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Centrifugation-Basic study
- 1. Centrifugation •Centrifugation is a procedure that involves the use of centrifugal force for the sedimentation of used in industry and in laboratory settings. •More dense components of the mixture move away from the axis of the centrifuge while less dense components of the mixture move towards the axis
- 2. What is centrifuge?? • Centrifuge is device for separating particles from a solution according to there size, shape, density, viscosity of the medium. • Centrifuge uses centrifugal force to separate phases of different densities. • The centrifugal force is proportional to the rotation rate of the rotor. • The centrifuge consists of a rotor enclosed in a refrigerated chamber spun by an electric motor
- 3. Principle • Centrifugation is based on the fact that any object moving in a circle at a steady angular velocity is subjected to an outward directed force , F. • The magnitude of this force depends on the angular velocity in radians ,omega , and the radius of rotation ,r, in cm. F= ω2r
- 4. Classification 75000rpm 20000~25000rpm 3000rpm Ultra- centrifuge High speed centrifuge Desk top centrifuge
- 5. Desk top clinical centrifuges • Simplest • Least expensive • Maximum speed is below 3000rpm • Ambient temperature
- 6. High-speed centrifuges • Speeds of 20000 to 25000rpm • Equipped with refrigeration equipment Refrigerated high-speed centrifuge Continuous flow centrifuge High speed centrifuges
- 7. Continuous flow centrifuge • Relatively simple • High capacity • Separating mixed liquids^
- 9. Ultracentrifugation Preparative ultracentrifugation Analytical ultracentrifugation Differential centrifugation Density gradient centrifugation Rate zonal centrifugation Isopycnic centrifugation Types of centrifugation techniques
- 10. Ultracentrifugation • Svedberg coined the term “ultracentrifuge". He was colloid chemist. • He used the ultracentrifuge to determine the MW and subunit structure of hemoglobin , studies which changed the ideas concerning the structure of proteins. • The first commercial ultracentrifuge was produced in 1940 by SPINCO.
- 11. The ultracentrifuge • Attain the speed of 75000rpm • Isolate viruse DNA RNA protein
- 12. Analytical ultracentrifuge • In an analytical ultracentrifuge, a sample being spun can be monitored in real time through an optical detection system, using ultraviolet light absorption and/or interference optical refractive index sensitive system • This allows the operator to observe the sample concentration versus the axis of rotation profile as a result of the applied centrifugal field.
- 13. Preparative ultracentrifuge 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. Preparative rotors are used in biology for pelleting of fine particulate fractions, such as cellular organelles mitochondria, microsomes,ribosomes and viruses.
- 14. Density gradient centrifugation • It allow separation of many or all components in a mixture and allows for measurement to be made • There are two forms of Density gradient centrifugation : Rate zonal centrifugation Isopynic or sedimentation equilibrium centrifugation
- 15. Rate zonal centrifugation • In Rate zonal centrifugation the solution have a density gradient. The sample has a density i.e. greater than all the layers in the solution. • The sample is applied in a thin zone at the top of the centrifuge tube on a density gradient. Under centrifugal force, the particles will begin sedimenting through the gradient.
- 17. Rate zonal centrifugation • The particles will begin sedimenting in separate zones according to their size shape and density.
- 18. Photograph of an actual tissue separation: sub fractionation of a rat hepatic light mitochondrial fraction by rate zonal centrifugation using a vertical rotor.
- 19. Isopynic or sedimentation equilibrium centrifugation • In this type of centrifugation , the solution contains a greater range of densities. • The density gradient contains the whole range of densities of the particles in the sample. • Each particle will sediment only to the position in the centrifuge tube at which the gradient density is equal to its own density.
- 20. Isopynic or sedimentation equilibrium centrifugation • In Isopycnic centrifugation separation of particles occurs into zones on the basis of their density differences, independent of time.
- 22. Viruses (rotaviruses) purified by centrifugation through a caesium chloride density gradient. The original tube was 5cm long.
- 23. Differential centrifugation • Differential centrifugation is a common procedure in microbiology and cytology used to separate certain organelles from whole cells for further analysis of specific parts of cells. • In the process, a tissue sample is first homogenized to break the cell membranes and mix up the cell contents. • The homogenate is then subjected to repeated centrifugations, each time removing the pellet and increasing thecentrifugal force.
- 25. Rotors • Two types: angle rotor, swinging bucket rotor Angle rotor: Consist of a solid piece of metal with 6 to 12 holes At an angle between 20° and 45° Sedimenting particles have only short distance to travel before pelleting. Shorter run time. The most widely used rotor type.
- 26. Swinging bucket rotor: Hang three to six free moving buckets Longer distance of travel may allow better separation, such as in density gradient centrifugation. Easier to withdraw supernatant without disturbing pellet.