Centrifugation basic principle & theory

Centrifugation is a process used to separate or concentrate materials suspended in a liquid medium. It is a method to separate molecules based on their sedimentation rate under the centrifugal field. It involves the use of centrifugal force for the sedimentation of molecules.

1. CENTRIFUGATION: Basic Principle & Theory By: Mayank Sagar

2. WHAT IS CENTRIFUGATION ? Centrifugation is a process used to separate or concentrate materials suspended in a liquid medium. It is a method to separate molecules based on their sedimentation rate under centrifugal field. It involves the use of the centrifugal force for the sedimentation of molecules. It is also used to measure physical properties such as molecular weight, density & shape of molecules.

3. For separation of one type of material from others For measurement of physical properties of macromolecules

4. PRINCIPLE OF CENTIFUGATION Particles suspended in a solution are pulled downward by Earth's gravitational force , In a solution, particles whose mass or density is higher than that of the solvent sink, and particles that are lighter than it float to the top. The greater the difference in mass or density, the faster they sink. This sedimentation movement is partially offset by the buoyant of the particle. Because the Earth’s gravitational field is weak , a solution containing particle of very small masses usually remain suspended due to the random thermal motion.

5. Gravitational force depends only on the mass and not on charge, shape and chemical composition. Forces much larger than Earth's gravitational force are required to cause appreciable sedimentation of such very small masses. Such forces can be obtained by subjecting particles to centrifugation. Sedimentation of these particles can be enhanced by applying centrifugal forces. A centrifuge does the same thing. It increases the sedimentation by generating centrifugal forces as great as 1,000,000 times the force of gravity.

6. CENTRIFUGAL FORCE A centrifugal force is a kind of force that is exerted on particle when they are subjected to circular motion. Lets consider a solution being spun at centrifuge. Where, m = Mass of particle ω = Angular velocity in radians per second r = Radius of a particle from the axis of rotation ω 2 r =Centrifugal acceleration

7. OTHER INTERACTING FORCES Other than, centrifugal force, there are some other interacting forces, namely buoyant and frictional force.

8. Buoyant force A buoyant force is an exerted force that causes movement of particles upwards and develops as a result of force applied to the particles by the fluid. The opposing buoyant force is smaller for a dense particle and can express as: Where ρ= Fluid density g = Gravitational or centrifugal acceleration V= Volume of fluid displaced So, Net force = centrifugal force – buoyant force

9. Frictional force A frictional force is a kind of force that restricts the movement of dispersed particles in the solution. The frictional coefficient is smaller for a minute particle than the dense particle. When particles move downward through the solution, the motion is also opposed by the frictional force. The frictional force is equal to the product of the frictional coefficient and the sedimentation velocity. It acts in the opposite direction to the net force: Where f= Frictional coefficient v= Sedimentation velocity

10. In a steady-state, the  frictional force  is equal to the  net force . fv= m ω 2 r – ω 2 rV ρ = m ω 2 r – ω 2 rmv ̅ ρ = m ω 2 r (1– v̅ ρ) The mass of 1mole of particles represented by, M= mN (Where N is the Avogadro’s number) fv= M ω 2 r (1– v̅ ρ) N

11. SEDIMENTATION COEFFICIENT The S edimentation coefficient (S) is defined as sedimetation rate per unit of centrifugal force. It is equals to the ratio of velocity to the gravitational acceleration. S= v/ ω2r =  M/ N. (1– v̅ ρ)/ f A sedimentation coefficient is measured by the units of a second . 1 S = 10 -13 second. This unit is named for The Svedberg, a pioneer in the field of centrifugation.

12. REALTIVE CENTRIFUGAL FORCE Also known as g- force. Relative centrifugal force is the force acting on samples during centrifugation. RCF is the ratio of the centrifugal acceleration at a specified radius and the speed to the standard acceleration of gravity. It is expressed as multiples of the earth's gravitational field ( g ). E.g , 500  g  = 500 RCF  It depends on the revolutions per minute (RPM) and radius of the rotor, and is relative to the force of Earth’s gravity. RCF = 11.2 × r (RPM/1000) 2   or RCF = 1.12 × 10 -5  (RPM) 2 RCF units are therefore dimensionless (denoting multiples of g).

