centrifugation and its type and techniques

This PowerPoint presentation provides a comprehensive overview of centrifugation, a fundamental technique used in laboratories for separating components based on density. The slides cover the basic principles of centrifugation, different types of centrifuges, operational parameters (e.g., RCF, RPM), and practical applications in biology, chemistry, and clinical diagnostics. Ideal for students, educators, and lab professionals, this presentation simplifies complex concepts with clear visuals and examples.

1. CENTRIFUGATIONAL Presented by : Sadik hussain M.Sc. 2 ND Sem Biotechnology

2. Contents Introduction Principle Centrifugal force Relative centrifugal force Type of centrifuge Part of centrifuge Type of rotors Type of centrifugation force Application Advantages Limitation

3. Introduction Biological centrifugation is a process that was centrifugal forces to separate and purify mixture of biological particle in a liquid medium. It is a key technique for isolating and analyzing the all-subcellular fractions, supramolecule complexes and isolated macromolecules such as proteins and nucleic acids. A centrifuge is a device for separating particles from an isolation according to their size, density, shape, viscosity of the medium and rotor speed.

4. Principle The centrifuge works on the principal of gravity of the generation of the centrifuge force to sediment different fractions. Rate of sedimentation depends on centrifugal field (G) being directed radially outwards. G depends on, Angular velocity Radial distance ( r in cm) particle from axis of rotation. G  

5. Centrifugal force Centrifugal force, word from Latin Centrum, meaning “Centre”, and fugere , means “ to flee”, is the apparent force that draws a rotating body away from the Centre of rotation. The term centrifugal force is used to refer to one of two distinct concepts: An inertial force observed in a non-inertial reference frame. A reaction force corresponding to a centripetal force. The concept of centrifugal force is applied in rotating devices such as centrifuges, centrifugal pumps. The two different forces are equal in magnitude, but centrifugal forces is opposite in direction to the centripetal force.

6. Relative Centrifugal Force(RCF) RCF, is the ratio of the centrifugal acceleration at a specified radius and the speed to the strand acceleration of gravity. Relative centrifugal force is defined as , F= Mw²r Where, F= intensity of centrifugal force M= mass of particle W= angular velocity of rotation R= distance of migrating particles from central axis of rotation

7. The RCF value for any centrifuge may be calculated from the following equation: RCF = 1.18* r * (rpm)2*10-5 Where, 1.18*10-5 = An empirical factor R is the radius in cm from the Centre of the centrifuge shaft to the external tip of centrifuge tube. Rpm is the number of revolution per minute of the centrifuge rotor.

8. The time required to separated particles depends on the rotor speed, radius of rotor and effective path length travelled by sedimented particles. The following formula useful for the calculation of the speed required for a rotor with radius that differ from the radius with which a prescribed, R.P.M = 1000* The length of time of centrifugation also can be calculated so, that running with an alternative rotor of a different size is equivalent to running with a original rotor.  

9. Types of Centrifuge depends on: Speeds of Sedimentation ( ultra centrifuge or high-speed centrifuge. Presence/ absence of vacuum ( ultra centrifuge or small bench top. Temperature control refrigeration. Volume of sample and capacity of centrifugation tubes. Depending on the particular application, centrifuge 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.

10. Low speed centrifuge High speed centrifuge Ultra centrifuge TYPE

11. Low Speed Centrifuge Operate at maximum speed of 4000-5000 rpm. There are few instances of temperature regulation and they are often operated at room temp. There centrifuge contain swinging bucket and fixed- angle rotor type

12. High speed centrifuge Refrigerated. Use for protein precipitates, large intact organelles cellular debris from tissue homogenization and microorganism. They operate maximal centrifugal force of approx. 50000 g. Use for research applications. Differential separation of nucleus, mitochondria, protein precipitate, etc.

13. Ultracentrifuge Speed range up top 75000 rpm providing centrifugal force. Include refrigeration system (0-4ºc). Rotor chamber is seated and evacuated by pumps to obtain vacuums. Rotor chamber is always enclosed in a heavy armor plate.

14. Micro centrifuge Small volume is small Eppendorf tubes Refrigerated with or without. Centrifuge maximum approx. 10000g. Take tube of small volume up to 2ml. Commonly used of concentration protein.

15. Dest top centrifuge Very simple and small. Maximum speed of 3,000 rpm. Do not have any temperature regulatory system. Used normally to collect rapidly sedimenting substances such as blood cells, yeast cell or bulky precipitates of chemical reaction.

