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1 Sedimentation Sedimentation describes the motion of molecules in solutions or particles in suspensions in response to an external force such as gravity, centrifugal force or electric force. 2 Sedimentation Theory Svedberg equations solvent FC: The Centrifugal Force solvent =M0* 2* r FB: the Buoyant Force (Archimedes) =Mw* 2* r Ff: the Frictional Force =f*v Centrifugal force = buoyant force + frictional force 3 1. The Centrifugal Force 2 Fc = M 0* w * r Mo is the particle weight, or molecular weight (omega)= angular velocity (radians/sec) r is the radius of rotation This equation says that the larger the molecule, or the faster the centrifugation, or the longer the axis of rotation, the greater the centrifugal force and the rate of sedimentation. 4 The Centrifugal Force A more common expression is the relative centrifugal force (RCF): rpm RCF 11.18 * r * 1000 2 r = Radial distance of particle from axis of rotation rpm = Revolutions ( round) per minute RCF reports centrifugal force relative to earth’s gravitational force; commonly refer to as “number of times or g.” A sample rotating at 20, 000 rpm with r = 7 cm will experience RCF= 33,000 x g. 5 2.The Buoyant Force FB = Mw * w * r 2 The buoyant force opposes the centrifugal force. where Mw is the mass of the solvent displaced by the particle. The net force= (Fc-FB) will determine whether a particle floats or sediments Particles with higher density will experience smaller buoyant force, and thus, sediment faster. 6 The Frictional force Ff = f V Frictional force (resistance of a molecule to movement) v = velocity relative to the centrifuge tube, f = frictional coefficient. 7 The frictional coefficient depends upon: 1. the size 2. shape of the molecule, 3. the viscosity of the gradient material. The frictional coefficient f of a compact particle is smaller than that of an extended particle of the same mass. 8 Centrifuge Centrifuge is a piece of equipment, generally driven by a motor, that puts an object in rotation around a fixed axis, applying a force perpendicular to the axis. The centrifuge works using the sedimentation principle 9 Applied Centrifugation Parameters you need to know: 1. Type of rotor: fixed angle, swinging bucket, vertical 2. Type of centrifuge: Low speed , high Speed, ultracentrifuge 3. Type of centrifugation Differential, preparative, or analytical Also, the Speed and duration of centrifugation 10 1. Types of Rotors swinging bucket rotors: fixed-angle rotors: * Longer distance of travel may allow better separation * Excellent for gradient centrifugation .* Easier to withdraw supernatant without disturbing pellet * Sedimenting particles have only short distance to travel before pelleting. * Excellent for fractionation purposes * The most widely used rotor type. Other types include vertical rotors and continuous-flow rotors 11 Dr Gihan Gawish 2. Type of Centrifuge 2-1.Low-speed centrifuges Also called: microfuge, Clinical, Table top or bench top centrifuges Max speed ~ 20,000 rpm Operate at room temperature Fixed angle or swinging bucket can be used Commonly used for rapid separation of coarse particles E.g. RBC from blood, DNA from proteins, etc. The sample is centrifuged until the particles are tightly packed into pellet at the bottom of the tube. Liquid portion, supernatant, is decanted. 12 2-2. High-speed Centrifuges Preparative centrifuges Max speed ~ 80,000 rpm Often refrigerated, vacuum to operate and requires Fixed angle or swinging bucket can be used Generally used to separate macromolecules (proteins or nucleic acids) during purification or preparative work. Can be used to estimate sedimentation coefficient and MW 13 2-3. Ultracentrifuge The most advanced form of centrifuges: (specialized and expensive) Used to precisely determine sedimentation coefficient and MW of molecules, Molecular shape,Protein-protein interactions Uses very high speed Uses small sample size (< 1 ml) Uses relatively pure sample Built in optical system to analyze movements of molecules during Analytical Ultracentrifuge centrifugation 14 3. Types of Centrifugation There are basically three modes of centrifugation 3-1.Differential or pelleting Cellular fractionation suspension and/or removal of precipitates crude purification step 15 separating coarse 3-2. Preparative or Density gradient centrifugation: Separation of complex mixtures Finer fractionation of cellular components Purification of proteins, nucleic acids, plasmids Characterization of molecular interactions 16 3-3. Analytical Determining hydrodynamic properties of bio molecules or Relative MW Molecular shape Aggregation behavior Protein-protein interactions 17 thermodynamic