Centrifugation

Centrifugal force, types of centrifuges, applications etc.

1. CENTRIFUGATION SUBMITTED BY- 1.Marzeea Ahmad Raka 2.Mir Tasnia Noshin 3.Suraiya Ahmed 4.Isfat Hasan Tomal 5.Nusrat Alam Mou 6.Atikur Rahman Nyeem

2. CONTENTS Definition of centrifugation History Principle of centrifugation Classification Operation Construction Application in Pharmaceutical industry Advantages & disadvantages Safety measures importance Summary

3. WHAT IS CENTRIFUGATION? Centrifugation is one of the most important and widely applied research cellular techniques in bio-chemistry and molecular biology, pharmacy and in medicine . Centrifugation is a process which involves the use of the centrifugal force for the sedimentation of heterogeneous mixtures with a  centrifuge. A centrifuge is a device that spins quickly to press objects outward with centrifugal force . Centrifugal force is an apparent force that acts outward on a body moving around a center, arising from the body's inertia.

4. CENTRIFUGATION A centrifuge is used to separate particles or even macro-molecules: Cells Subcellular components Proteins Nucleic acid Basis of separation- Size Shape Density

5. HISTORY English military engineer Benjamin Robins (1707–1751) 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. The potential of the centrifuge in the laboratory setting was first exploited by Friedrich Miescher. In 1869, Miescher used a crude centrifuge system to isolate a cell organelle. This process led to the discovery of an important new class of biological constituents, later to be known as nucleic acids. The work of Miescher was quickly recognized and developed further by others. In 1879, the first continuous centrifugal separator was demonstrated by Gustaf de Laval. This development made the commercialization of the centrifuge a possibility for the first time.

6. PRINCIPLE OF CENTRIFUGATION Methodology- Utilizes density difference between the particles and the medium in which these are dispersed Dispersed systems are subjected to artificially induced gravitational fields. The centrifugal force causes the sedimentation of heavier solid particles.

7. PRINCIPLE OF CENTRIFUGATION A particle is subjected to centrifugal force when it is rotated at high rate of speed. The centrifugal force, F is defined by the equation- F= m ω 2 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

8. PRINCIPLE OF CENTRIFUGATION 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 .”

9. CLASSIFICATION OF CENTRIFUGATION

10. TYPES OF CENTRIFUGATION Depend on the basis of : -speed -temperature

11. CONTINUED……. There are various types of centrifugation : 1.Differential Centrifugation 2. Isopycnic Centrifugation 3. Sucrose Gradient Centrifugation 4. Ultracentrifuges

12. 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 Undergoes tissue disruption and cell lysis. Finally centrifugation

13. ISOPYCNIC CENTRIFUGATION In isopycnic separation, also called equilibrium separation particles are separated solely on the basis of their density. Particle size only affects the rate at which particles move until their density is the same as the surrounding gradient medium Particles separate out according to their buoyancy. used to isolate nucleic acids such as DNA

14. SUCROSE GRADIENTCENTRIFUGATION A homogenate is placed on top of a special medium e.g. sucrose solution that progressively increases in concentration density also when this sucrose gradient is centrifuged at high speed each particle in the homogenate will move down in the tube and will come to rest at the point in the gradient where a density equals that of the sucrose solution. often used to purify enveloped viruses (with densities 1.1-1.2 g/cm³), ribosomes, membranes and cell organelles from crude cellular extract.

15. ULTRACENTRIFUGE The ultracentrifuge is a centrifuge optimized for spinning a rotor at very high speeds, capable of generating acceleration as high  19 600 km/s². There are two kinds of ultracentrifuges: 1.the preparative and 2. the analytical ultracentrifuge. Both classes of instruments find important uses in molecular biology , biochemistry, and polymer science

16. LABORATORY CENTRIFUGES There are different types of laboratory centrifuges: Micro Centrifuges Clinical Centrifuges Multipurpose High-Speed Centrifuges

17. INDUSTRIAL CENTRIFUGATION Definition: An industrial centrifuge is a machine used for fluid/particle separation. Types: Industrial centrifuges can be classified into 3 main types: (1)Filtration centrifuge: Those using perforated baskets, which perform a filtration-type operation (work like a spin-dryer). E.g. perforated basket centrifuge. (2) Sedimentation centrifuge: Those with a solid walled vessel, where particles sediment towards the wall under the influence of the centrifugal force. E.g. Tubular bowl centrifuge (3) Continuous centrifuge: Those where a continuous process or very high capacity is required.

18. 1. Perforated-basket centrifuges (centrifugal filters)

19. Fig. Perforated basket centrifuge

20. 2. Tubular-bowl centrifuges (centrifugal sedimenters)

21. Fig: Tubular-bowl centrifuge

22. 3. Continuous centrifuge

23. It is fitted with a conical basket to allow continuous feeding of a slurry and discharge of the separated solids. Continuous operations are best suited for applications that require high capacity and large (greater than 300 micron) particle size. These centrifuges can provide excellent washing and low final moisture.

