Centrifugation Methods & Techniques PDF

CENTRIFUGATION  Contents: Basic principle Operation Types Application  Definition:  Biological centrifugation is a process that uses centrifugal forces to separate and purify mixture of biologic...

CENTRIFUGATION  Contents: Basic principle Operation Types Application  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.  History:  The first analytical ultracentrifuge was developed by Svedberg in 1920  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 1. Angular velocity (ω in radians / sec). 2. Radial distance (r in cms)of particle from axis of rotation G = ω2r Rate of sedimentation  Depends on factors other than CF  Mass of particle ---Density & Volume  Density of medium  Shape of particle  Friction  Sedimentation time  Depends on , 1. Size of particle 2. Density difference b/w particle and medium 3. Radial distance from the axis of rotation to liquid meniscus (rt) 4. Radial distance from the axis of rotation to the bottom of the tube(rb) THE 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.  Common feature of all centrifuges is the central motor that spins a rotor containing the samples to be separated.  Types of centrifuge Desk top High speed Ultracentrifuges centrifuges centrifuges Analytical Preparative  Types of rotor Fixed Vertical angle tube rotors rotors Swinging- bucket rotors  Types of rotor Fixed angle rotors Vertical tube rotors  Tubes are held at angle of  Held vertical parallel to rotor 14 to 400 to the vertical. axis.  Particles move radially  Particles move short outwards, travel a short distance. distance.  Time of separation is  Useful for differential shorter. centrifugation  Disadvantage: pellet may  Reorientation of the tube fall back into solution at end occurs during acceleration of centrifugation. and deceleration of the rotor.  Types of rotor Swinging-bucket rotors  Sing 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. Separation  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  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.  High speed centrifuges  Maximum speed of 25000rpm, providing 90000g centrifugal forces.  Equipped with refrigeration to remove heat generated.  Temperature maintained at 0-40C 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)  Ultracentrifuges  Operate at speed of 75,000rpm, providing the centrifugal force of 500,000g.  Rotor chamber is sealed and evacuated by pump to attain vacuum.  Refrigeration system (temp 0-40C).  Rotor chamber is always enclosed in a heavy armor plate.  Centrifugation for isolation and purification of components is known as preparatory centrifugation, while that carried out with a desire for characterization is known as analytical centrifugation.  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  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)  During centrifugation, larger particles sediment faster than the smaller ones.  At a series of progressive higher g-force generate partially purified organelles.  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 and fact that preparation obtained never pure. 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 in tube, with heaviest layer at the bottom and lightest at the top in either.  Classified into two categories: Rate-zonal Isopycnic (size) (density) separation separation  Gradient material used are: Sucrose (66%, 50C) Silica sols Glycerol CsCl Cs Acetate Ficol (high molecular wgt sucrose polymer & epichlorhydrin) Sorbitol Polyvinylpyrrolidone  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)  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.  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. ‘  Once quasi-equilibrium is reached, the length of centrifugation doesnot have any influence on the migration of particle.  Ex: separation of Nucleic acid in CsCl (Caseium chloride) gradient.  Rate-Zonal Isopycnic Synonym S-zonal, sedimentation velocity Density equilibrium, sedimentation equilibrium Gradient Shallow, Steep, Maximum gradient density less Maximum gradient density greater than the least dense sedimenting than that of the most dense specie, sedimenting specie, Gradient continuous. Continuous or discontinuous gradients. Centrifuga- Incomplete sedimentation, Complete sedimentation till tion Low speed, equilibrium is achieved, Short time High speed, Long time. Separation RNA- DNA hybrids, ribosomal DNA, plasma lipoproteins, subunits, etc., lysosomes, mitochondria, peroxisomes, etc.,  Operation 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 are available). Should centrifuge before unstopper the tubes. Cleanliness –minimizing the possible of spread of infection (hep Virus). Spillage and break of tube should be considered as the bloodborne pathogen hazard. Speed of centrifuge should be checked once 3m.  Application  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.  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