13. FACTORS AFFECTING CENTRIFUGATION

14. FIXED- ANGLE ROTOR cross-sectional diagram of a fixed -angle rotor cross-sectional diagram of a centrifuge tube positioned in a fixed -angle rotor

15. VERTICAL- TUBE ROTOR cross-sectional diagram of a vertical tube rotor cross-sectional diagram of a centrifuge tube positioned in a vertical tube rotor

16. SWINGING- BUCKET ROTOR cross-sectional diagram of a swinging-bucket rotor cross-sectional diagram of a centrifuge tube positioned in a swinging-bucket rotor

17. FIXED-ANGLE ROTOR VERTICAL TUBE ROTOR SWINGING BUCKET ROTOR Tubes are held at angle of 14° to 40° to the vertical Held vertical parallel to rotor axis (7° – 10°) Sing out to horizontal position when rotor accelerate Particles moves radially outwards, travel a short distance. Particles move short distance Longer distance of travel, may allow better separation Useful for differential centrifugation Used in density gradient centrifugation Used in density gradient centrifugation Comparatively low resolution Intermediate Best resolution Take more separation time Very less time More time than fixed angle . Pellet is formed at outermost points of the tube along outer wall of the tube across diameter At the bottom of the tube RCF is intermediate Highest lower

18. CENTRIFUGES Centrifuge is primarily used to separate biological components based upon differential sedimentation properties. Centrifugation is a technique for the separation of the components of an analyte based on differences in the rate of migration under the influence of a centrifugal field. All centrifuges basically consist of a motor which spins a rotor containing the experimental sample. The most obvious differences between centrifuges are: speed at which biological specimens are subjected to increased sedimentation. presence or absence of a vacuum potential for refrigeration or general manipulation of the temperature during a centrifugation run maximum volume of samples and capacity for individual centrifugation tubes

19. TYPES OF CENTRIFUGES Based on the speeds at which the samples are centrifuged and the volumes of samples following are the types of centrifuge: Microfuge so called because they centrifuge small volume samples in Eppendorf tubes 0.5 to 1.5 cm 3 volumes. centrifugal fields of approximately 10000×g sediment biological samples in minutes can also be used to concentrate protein samples

20. Table/bench (/large preparative) top centrifuge : vary in design and are mainly used to collect small amounts of biological material, such as blood cells. To prevent denaturation of sensitive protein samples, refrigerated centrifuges should be employed. maximum centrifugal fields of 3000×g to 7000×g used for the spinning of various types of containers like (depending on the range of available adapters), Volume of 5 to 250 cm3 plastic tubes, 96-well ELISA simple and relatively inexpensive central place in many high-throughput biochemical assays, where the quick and efficient separation of coarse precipitates or whole cells is of importance.

21. High-speed refrigerated centrifuges for the sedimentation of protein precipitates, large intact organelles, cellular debris derived from tissue homogenisation and microorganisms. centrifugal fields of approximately 100 000×g. can be employed to differentially separate nuclei, mitochondria or chloroplasts , bulky protein aggregates. example separation of myosin and actin macromolecules filaments aggregates from muscle fibres lysate . Can be used to harvest yeast cells or bacteria from large volumes of culture media, in a continuous flow mode with zonal rotors. but not sufficient to sediment smaller microsomal vesicles or ribosomes .

22. Ultracentrifugation can be operated at relative centrifugal fields of up to 900 000×g -1 000 000 g the rotor chamber is sealed, evacuated and refrigerated to minimize excessive rotor temperatures generated by frictional resistance Sophisticated instrument though very expensive Consists of specialised optical system enabling the sedimenting material to be observed throughout the duration of a centrifuge run. detailed biochemical analysis of subcellular structures and isolated biomolecules can be studied. concentration distributions can be recorded at any time during ultracentrifugation information about the purity/heterogeneity, sedimentation coefficient distribution, average molar mass and molar mass distributions, and ligand interaction information can be obtained.

23. SAFETY ASPECTS OF CENTIFUGES Always close lid of centrifuge during operation. Always balance centrifuge. Do not open the lid while the rotor is moving Disinfect weekly and after all spills or breakages. Check tubes for cracks because they can break in centrifuge during operation. The work surface must be level and firm . Do not use the centrifuge on an uneven or slanted work surface.

24. ROTOR BALANCE The mass of a properly loaded rotor will be evenly distributed on the centrifuge drive hub, causing the rotor to turn smoothly with the drive An improperly loaded rotor will be unbalanced; consistent running of unbalanced rotors will reduce ultracentrifuge drive life. To balance the rotor load , fill all opposing tubes to the same level with liquid of same density. Weight of opposing tubes must be distributed equally. Place tubes in the rotor symmetrically.

25. REFERENCES Principles and techniques of Biochemistry and Molecular biology(7 th ed.) – Wilson and Walker Biophysical chemistry : principles and techniques- Avinash Upadhyay , Kakoli Upadhyay . The cell: a molecular approach (4 th ed.)- Geoffrey M.Cooper , Robert E. Hausman http://www.biologydiscussion.com/biochemistry/centrifugation/centrifuge-introduction-types-uses-and-other-details-with-diagram/12489 https://en.wikipedia.org/wiki/Centrifugation . Image courtesy- google images.