centrifugation and its applications.pptx

The presentation deals with physics, different factors affecting sedimentation, types of centrifuges and their application

1. CENTRIFUGATION Presenter : D evlina Sengupta . Kanchrapara College , M icrobiology Depatment

2. Contents: Basic principle T y pes Application O peration

3. Definition: Biological centrifugation is a process that uses centrifugal forces to separate and purify mixture of biological particle in a liquid medium. It is key technique for isolating and analysing the cells, subcellular fractions, supramolecule complexes and isolated macromolecules such as proteins and nucleic acids.

4. History: The first analytical ultracentrifuge was developed by Svedberg in 1920

5. Basic principle The basic physics on which the centrifuge works is gravity and generation of the centrifugal force to sediment different fractions. Rate of sedimentation depends on ------ applied centrifugal field (G) being directed radially outwards G depends on Angular velocity (ω in radians / sec) . Radial distance (r in cms)of particle from axis of rotation G = ω 2 r

6. Rate of sedimentation Depends on factors other than CF Mass of particle ---Density & Volume Density of medium Shape of particle Friction S = m( 1- ṽ ρ ) f Where S= Sedimentation Coefficient ( given in vedberg unit) m = mass of particle ṽ = Partial Specific Volume (1/ ρ molecule ) ρ = Density of medium F = Frictional Coefficient of particle

7. How different factors influence the movement of a particle? Mass Of Particle: Greater the mass, faster the particle travels down. Eg if both the particle A & B have equal densities & shape, but M B > M A , so particle B travels fatser than A. Shape of Particle: More Spherical the shape, less frictional coefficient and hence faster sedimentation. The more flattened the shape, more frictional coefficient and hence slower sedimentation. Eg if both particles A & B have M B =M A but M B has greater spherical shape than M A , then particle B will face less frictional coefficient and hence travel faster than A. Partial Specific Volume : Partial specific volume is inverse of density of particle. So, greater the density of particle, lesser is the partial specific volume and greater Sedimentation coefficient value. (As in the sedimentation coefficient equation if the right side ṽ ρ becomes large, S value becomes – ve . So, the particle would not travel downward or rather would float the above. Density of fluid: If the density of the fluid (medium) is greater than the density of the molecule, then ṽ ρ > 1, S becomes – ve and particle floats. If the density of the fluid (medium) is lesser than the density of the molecule, then ṽ ρ < 1, S becomes highly + ve and particle sediments. If density of fluid equals density of molecule, then ṽ ρ =1. So, particle does not move.

8. Movement of particle based on density of fluid & particle If ρ medium > ρ molecule, ṽ ρ > 1 Particle floats; S = - ve If ρ medium < ρ molecule, ṽ ρ > 1 Particle sediments; S = + ve If ρ medium = ρ molecule, ṽ ρ > 1 Particle does not move ; S = 0

9. Sedimentation time Depends on , Size of particle Density difference b/w particle and medium Radial distance from the axis of rotation to liquid meniscus (rt) Radial distance from the axis of rotation to the bottom of the tube (rb)

10. T HE FACTORS ON WHICH THESE WORKS ARE More dense a biological structure, faster it sediments in centrifugal force. More massive biological particle, faster it moves in centrifugal field. Dense the buffer system, slower particle moves. Greater the frictional coefficient, slower a particle will move Greater the centrifugal force, faster particle sediments Sedimentation rate of a given particle will be zero when density of particle and the surrounding medium are equal.

11. Common feature of all centrifuges is the central motor that spins a rotor containing the samples to be separated . Rotor shaft placed centrally along which the rotor is attached A metal rotor with holes in it to accommodate a vessel of liquid. Additionally some centrifuges are accommodated with refrigeration & evacuation pump system (to reduce heat generated as a result of friction), optical system (in analytical centrifuges to monitor progress of centrifugation).

