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A presentation on the principles and uses of the technique of Ultracentrifugation

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  1. 1. ULTRACENTRIFUGATION GVM/16– 046 I M.V.Sc Departmentof VeterinaryPublic Healthand Epidemiology NTR CVSc,Gannavaram
  2. 2. CENTRIFUGE A centrifuge is a device for separating particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed.
  3. 3. HISTORY  Swedish Biochemist Theoder Svedberg invented the Ultracentrifuge in 1923.  Won the Nobel Prize in chemistry in 1926 for his research on colloids and protein using the ultracentrifuge.
  5. 5. ULTRACENTRIFUGATION • It is an important tool in biochemical research. • Rapid spinning imposes high centrifugal forces on suspended particles, or even molecules in solution • Causes separations of such matter on the basis of differences in weight. • Its rotational speed up to 150,000 rpm. • It creates a centrifugal force up to 900,000 g.
  6. 6. What happens to a particle in a centrifugal field??? The particle (m) is acted on by three forces: FC: the centrifugal force FB: the buoyant force Ff: the frictional force between the particle and the liquid
  7. 7. THE PHYSICS OF ULTRACENTRIFUGATION 1.Centrifugal force:- • The outward force experienced by a particle in circular motion • The tube containing the suspension of particles is rotated at a high speed, which exerts a centrifugal force directed from the centre of the rotor towards the bottom of the tube.
  8. 8. Centrifugal force: M: mass of particle r: radius of rotation (cm) (ie distance of particle from axis of rotation) ω :Average angular velocity (radians/sec)
  9. 9. Centrifugal field :- • field where centrifugal force is experienced. • Depends on the radical distance of the particle from the rotation axis and the square of the angular velocity.
  10. 10. Angular Velocity:- • Rate of rotation around an axis • Detect to revolution per minute (r.p.m)
  11. 11. 2.Sedimentation rate:- This force acts on the suspended particles pushing them towards the bottom of the tube at a rate determined by the velocity of the spinning rotor. Rate of Sedimentation: Where, r = radius at which the organelle is located t = time M = molecular weight ν = partial specific volume of the molecule; inverse of the density ρ = density of the solvent f = translational frictional coefficient ω = angular velocity N = Avagadro’s number
  12. 12. 3.Sedimentation coefficient:- Centrifugation separates particles in a suspension based on differences in size, shape and density that together define their sedimentation coefficient. Sedimentation Coefficient:  This is known as the Svedberg equation and is usually expressed in Svedberg units, S (= 10-13 second).  This equation indicates that ‘S’ is dependent upon the molecular weight, the density and the frictional coefficient.
  13. 13. STOKES EQUATION Frictional coefficient, f = 6πηr where, r : particle radius η : viscosity of solution • f is minimal when particle is a sphere • Non spherical particle has larger surface area and thus greater value of f p p
  14. 14. 1. More density  Faster sedimentation 2. More massive  Faster sedimentation 3. Denser biological buffer system slower movement of particle 4. More frictional coefficient  slower movement 5. More centrifugal force  faster sedimentation 6. Sedimentation rate = 0, if density of particle = density of the surrounding medium PRINCIPLESOF SEDIMENTATION
  15. 15. 1. Analytical ultracentrifugation:- The aim of Analytical ultracentrifugation is use to study molecular interactions between macromolecules or to analyse the properties of sedimenting particles such as their apparent molecular weight. 2. Preparative ultracentrifugation:- The aim of Preparative ultracentrifugation to isolate and purify specific particles such as subcellular organelles. TYPES OF ULTRACENTRIFUGATION:
  16. 16. ANALYTICAL CENTRIFUGE • Used for performing physical measurements on sample during sedimentation. • Sedimentation coefficient used to characterize changes in the size and shape of macromolecules with changing experimental conditions. • Concentration distributions measured by Schlieren system or Raleigh interferometric system.
  17. 17. ANALYTICAL CENTRIFUGE Two kinds of experiments are commonly performed on these instruments: 1. Sedimentation velocity experiments 2. Sedimentation equilibrium experiments
  18. 18. To estimate sample purity • Aim of SVEs to interpret the entire time-course of sedimentation, and report on the shape and molar mass of the dissolved macromolecules, as well as their size distribution. • Components observed as peaks. SEDIMENTATIONVELOCITY EXPERIMENTS
  19. 19. SEEs are concerned only with the final steady-state of the experiment, where sedimentation is balanced by diffusion opposing the concentration gradients, resulting in a time- independent concentration profile. SEDIMENTATIONEQUILIBRIUM EXPERIMENTS
  20. 20. • Designed for sample preparation • Lack sample observation facility PREPARATIVE ULTRACENTRIFUGATION Types of preparative ultracentrifugation: • Differential ultracentrifugation • Density gradient ultracentrifugation
  21. 21. • Used to separate certain organelles from whole cells for further analysis of specific parts of cells. • Based on differences in sedimentation rate of particles. • Crude tissue homogenate divided into different fractions by stepwise increase in applied centrifugal field. • Largest sediment faster followed by smaller particles. • Rpm gradually increased to sediment particles. DIFFERENTIAL ULTRACENTRIFUGATION
  22. 22. • Based on density difference. • Sample layered on top of preformed density gradient. • Caesium chloride density gradient is widely used for DNA, isolation of plasmids, nucleoproteins and viruses. • Sodium bromide and sodium iodide for fractionation of lipoproteins. • Max density of gradient must exceed density of most dense particle of the sample. • Step wise gradient and continuous gradient applied. DENSITYGRADIENT ULTRACENTRIFUGATION
  23. 23. • Sucrose – a sugar • Glycerol • Ficoll – a polysaccharide • Percoll – a colloidal silica • Caesium chloride – chemical GRADIENTS
  25. 25. • Mixture to be separated is layered on top of a gradient (increasing concentration down the tube). • Provides gravitational stability as different species. • Move down tube at different rates. • Sucrose gradient is commonly used to create zones of different gradient. • Separation based on molecular masses. • Fractionation achieved by puncturing bottom of celluloid centrifuge tube. ZONAL OR RATE CENTRIFUGATION
  26. 26. • Isopycnic means “of the same density”. • Molecules separated on equilibrium position. • Sample dissolved in relatively concentrated solution of dense, fast diffusing substance and spun at high speeds until solution achieves equilibrium. • Caesium chloride or Caesium sulphate used. • High centrifugal field causes low molecular mass solute to form a steep density gradient in which the sample components band at positions where their densities are equal to that of solution. ISOPYCNICCENTRIFUGATION
  27. 27. • Bands collected as separate fractions. • Used for separating sample whose components have a range of densities. • Used for nucleic acids, viruses and certain subcellular organelles. • Not used for proteins as they have similar densities. • Used to show semi conservative replication of DNA. ISOPYCNICCENTRIFUGATION
  28. 28. Schematic presentation ofa ultracentrifuge:
  29. 29. Schematic presentation ofa ultracentrifuge: Fig; A Beckman Ultracentrifugation.