16. Parts of centrifuge Motor Control Panel Chamber Rotors Sample Latch lid

17. Motor- The motor is the powerful central component of the centrifuge that creates the spin Control Panel:  It serves the purpose of controlling different parameters such as temperature, rotational speed ( rcf or rpm), etc. Latch:  When a tube breaks, or there are other issues with the centrifuge while running, the latch keeps the lid closed. Lid:  The centrifuge will only spin if the lid is closed and locked to prevent mishaps. Containers:  Several types of containers, such as test tubes, blood bags, cuvettes, centrifuge tubes, etc., are held in the rotors such that the sample rotates along as the rotor rotates.

18. Type of rotor Fixed angle rotor Vertical rotor Swinging bucket rotor

19. Fixed angle rotor Ideally tool for pelleting. Centrifugation tubes are held at fixed angle of between 14’-40’ to vertical axis of rotation. Start of centrifugation particles are driven outward horizontally but strike side of the tube so that sediment pack against the side & bottom of the tube, with the surface of the sediment parallel to the shaft of centrifuge.

20. Swinging bucket rotors: Sample tube are loaded into individual buckets that hand vertically while the rotor is at rest. When the rotor begins to rotate the buckets swing out to a horizontal position. Useful when sample are to be resolved in density gradients. The longer path length permits better separation of individual particle types from a mixture. This rotor is relatively inefficient for pelleting.

21. Types of centrifugation techniques There are two types of centrifugation techniques, 1 .preparatory centrifugation    2. A nalytical centrifugation . Preparatory centrifugation deals with the isolation and purification of components such as tissue, cells, subcellular structure, membrane vesicles, and other particles of biochemical interest. In contrast, analytical centrifugation is carried out to characterize purified biomolecules.

22. Preparative Centrifugation Based on suspension, preparative centrifugation is divided into two different types. They are: 1.Differential centrifugation 2.Density gradient centrifugation

23. Differential centrifugation It separates particles based on shape, size, and density. A suspension of particles with varying densities or sizes will sediment at varying speeds, with the larger and denser particles sedimenting more quickly. Following a series of rising centrifugal force cycles on a suspension of cells, a series of pellets containing cells with a decreasing sedimentation rate will result.

24. Density gradient centrifugation It separates particles based on their buoyant density or sedimentation rate. A sample mixture is placed on the top of a preformed liquid density gradients such as CsCl for DNA banding and isolation of plasmids, nucleoproteins, and viruses; NaBr and NaI for fractionation of lipoprotein; Per coll , Ficoll , Metrizamide, Dextran for separation of whole cells and sucrose solution for the separation of DNase, RNase and Protease

25. S ubtypes of density gradient centrifugation 1. R ate-zonal 2.I sopycnic centrifugation

26. Rate-zonal centrifugation On top of a density gradient, the sample is overlaid as a small zone. Depending on their mass, particles travel under centrifugal force at various speeds. Size and mass are the main determinants of how quickly particles settle. As the band of particles descends through the density medium, zones with particles of comparable size develop as the faster sedimenting particles pass the slower ones

27. Isopycnic centrifugation Particles are separated exclusively based on their density in an isopycnic separation, also known as buoyant or equilibrium separation. It is necessary for the gradient medium to have a higher density than the particles that need to be separated. Particles migrate under the influence of centrifugal force from a uniformly mixed sample and density gradient until their densities are equal to those of the surrounding medium. After centrifugation, particles of a certain density settle until their density equals that of the gradient media (i.e., the equilibrium position

28. Analytical Centrifugation It aims to collect information to characterize the spun sample (sedimentation velocity, viscosity, concentration, etc.), determine the relative molecular weight of the solutes, purity of biomolecules, detect conformational changes of protein structure, etc.

29. Application In laboratory , centrifugation is used to ; Remove cellular elements from blood to provide cell free plasma or serum for analysis. Remove chemically precipitated protein from an analytical specimen. Separated protein bound from free ligand in immunochemical and other assay. Separation of the subcellular organelle, DNA, RNA. Extract solutes in biological fluids from aqueous to organic solvents. Separate lipid components.

30. Advantages of Centrifuge Enclosed operation and consequently clean appearance Quick start-up and shutdown Easy automation and continuous operation if necessary Low capital cost-to-capacity ratio Quick adjustment of operating parameters High flexibility and outstanding performance Simple operation and easy installation

31. Limitations of Centrifuge Separating light particles (with almost negligible mass) is exceedingly difficult.  A centrifuge, despite its kind, is a very sophisticated electrical device that requires specialist servicing in times of malfunction, making device maintenance extremely difficult and expensive. High energy usage because it has so many power-consuming features such as temperature control and rotor spinning. Due to their high rotation rates per minute, centrifuges produce noise disruptions. Since it uses rotations, vibrations are unavoidable, which will cause noise pollution and prevent its use in some areas. Most often, a backup machine is required to step in when the primary equipment breaks down.  Extensive use of polymers

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