12. Types of centrifuge Desk top C entrifuge s 3000 rpm High speed C entrifuge s 25,000 rpm Ultracentrifuges 75,000 rpm Ana l ytical Prep a rati v e

13. Types of rotor V e r t ic al tube rotors Swinging- bucket rotors Fixed angle ro t ors

14. c e ntrifugation Re o rie n ta t ion of the tu b e occurs during acceleration and deceleration of the rotor. Particles move radially Particles move short outwards, travel a sh o rt distance. Time of separation is shorter. Disadvantage: pellet may fall back into solution at end of centrifugation. Fixed angle rotors Tubes are held at angle of 14 to 40 to the vertical. distance. Useful for differential Vertical tube rotors Held vertical parallel to rotor axis. Types of rotor

15. Swinging-bucket rotors S w ing out to horizontal position when rotor accelerates. Longer distance of travel may allow better separation, such as in density gradient centrifugation. Easier to withdraw supernatant without disturbing pellet. Normally used for density-gradient centrifugation. Types of rotor

16. Wall Effect When performing centrifugation in a fixed angle rotor. The particle does not travel in a straight line towards the bottom of the tube. Rather it moves outward within the tube till it hits its walls and then slides down this wall to be pelleted at the bottom. This is called Wall effect. This results in improper resolution of particles in case they vary very little in their sedimentation characteristics.

17. Separation

18. Small microfuges work with speed- 8000- 13000 rpm & RCF 10000g for rapid sedimentation of small volumes (1-2 min) Eg : Blood , Synaptosomes ( effect of drugs on biogenic amines) 2

19. Desk top centrifuge Very simple and small. Maximum speed of 3000rpm Do not have any temperature regulatory system. Used normally to collect rapidly sedimenting substances such as blood cells, yeast cells or bulky precipitates of chemical reactions.

20. High speed centrifuges Maximum speed of 25000rpm, providing 90000g centrifugal forces. Equipped with refrigeration to remove heat generated. Temperature maintained at 0-4 C by means of thermocouple. Used to collect microorganism, cell debris, cells, large cellular organelles, precipitates of chemical reactions. Also useful in isolating the sub- cellular organelles(nuclei, mitochondria, lysosomes ) .

21. Ultracen t rifuges at speed of 75,000rpm, t he c en t rif u gal fo r ce of Operate providing 500 , 000g. Rotor chamber is sealed and evacuated by pump to attain vacuum. Refrigeration system (temp 0-4 C). Rotor chamber is always enclosed in a heavy armor plate . Cen t rifugati o n for i s o lation a nd purification of components is known as ca r ried out with a de s i r e pr e p a ra t ory cen t rifu g at i o n , while th a t f or c h a ract e rizat i on i s known as a naly t ical centrifugation .

22. Preparative centrifugation Is concerned with the actual isolation of biological material for subsequent biochemical investigations. Divided into two main techniques depending on suspension medium in which separation occur. Homogenous medium – differential centrifugation Density gradient medium – density gradient centrifugation

23. 1. Differential centrifugation Separation is achieved based in the size of particles in differential centrifugation. Commonly used in simple pelleting and obtaining the partially pure separation of subcellular organelles and macromolecules. Used for study of subcellular organelle, tissues or cells (first disrupted to study internal content )

24. During centrifugation, larger particles sediment faster than the smaller ones. At a series of progressive higher g-force generate partially purified organelles.

25. Inspite of its reduced yield differential centrifugation remains probably the most commonly used method for isolation of intracellular organelle from tissue homogenates because of its ; relative ease Convenience Time economy Drawback is its poor yield due to repeated washing for obtaining pure pellet and fact that preparation obtained is never pure.