  30. 30. Analytical:  Uses small sample size (less than 1 ml).  Built in optical system to analyze progress of molecules during centrifugation.  Uses relatively pure sample.  Used to precisely determine sedimentation coefficient and MW of molecules.  Beckman Model E is an example of centrifuge used for these purposes. FUNCTIONSOF ANALYTICAL AND PREPARATIVE ULTRACENTRIFUGATION
  31. 31. Preparative:  Larger sample size can be used.  No optical read-out collect fractions and analyze them after the run.  Less pure sample can be used.  Can be used to estimate sedimentation coefficient and Molecular weight.  Generally used to separate organelles and molecules. Most centrifugation work done using preparative ultracentrifuge
  32. 32. ROTOR  Four types of rotors are available for ultracentrifugation, 1. Fixed-angle rotor, 2. Swinging-bucket rotor, 3. Vertical rotor and 4. Near-vertical rotor. .
  33. 33. ROTOR  Rotors are made from either aluminium or titanium, or from fiber-reinforced composites.  A titanium rotor is designated by T or Ti, as in the Type 100 Ti, the SW 55 Ti, or the NVT 90 rotor.  A composite rotor (fiber) is designated by C, as in VC 53.  A aluminium rotor is designated by AC, as in VAC 50.  Rotors without the T, Ti, C, or AC designation (such as the Type 25) are fabricated from an aluminium alloy.
  34. 34.  Titanium rotors are stronger and more chemical resistant than the aluminium rotors.  Exterior surfaces of titanium and composite rotors are finished with black polyurethane paint.  Titanium buckets and lids of high-performance rotors are usually painted red for identification.
  35. 35. FIXED ANGLE ROTOR  Fixed-angle rotors are general- purpose rotors that are especially useful for pelleting subcellular particles and in short column banding of viruses and subcellular organelles.  Tubes are held at an angle (usually 20 to 45 degrees) to the axis of rotation in numbered tube cavities.
  36. 36. SWINGINGBUCKET ROTOR  Swinging-bucket rotor are used for pelleting, isopycnic studies and rate zonal studies.  Tubes are attached to the rotor body by hinge pins or a crossbar. The buckets swing out to a horizontal position.
  37. 37. VERTICAL ROTOR  Vertical rotors hold tubes parallel to the axis of rotation; therefore, bands separate across the diameter of the tube rather than down the length of the tube.  Vertical rotors are useful for isopycnic and, in some cases, rate zonal separations when run time reduction is important.
  38. 38. NEAR VERTICAL ROTOR  Near-vertical rotors are designed for gradient centrifugation when there are components in a sample mixture that do not participate in the gradient.  Tubes are held at an angle (typically 7 to 10 degrees) to the axis of rotation in numbered tube cavities.  In this rotor used only Quick-Seal and Opti-Seal tubes.
  39. 39. Common Centrifuge Classesand TheirApplications ( ) = can be done but not usually used for this purpose.
  40. 40. Tube Type and Rotor Compatibility Rotor Types Tube Types Fixed-Angle Swinging-bucket Vertical Thin wall open top No Yes No Thick wall open top Yes Yes No Thin wall sealed Yes Some tubes Yes Oak ridge Yes No No Types of Rotors and Theirs Applications Rotor Types Pelleting R or Z-Sedimentation Isopycnic Fixed-angle Excellent Limited Variable S-bucket Inefficient Good Good Vertical Not suitable Good Excellent N-vertical Not suitable Excellent Good
  41. 41. ROTOR BALANCE  The mass of a properly loaded rotor will be evenly distributed on the ultracentrifuge 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 the same density.  Weight of opposing tubes must be distributed equally.  Place tubes in the rotor symmetrically.
  42. 42. CARE OF CENTRIFUGESAND ROTORS  Select the proper operating conditions on the instrument.  Check the rotor chamber for cleanliness and for damage.  Select the proper rotor. Many sizes and types are available.  Be sure the rotor is clean and undamaged.  Filled centrifuge tubes or bottles should be weighed carefully and balanced before centrifugation.
  43. 43. CARE OF CENTRIFUGESAND ROTORS  Rotor manufactures provide a max. allowable speed limit for each rotor. Do nor exceed that limit.  Keep an accurate record of centrifuge and rotor use.  If an unusual noise or vibration develops during centrifugation, immediately turn the centrifuge off.  Carefully clean the rotor chamber and rotor after centrifugation.