Centrifugation sunwei 2011.3.17

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presented by sun wei, talk about basic concepts of centrifuge

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Centrifugation sunwei 2011.3.17

  1. 1. Centrifugation
  2. 2. Contents 1. Definition 2. Classification 3. Composition 4. Relative force & application
  3. 3. Centrifugation <ul><li>Use of the centrifugal force for the separation of mixtures </li></ul><ul><li>More-dense components migrate away from the axis of the centrifuge </li></ul><ul><li>less-dense components of migrate towards the axis </li></ul>
  4. 4. Classification 75000rpm 20000~25000rpm 3000rpm Ultra- centrifuge High speed centrifuge Desk top centrifuge
  5. 5. Desk top clinical centrifuges <ul><li>Simplest </li></ul><ul><li>Least expensive </li></ul><ul><li>Maximum speed is below 3000rpm </li></ul><ul><li>Ambient temperature </li></ul>
  6. 6. High-speed centrifuges <ul><li>Speeds of 20000 to 25000rpm </li></ul><ul><li>Equipped with refrigeration equipment </li></ul>Refrigerated high-speed centrifuge Continuous flow centrifuge High speed centrifuges
  7. 7. Continuous flow centrifuge <ul><li>Relatively simple </li></ul><ul><li>High capacity </li></ul><ul><li>Separating mixed liquids^ </li></ul>
  8. 8. Refrigerated high-speed centrifuge <ul><li>Lower capacity </li></ul><ul><li>Collect microorganisms O </li></ul><ul><li>cellular debris O </li></ul><ul><li>cells O </li></ul><ul><li>large cellular organelles O </li></ul><ul><li>ammonium sulfate precipitates O </li></ul><ul><li>immunoprecipitates O </li></ul><ul><li>viruses X </li></ul><ul><li>small organells X </li></ul>
  9. 9. Refrigerated high-speed centrifuge
  10. 10. The ultracentrifuge <ul><li>Attain the speed of 75000rpm </li></ul><ul><li>Isolate viruse </li></ul><ul><li>DNA </li></ul><ul><li>RNA </li></ul><ul><li>protein </li></ul>
  11. 11. Composition <ul><li>Centrifuge consist of four parts: </li></ul><ul><li>Drive and speed control </li></ul><ul><li>Temperature control </li></ul><ul><li>Vacuum system </li></ul><ul><li>Rotors </li></ul>
  12. 12. Drive & Speed control <ul><li>Drive: water-cooled electric motor </li></ul><ul><li>Speed control: </li></ul><ul><li>1.selected by rheostat </li></ul><ul><li>2.monitored with a tachometer </li></ul>
  13. 13. Overspeed system <ul><li>Prevent operation of a rotor above its maximum rated speed </li></ul><ul><li>Consist of ^ </li></ul><ul><li>1.a ring of alternating reflecting and nonreflecting surfaces attached to the bottom of the rotor. </li></ul><ul><li>2.a small but intense point source of light </li></ul><ul><li>3.a photocell </li></ul>
  14. 14. Temperature control <ul><li>highspeed centrifuge: </li></ul><ul><li>placing a thermocouple in the rotor chamber </li></ul><ul><li>monitoring only the rotor chamber temperature </li></ul><ul><li>Ultracentrifuge: </li></ul><ul><li>an infrared radiometric sensor placed beneath the rotor </li></ul><ul><li>continuously monitors the rotor temperature </li></ul>
  15. 15. Vacuum system <ul><li>The speed of centrifuge < 15000 to 20000rp Not required </li></ul><ul><li>The speed of centrifuge > 4000rpm </li></ul><ul><li>Required </li></ul>
  16. 16. Rotors <ul><li>Two types: angle rotor </li></ul><ul><li>swinging bucket rotor </li></ul><ul><li>Angle rotor: </li></ul><ul><li>Consist of a solid piece of metal with 6 to 12 holes </li></ul><ul><li>At an angle between 20° and 45° </li></ul>
  17. 17. <ul><li>Swinging bucket rotor: </li></ul><ul><li>Hang three to six free moving buckets </li></ul>
  18. 18. Relative centrifugal force <ul><li>Object moving in circle at a steady angular velocity -> an outward directed force F </li></ul><ul><li>Depend on ω ,and r </li></ul><ul><li>F = ω 2 r </li></ul><ul><li>F is expressed in terms of the earth’s gravitational force, referred to as the relative centrifugal force , RCF (× g) </li></ul><ul><li>RCF = ω 2 r / 980 </li></ul>
  19. 19. <ul><li>To be of use, these relationships must be expressed in terms of “revolutions per minute” , rpm </li></ul><ul><li>Rpm values may be converted to radians </li></ul><ul><li>ω = π (rpm) /30 & F = ω 2 r </li></ul><ul><li>-> RCF = ( π (rpm) /30) 2 × r/ 30 2 /980 </li></ul><ul><li>=(1.119 ×10 -5 )(rpm) 2 r </li></ul>
  20. 20. <ul><li>So, RCF is related to r </li></ul><ul><li>The sample is located at a fixed distance r </li></ul><ul><li>The problem is illustrated in the following example </li></ul>
  21. 21. Example <ul><li>Calculate the RCF exerted at the top an bottom of a sample vessel spinning in a fixed angle rotor.^ Assume that the rotor dimensions , r min and r max , are 4.8 and 8.0cm , spinning at a speed of 12000rpm. </li></ul><ul><li>Calculate RCF top and RCF bottom </li></ul>
