, preventing damage to the structure of proteins including enzymes (which could affect ionic bonds) Isotonic (of equal water potential) - this prevents water gain or loss by the organelles Cool - reducing the overall activity of enzyme released later in the procedure Cells are homogenised in a blender and filtered to remove debris The homogenised 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[edit] 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[edit] 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.[6] 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.[7] 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.[6] 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.[7] 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.[6] Differential Centrifugation[edit] 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.[8]


Other applications[edit] 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.[9]


History[edit] By 1923 Theodor Svedberg and his student H. Rinde had successfully analyzed large-grained sols in terms of their gravitational sedimentation.[10] Sols consist of a substance evenly distributed in another substance, also known as a colloid.[11] However, smaller grained sols, such as those containing gold, could not be analyzed.[10] To investigate this problem Svedberg developed an analytical centrifuge, equipped with a photographic absorption system, which would exert a much greater centrifugal effect.[10] In addition, he developed the theory necessary to measure molecular weight.[11] During this time, Svedberg’s attention shifted from gold to proteins.[10] 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.[12] 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).[13] 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.[10][11] 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.[12] 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.[10] The same molecular mass was determined, and the presence of a spreading boundary suggested that it was a single compact particle.[10] Further application of centrifugation showed that under different conditions the large homogeneous particles could be broken down into discrete subunits.[10] The development of centrifugation was a great advance in experimental protein science.


See also[edit] Centrifuge


Sources[edit] Harrison, Roger G., Todd, Paul, Rudge, Scott R., Petrides D.P. Bioseparations Science and Engineering. Oxford University Press, 2003. Dishon, M., Weiss, G.H., Yphantis, D.A. Numerical Solutions of the Lamm Equation. I. Numerical Procedure. Biopolymers, Vol. 4, 1966. pp. 449–455. Cao, W., Demeler B. Modeling Analytical Ultracentrifugation Experiments with an Adaptive Space-Time Finite Element Solution for Multicomponent Reacting Systems. Biophysical Journal, Vol. 95, 2008. pp. 54–65. Cole, J.L., Hansen, J.C. Analytical Ultracentrifugation as a Contemporary Biomolecular Research Tool. Methods and Reviews, 1999/2000. Howlett, G.J., Minton, A.P., Rivas, G. Analytical Ultracentrifugation for the Study of Protein Association and Assembly. Current Opinion in Chemical Biology, Vol. 10, 2006. pp. 430–436. Dam, J., Velikovsky, C.A., Mariuzza R.A., et al. Sedimentation Velocity Analysis of Heterogeneous Protein-Protein Interactions: Lamm Equation Modeling and Sedimentation Coefficient Distributions c(s). Biophysical Journal, Vol. 89, 2005. pp. 619–634. Berkowitz, S.A., Philo, J.S. Monitoring the Homogeneity of Adenovirus Preparations (a Gene Therapy Delivery System) Using Analytical Ultracentrifugation. Analytical Biochemistry, Vol. 362, 2007. pp. 16–37.


References[edit] ^ Garrett, Reginald H.; Grisham, Charles M. (2013). Biochemistry (5th ed.). Belmont, CA: Brooks/Cole, Cengage Learning. p. 111. ISBN 9781133106296.  ^ Frei, Mark. "Centrifugation Basics". Sigma-Aldrich. Retrieved 10 May 2016.  ^ Article on "Centrifugation" retrieved on 15 October 2013 from http://www.lenntech.com/library/clarification/clarification/centrifugation.htm ^ Ballou, David P.; Benore, Marilee; Ninfa, Alexander J. (2008). Fundamental laboratory approaches for biochemistry and biotechnology (2nd ed.). Hoboken, N.J.: Wiley. p. 43. ISBN 9780470087664.  ^ Ballou, David P.; Benore, Marilee; Ninfa, Alexander J. (2008). Fundamental laboratory approaches for biochemistry and biotechnology (2nd ed.). Hoboken, N.J.: Wiley. p. 235. ISBN 9780470087664.  ^ a b c Brakke, Myron K. (April 1951). "Density Gradient Centrifugation: A New Separation Technique". J. Am. Che. Soc. 73 (4): 1847–1848. doi:10.1021/ja01148a508.  ^ a b Oster, Gerald; Yamamoto, Masahide (June 1963). "Density Gradient Techniques". Chem. Rev. 63 (3): 257–268. doi:10.1021/cr60223a003.  ^ Ballou, David P.; Benore, Marilee; Ninfa, Alexander J. (2010). Fundamental Laboratory Approaches for Biochemistry and Biotechnology (seconde ed.). University of Michigan: John Wiley & Sons, Inc. p. 213. ISBN 978-0-470-08766-4.  ^ Ballou, David P.; Benore, Marilee; Ninfa, Alexander J. (2008). Fundamental laboratory approaches for biochemistry and biotechnology (2nd ed.). Hoboken, N.J.: Wiley. pp. 238–239. ISBN 9780470087664.  ^ a b c d e f g h Van Holde, K. E. (1998). Analytical ultracentrifugation from 1924 to the present: A remarkable history. Chemtracts – Biochemistry and Molecular Biology. 11:933-943 ^ a b c Svedberg, T. (1927). The Ultracentrifuge Nobel Lecture ^ a b Tanford, C., and Reynolds, J. 2001. Nature’s robots: A history of proteins. Oxford University Press. pp. 303-305 ^ Simoni, D. S., Hill, R. L., and Vaughan, M. (2002). The structure and function of hemoglobin: Gilbery Smithson Adair and the Adair equations. The Journal of Biological Chemistry. 277(31): e1-e2 v t e Separation processes Processes Absorption Acid-base extraction Adsorption Chromatography Cross-flow filtration Crystallization Cyclonic separation Dialysis (biochemistry) Dissolved air flotation Distillation Drying Electrochromatography Electrofiltration Filtration Flocculation Froth flotation Gravity separation Leaching Liquid–liquid extraction Electroextraction Microfiltration Osmosis Precipitation (chemistry) Recrystallization Reverse osmosis Sedimentation Solid phase extraction Sublimation Ultrafiltration Devices API oil-water separator Belt filter Centrifuge Depth filter Electrostatic precipitator Evaporator Filter press Fractionating column Leachate Mixer-settler Protein skimmer Rapid sand filter Rotary vacuum-drum filter Scrubber Spinning cone Still Sublimation apparatus Vacuum ceramic filter Multiphase systems Aqueous two-phase system Azeotrope Eutectic Concepts Unit operation Retrieved from "https://en.wikipedia.org/w/index.php?title=Centrifugation&oldid=820332602" Categories: CentrifugationHidden categories: Use dmy dates from November 2017Articles lacking in-text citations from November 2010All articles lacking in-text citationsAll articles with unsourced statementsArticles with unsourced statements from December 2010


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