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# centrifugation.pptx

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# centrifugation.pptx

Centrifugation

Centrifugation

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### centrifugation.pptx

1. 1. Centrifugation
2. 2. WHAT IS CENTRIFUGE? Centrifuge is device for separating particles from a solution according to there size, shape, density, viscosity of the medium. WHAT IS CENTRIFUGATION? Centrifuge uses centrifugal force to separate phases of different densities.
3. 3. Centrifugation is a procedure that involves the use of centrifugal force for the sedimentation of mixture with a centrifuge 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.
4. 4. A particle whether it is a precipitate a macromolecule or a cell organelle is subjected to a centrifugal force when it is rotated at a high rate of speed. The centrifugal force F is denoted by equation F=mω2 r Where F= intensity of the centrifugal force m= effective mass of the sedimenting particle ω= angular velocity of rotation r= distance of the migrating particles from the central axis of rotation A more common measurement of F in terms of the earths gravitation force , g, is relative centrifugal force, RCF its defined by RCF = 1.119 x 10-5 (rpm)2 (r) This equation relates RCF to revolutions per minute of the sample . Equation dictates that the RCF on a sample will vary with r, the distance of the sedimenting particles from the axis of rotation . The RCF value is reported as “ a number times gravity ,g .”
5. 5. On and off control, operation time control (timer), rotation speed control, temperature control (in refrigerated centrifuges), vibration control (safety mechanism) and brake system Refrigeration system (in refrigerated centrifuges). Base Lid/cover Casing Electric motor Rotor. There are different types of rotors. The most common are the fixed angle, the swinging buckets, the vertical tube and the almost vertical tube types.
6. 6. 1. Low –speed centrifuges . Its also called as clinical centrifuge. The common centrifuge has a maximum speed in the range of 4000 to 5000 rpm with RCF value approx. up to 3000Xg. These instruments usually operate at low temperature with no means of temperature control of the samples. Two types of rotors fixed angle and swinging bucket may be used in this instruments.
7. 7. a)Preparative 1. Differential centrifugation 2. Density gradient centrifugation 3. Rate-zonal density gradient centrifugation. b) Analytical 4. Isopycnic centrifugation
8. 8. based on the size of the particles used for simple pelleting, for the separation of subcellular organelles and macromolecules first, sample must be homogenised ultra centrifugation sedimentation depends on mass, shape and partial specific volume of a macromolecule, as well as solvent density, rotor size, rate of rotation. Usually uses a fixed angle rotor
9. 9. method to purify subcellular organelles and macromolecules. Density gradients generated by placing layer after layer of gradient media Particles are placed on top of gradient and centrifuge . Particles travel through gradient until density matches with sucrose. Now fraction is removed and analyzed.
10. 10. use of continuous density gradient of solvent such as sucrose. density increases towards the bottom of the tube sample layered on the top molecules form discrete bands after centrifugation separation based on size of the molecules Swinging bucket rotors 2.Rate- zonal Density Gradient centrifugation
11. 11. based on the density of the molecules Mix gradient material with the sample molecule (CsCl) molecules move to the position where their density is same as the gradient material (isopycnic position) inorder to generate a gradient, we select a CsCl concentration that will give a range of densities that includes the range of molecules that have to be separated. used for the separation of DNA Swinging bucket or fixed angle rotor 3. Isopycnic Centrifugation
12. 12. USES SMALL SIZE SAMPLES BUILT-IN OPTICAL SYSTEM USES RELATIVELY PURE SAMPLE
13. 13. Types centrifuges depends upon Maximum speed of sedimentation Presence /absence of vacuum Temperature control refrigeration. Volume of sample and capacity of centrifugation tubes Depending on the particular application, centrifuges differ in their overall design and size. a common feature in all centrifuges is the central motor that spins a rotor containing the samples to be separated
14. 14. With or without refrigeration Slow speed (eg up to 4000 RPM) Common in clinical lab (blood/plasma/serum separation) can take approx (up to) 100 tubes, depending on diameter
15. 15. (“MICROFUGE”, “EPPENDORF”) Sample volume is small in tubes Refrigerated with or without can generate forces up to ~15,000 x g Take tube of small volume up to 2ml. Commonly used of concentration protein very common in biochemistry/molecular biology/biological labs
16. 16. Refrigerated use for protein precipitates, large intact organelles cellular debris from tissue homogenisation and micoorganism They operate maximal centrifugal force of approx 10000g Use for research applications differntial sepration of nucleus, mitrochondrial, protein precipitate,etc. 15000-20000 RPM
17. 17. Refrigerated and evacuated The detail biochmestry analysis of subcellular structures and isolate biomolecules. Operate at upto 90000 g limited lifetime expensive require special rotors care in use – balance critical! research applications
18. 18. FIXED ANGLE SWINGING BUCKET ROTORS (VERTICAL/ HORIZONTAL)
19. 19. THE ROTOR (MAINLY MADE OF ALUMINIUM) IS VERY COMPACT. THERE ARE BOREHOLES WITH A SPECIFIC ANGLE (LIKE 45°) WITHIN THE ROTOR. THESE BOREHOLES ARE USED FOR THE SAMPLE TUBES.
20. 20. a. Vertical rotors Vertical rotors Sample tubes are held in vertical position during rotation This type of rotor is not suitable for pelleting applications but is most efficient for isopycnic (density) separations due to the short pathlength . Applications include plasmid DNA, RNA, and lipoprotein isolations. b. horizontal rotors The rotor looks like a cross with bucket. Within these buckets, different tubes can be centrifuged. For a safe centrifugation, a specific adadpter for every tube shape is mandatory.
21. 21. Centrifugal field from the center of rotation may be calculated using the equation G=ω2 r Angular velocity ω may be calculated by using this equation ω= 2 π rev min-1 60
22. 22. Lubricate and clean motor. Clean case. Inspect power cords and plugs. Inspect controls and switches. Ensure appropriate menu settings for proper use. Ensure tightness of rotor. Check lights and indicators. Verify that alarms are operating properly. Ensure safety switch is functioning. If refrigerated, ensure temperature reading is working. Replace/repair gaskets, seals, and vacuum pump (if applicable).
23. 23. Application s • To separate two miscicible substances • To analyze hydrodynamic properties of macrmolecules . • Purification of mammalian cell. • Fractionation of subcellular organelles and membrane vesical. • Separation of chalk from water. • To separate fat from milk . • Particulate matter from air flow using cyclonic precipitator.
24. 24. Thankyou
25. 25. Thankyo u