24. Fabrication: Carbon fiber, Aluminum and Titanium.

25. Fixed Angle Rotor Fixed-angle rotors are the most ubiquitous rotors used in centrifugation. Angle: varies in 20 – 45⁰. Speeds range: 0 - 1,000,000 × g The cavities range: In volume from 0.2 mL to 1 L. Factors determine the type: Desired g-force (RCF) Desired volume Mechanism: The rotor begins to rotate the solution in the tubes reorients. This reorientation cause centrifugation. Applications: Pellet separation Discard the. Excess debris Isopycnic separation of macromolecules Fig: Fixed angle rotor

26. SWING BUCKET ROTOR Ideal for separating large-volume samples up to 12 L at low speeds. Consists of three parts: (1)The rotor body attaches to the centrifuge drive and has four or six arms to support the buckets 2) The buckets are placed onto the arms of the rotor body 3) Pins are used to hold the buckets in place. Fig: Swing bucket rotor

27. MECHANISM Sample tubes are loaded into individual buckets that hang vertically while the rotor is at rest. When the rotor begins to rotate the buckets swing out to a horizontal position. The longer path length permits better separation of individual particle types from a mixture. However, this rotor is relatively inefficient for pelleting.

28. VERTICAL ROTOR Tubes are held in vertical position during rotation. Use: Ultracentrifugation for iso-pycnic separations, specifically for the banding of DNA in cesium chloride. Mechanism: In this type of separation, the density range of the solution contains the same density as the particle of interest; thus the particles will orient within this portion of the gradient. The particles to orient at the proper position within the gradient travel a short distance to pellet; therefore run time is minimized. Application: Include plasmid DNA, RNA, and lipoprotein isolations.

29. ROTOR CARE Proper rotor care is essential for ensuring safety and longevity. In addition to reducing the risk of accidents, regular rotor maintenance can save time and money and greatly extend rotor life span. All the rotors must be protected by rinse, disinfectant and must be sterilize.

30. Applications in Industry There are techniques on which the application of centrifuge is based on- preparative technique-

31. Application in Water Treatment

32. Pharmaceutical Industrial Application Production of bulk drugs: aspirin is separated from its mother liquor by centrifugation. Biopharmaceutical analysis of drugs: pharmacokinetic parameters and bioequivalence studies. Determination of molecular weight of colloids: Polymers , proteins and such macromolecules often form colloidal dispersions. The molecular weights of those molecules can be determined by ultracentrifugation .

33. Pharmaceutical Industrial Application Evaluation of suspensions and emulsions: Centrifugation method is used as a rapid empirical test parameter for the evaluation of suspension and emulsion. Production of biological products: a) Separation of blood cells. b) Purification of insulin by selectively precipitating other fraction of proteins. c) Separation of most of the proteinaceous drugs and macromolecules.

34. Some Other Commercial applications Large industrial centrifuges are commonly used in water and wastewater treatment to dry sludge. In sugar industry to separate the sugar crystals from the mother liquor . Standalone centrifuges for drying (hand-washed) clothes – usually with a water outlet. Large industrial centrifuges are also used in the oil industry to remove solids from the drilling fluid. In soil mechanics. Centrifuges are used in the attraction Mission

35. ADVANTAGES AND DISADVANTAGES: Advantages: Centrifuges have a clean appearance and have little to no odor problems. Not only is the device easy to install and fast at starting up and shutting down, but also only requires a small area for operation. They can be selected for different applications. . The device is simple to operate . Centrifuge has more process flexibility and higher levels of performance.

36. DISADVANTAGES : The machine can be very noisy and can cause vibration. The device has a high-energy consumption due to high G-forces . High initial capital costs

37. SAFETY MEASURES A centrifuge user should strictly observe the following precautions : Manufacturer’s manual should be strictly followed. Rotor should be stored in proper containers. Attention should be given to imbalance detectors. Rotor speed should not exceed the assigned speed. Lid of the rotor chamber should remain locked during operation. To avoid the rotor failure, manufactures instructions regarding rotor care and use should always be followed.

38. SUMMARY The centrifugation is a modern & easy technique of separation and sedimentation on the basis of shape, size and density of macromolecules and other particles. In the centrifugation there are different types of forces are applied like as centrifugal force, gravitational force and centripetal force etc. and also different types of rotors are to be used that is; Swinging Bucket Rotor and fixed angle rotors at different RPM/RCF.

39. IMPORTANCE Centrifugation play an important role in both biological sectors as well as industrial sectors. In biological sector: Medical laboratories use centrifuges to separate plasma from heavier blood cells . Modern centrifuges can even separate mixtures of different sized molecules or microscopic particles such as parts of cells.

40. Continued….. In Industrial Sector: Cream separation from milk. Separating textiles Separating particles from an air flow using cyclonic separation Clarification and stabilization of wine Centrifugation is the most common method used for uranium enrichment.

41. REFERENCES http://www.labmanager.com/lab-product/2010/05/evolution-of-the-lab-centrifuge?fw1pk=2#. Viz08yskqxn http:// www.westernstates.com/continuous-centrifuges https:// en.wikipedia.org/wiki/Centrifuge https:// en.wikipedia.org/wiki/Differential_centrifugation http:// cellbiologyolm.stevegallik.org/node/74 D. Rickwood and J. M. Graham (2001); Biological Centrifugation, Springer Verlag ; ISBN: 0387915761. D. Rickwood, T. Ford, J. Steensgaard (1994): John Wiley & Son Ltd. ISBN: 0471942715. T. C. Ford and J. M. Graham (1991): An Introduction to Centrifugation, BIOS Scientific Publishers, Ltd. ISBN 1 872748 40 6. http://www.thermoscientific.com/content/dam/tfs/LPG/LED/LED%20Documents/Third-Party%20Papers/Centrifuges/Centrifuge%20Rotors/Ultracentrifuge%20Rotors/D20940 ~. pdf