26. 2. Density gradient centrifugation It is the preferred method to purify subcellular organelles and macromolecules. Density gradient can be generated by placing layer after layer of gradient media such as sucrose , Cscl , ficol in tube, with heaviest layer at the bottom and lightest at the top in either. Classified into two categories: Rat e - zo n al (size) separation Isopycnic (density) separa t ion

27. Gradient material used are: Sucrose (66%, 5 C) Silica sols Glycerol CsCl Cs Acetate Ficol (high molecular wgt sucrose polymer & epichlorhydrin) Sorbitol Polyvinylpyrrolidone

28. 2.1 Rate zonal centrifugation Gradient centrifugation. Take advantage of particle size and mass instead of particle density for sedimentation. Ex: for common application include separation of cellular organelle such as endosomes or proteins ( such as antibodies ) , RNA-DNA hybrids, ribosomal subunits.

29. 2.1 Rate zonal centrifugation Criteria for successful rate-zonal centrifugation: Density of sample solution must be less than that of the lowest density portion of the gradient. Density of sample particle must be greater than that of highest density portion of the gradient. Path length of gradient must be sufficient for the separation to occur. Time is important, if you perform too long runs, particles may all pellet at the bottom of the tube.

30. 2.2 Isopycnic centrifugation Particle of a particular density will sink during centrifugation until a position is reaches where the density of the surrounding solution is exactly the same as the density of the particle. ‘ On c e qua s i-equilib r ium is rea c hed, the length of centrifugation doesnot have any influence on the migration of particle. Ex: separation of Nucleic acid in CsCl (Caseium chloride) gradient.

31. Rate-Zonal Is o p y cn i c Synonym S-zonal, sedimentation velocity Density equilibrium, sedimentation equilibrium Gradient Shallow, Maximum gradient density less than the least dense sedimenting specie, Gradient continuous. Steep, Maximum gradient density greater than that of the most dense sedimenting specie, Continuous or discontinuous gradients. C e ntrif u g a - tion Incomplete sedimentation, Low speed, Complete sedimentation till equilibrium is achieved, Short time High speed, Long time. Separation RNA- DNA hybrids, ribosomal subunits, etc., DNA, plasma lipoproteins, lysosomes, mitochondria, peroxisomes, etc.,

32. Analytical centrifugation Speed – 70000 rpm, RCF – 5 lakh g Motor, rotor ,chamber that is refrigerated and evacuated and optical system Optical system has light absorption system ,schleiren system & Rayleigh inferometric system 2 cells – analytical cell and counterpoise cell

33. Optics used – schlieren optics or Rayleigh interference optics At beginning , peak of refractive index will be at meniscus. With progress of sedimentation, macromolecules move down – peak sh i fts givi n g direct inf o r m a tion about t he sedimentation characteristics.

34. Analytical centrifugation Purity of macromole Relative molecular mass of solute (within 5% SD) Change in relative molecular mass of supermolecular complexes Conformational change of protein structure Ligand-binding study

35. Types of Centrifuges & applications Types of centrifuge Characteristic Low Speed High Speed Ultracentrifuge Range of Speed (rpm) 1 - 6000 1000-25,000 20-80,000 Maximum RCF (g) 6000 50,000 6,00,000 Refrigeration some Yes Yes Applications Pelleting of cells Yes Yes Yes Pelleting of nuclei Yes Yes Yes Pelleting of organelles No Yes Yes Pelleting of ribosomes No No Yes Pelleting of Macromolecules No No Yes

36. Operati o n Tubes recommended by their manufacturer should be used. Top of tube should not protrude so far above the bucket. Properly balanced- weight of racks, tubes, and content on opposite side of a rotor should not differ by more than 1%. ( Centrifuges auto balance is available ). Should centrifuge before unstopper the tubes. Cleanliness –minimizing the possible of spread of infection (hep Virus ). Spillage / break of tube to be considered as hazard for pathogenic sample Speed of centrifuge should be checked once 3m . Centrifuge timer to be checked per week.

37. Applic a tion In clinical 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. Separate 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.

38. References : Tietz – Clinical Chemistry And Molecular Diagnostic Keith Wilson and John Walker – Principle And Technique In Biochemistry And Molecular Biology. Avinash Upadhyay – Biophysical Chemistry. Internet sources.