  22. 22. <ul><li>Centrifugal force exerted at the top and bottom of the sample tube differs by nearly twofold </li></ul><ul><li>To account for this , RCF values may be expressed as an average RCF value(RCFave) </li></ul><ul><li>RCFave = (1.119 ×10 -5 )(12000) 2 6.4 </li></ul><ul><li>=10313 × g </li></ul>
  23. 23. Application <ul><li>Zone Centrifugation or Sedimentation velocity </li></ul><ul><li>Isopycnic Centrifugation or Sedimentation equilibrium </li></ul>
  24. 24. Sedimentation velocity <ul><li>v =dr / dt = Φ ( ρ p - ρ m ) ω 2 r /f </li></ul><ul><li>r(cm), the distance from the axis of rotation to the sedimenting particle or molecule </li></ul><ul><li>Φ (cm 3 ), volume of the particle </li></ul><ul><li>ρ p (g/cm 3 ), the density of the particle </li></ul><ul><li>ρ m (g/cm 3 ), the density of the medium </li></ul><ul><li>f(g/sec), the frictional coefficient </li></ul><ul><li>v(cm/sec), the radial velocity of sedimentation of the particle </li></ul>
  25. 25. Sedimentation coefficient <ul><li>s = (dr / dt) • (1 / ω 2 r) </li></ul><ul><li>Or s = Φ ( ρ p - ρ m ) f </li></ul><ul><li>S(s), unit:10 -13 seconds </li></ul><ul><li>18 ×10 -13 seconds = 18s </li></ul>
  26. 26. Frictional coefficient <ul><li>f = 6 πη r m </li></ul><ul><li>r m (cm), the molecule or particle radius </li></ul><ul><li>η (g/cm•sec) , the viscosity of the medium in poises </li></ul><ul><li>So, the rate of sedimentation is governed by the size , shape , and density of the sedimenting particle or molecule, as well as by the viscosity and density of the medium </li></ul>
  27. 27. <ul><li>Most often the sedimentation coefficient is corrected to the value that would be obtained in a medium with a density and viscosity of water at 20℃ </li></ul><ul><li>S 20 , w = s t,m • η t,m ( ρ p - ρ 20,w )/ η 20,w ( ρ p - ρ t,m ) </li></ul><ul><li>s t,m , the uncorrected sedimentation coefficient determined in medium m, and temperature t </li></ul><ul><li>η t,m , the viscosity of the medium at the temperature of centrifugation </li></ul><ul><li>η 20,w ,the viscosity of water at 20℃ </li></ul><ul><li>ρ p ,the density of the particle or molecule in solution </li></ul><ul><li>ρ t,m , the density of the medium at the temperature of centrifugation </li></ul><ul><li>ρ 20,w , the density of water at 20℃ </li></ul>
  28. 28. Time <ul><li>s = (d r / d t ) • (1 / ω 2 r) </li></ul><ul><li>-> s = (ln rt –ln ro ) / ( ω 2 (t t –t 0 )) </li></ul><ul><li>-> t t –t 0 = 1/s • (ln rt –ln ro ) / ω 2 = Δ t </li></ul><ul><li>r t , the radii at the top of the spinning centrifuge tube </li></ul><ul><li>r 0 , the radii at the bottom of the spinning centrifuge tube </li></ul><ul><li>Δ t is the time required to bring about total sedimentation or pelleting of the sedimenting species </li></ul>
  29. 29. The density gradient <ul><li>The solution is most dense at the bottom of the tube and decreases in density up to the top of the tube. </li></ul><ul><li>Two major types of techniques are commonly used: </li></ul><ul><li>Zone centrifugation </li></ul><ul><li>Isopycnic centrifugation </li></ul>
  30. 30. Example^ <ul><li>One method for further purifying fractions is  equilibrium density-gradient centrifugation,  which  separates cellular components according to their density </li></ul>at a high speed (about 40,000 rpm) for several hours
  31. 32. Sedimentation velocity Sedimentation equilibrium synonym Zone centrifugation Isopycnic , equilibrium density-gradient centrifugation gradient Shallow, stabilizing – maximum gradient density below that of least dense sedimenting species Steep – maximum gradient density greater than that of most dense sedimenting species centrifugation Incomplete sedimentation , Short time , Low speed Complete sedimentation to equilibrium position, Prolonged time , High speed
  32. 33. Sedimentation velocity <ul><li>Maximum gradiet density < the least dense sedimenting species </li></ul><ul><li>During centrifugation sedimenting material moves through the gradient at a rate determined by its sedimentation coefficient </li></ul><ul><li>It is important to terminate centrifugation before the first species reaches the bottom of the tube </li></ul><ul><li>This method works well for species that differ in size but not in density </li></ul>
  33. 35. Sedimentation equilibrium <ul><li>Allowing the sedimenting species to move through the gradient until they reach a point </li></ul><ul><li>no further sedimentation occurs because they are floating on a “cushion” of material that has a density greater than their own </li></ul><ul><li>Maximum gradient density > the most dense sedimenting species </li></ul><ul><li>prolonged periods and at relatively higher speeds </li></ul><ul><li>This technique is used to separate particles similar in size but of differing densities </li></ul>
  34. 36. SUN WEI Pharmacy of woosuk university [email_address] Thank You !

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