Centrifugation

Contents 1. Definition 2. Classification 3. Composition 4. Relative force & application

Use of the centrifugal force for the separation of mixtures Centrifugation Use of the centrifugal force for the separation of mixtures More-dense components migrate away from the axis of the centrifuge less-dense components of migrate towards the axis

Classification High speed Ultra- Desk top centrifuge centrifuge 75000rpm 20000~25000rpm 3000rpm

Desk top clinical centrifuges Simplest Least expensive Maximum speed is below 3000rpm Ambient temperature

High-speed centrifuges Speeds of 20000 to 25000rpm Equipped with refrigeration equipment High speed centrifuges Continuous flow centrifuge Refrigerated high-speed centrifuge

Continuous flow centrifuge Relatively simple High capacity Separating mixed liquids^

Refrigerated high-speed centrifuge Lower capacity Collect microorganisms O cellular debris O cells O large cellular organelles O ammonium sulfate precipitates O immunoprecipitates O viruses X small organells X

Refrigerated high-speed centrifuge

Attain the speed of 75000rpm Isolate viruse DNA RNA protein The ultracentrifuge Attain the speed of 75000rpm Isolate viruse DNA RNA protein

Centrifuge consist of four parts: Drive and speed control Composition Centrifuge consist of four parts: Drive and speed control Temperature control Vacuum system Rotors

Drive: water-cooled electric motor Speed control: Drive & Speed control Drive: water-cooled electric motor Speed control: 1.selected by rheostat 2.monitored with a tachometer

Prevent operation of a rotor above its maximum rated speed Overspeed system Prevent operation of a rotor above its maximum rated speed Consist of ^ 1.a ring of alternating reflecting and nonreflecting surfaces attached to the bottom of the rotor. 2.a small but intense point source of light 3.a photocell

highspeed centrifuge: Temperature control highspeed centrifuge: placing a thermocouple in the rotor chamber monitoring only the rotor chamber temperature Ultracentrifuge: an infrared radiometric sensor placed beneath the rotor continuously monitors the rotor temperature

The speed of centrifuge < 15000 to 20000rp Not required Vacuum system The speed of centrifuge < 15000 to 20000rp Not required The speed of centrifuge > 4000rpm Required

Two types: angle rotor swinging bucket rotor Angle rotor: Rotors Consist of a solid piece of metal with 6 to 12 holes At an angle between 20° and 45°

Swinging bucket rotor: Hang three to six free moving buckets

Relative centrifugal force Object moving in circle at a steady angular velocity → an outward directed force F Depend on ω ,and r F = ω2 r F is expressed in terms of the earth’s gravitational force, referred to as the relative centrifugal force , RCF (× g) RCF = ω2 r / 980

ω = π (rpm) /30 & F = ω2 r → RCF = (π (rpm) /30)2 × r/ 302/980 To be of use, these relationships must be expressed in terms of “revolutions per minute” , rpm Rpm values may be converted to radians ω = π (rpm) /30 & F = ω2 r → RCF = (π (rpm) /30)2 × r/ 302/980 =(1.119 ×10-5)(rpm)2r

So, RCF is related to r The sample is located at a fixed distance r The problem is illustrated in the following example

Calculate RCFtop and RCFbottom Example Calculate the RCF exerted at the top an bottom of a sample vessel spinning in a fixed angle rotor.^ Assume that the rotor dimensions , rmin and rmax , are 4.8 and 8.0cm , spinning at a speed of 12000rpm. Calculate RCFtop and RCFbottom

Centrifugal force exerted at the top and bottom of the sample tube differs by nearly twofold To account for this , RCF values may be expressed as an average RCF value(RCFave) RCFave = (1.119 ×10-5)(12000)2 6.4 =10313 × g

Zone Centrifugation or Sedimentation velocity Application Zone Centrifugation or Sedimentation velocity Isopycnic Centrifugation or Sedimentation equilibrium

Sedimentation velocity v =dr / dt = Φ(ρp - ρm) ω2r /f r(cm), the distance from the axis of rotation to the sedimenting particle or molecule Φ(cm3), volume of the particle ρ p(g/cm3), the density of the particle ρ m(g/cm3), the density of the medium f(g/sec), the frictional coefficient v(cm/sec), the radial velocity of sedimentation of the particle

Sedimentation coefficient s = (dr / dt) • (1 / ω2r) Or s = Φ (ρp-ρm) f S(s), unit:10-13 seconds 18 ×10-13 seconds = 18s

Frictional coefficient f = 6 πηrm rm (cm), the molecule or particle radius η(g/cm•sec) , the viscosity of the medium in poises 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

S20，w = st,m • ηt,m(ρp- ρ20,w)/ η20,w (ρp- ρt,m) 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℃ S20，w = st,m • ηt,m(ρp- ρ20,w)/ η20,w (ρp- ρt,m) st,m, the uncorrected sedimentation coefficient determined in medium m, and temperature t ηt,m , the viscosity of the medium at the temperature of centrifugation η20,w ,the viscosity of water at 20℃ ρp ,the density of the particle or molecule in solution ρt,m , the density of the medium at the temperature of centrifugation ρ20,w , the density of water at 20℃

→ s = (lnrt –lnro) / (ω2(tt –t0)) Time s = (dr / dt) • (1 / ω2r) → s = (lnrt –lnro) / (ω2(tt –t0)) → tt –t0 = 1/s • (lnrt –lnro) / ω2 =Δt rt , the radii at the top of the spinning centrifuge tube r0 , the radii at the bottom of the spinning centrifuge tube Δt is the time required to bring about total sedimentation or pelleting of the sedimenting species

Two major types of techniques are commonly used: Zone centrifugation The density gradient The solution is most dense at the bottom of the tube and decreases in density up to the top of the tube. Two major types of techniques are commonly used: Zone centrifugation Isopycnic centrifugation

Example^ One method for further purifying fractions is equilibrium density-gradient centrifugation, which  separates cellular components according to their density at a high speed (about 40,000 rpm) for several hours

Testube

Sedimentation velocity Sedimentation equilibrium table 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

Sedimentation velocity Maximum gradiet density < the least dense sedimenting species During centrifugation sedimenting material moves through the gradient at a rate determined by its sedimentation coefficient It is important to terminate centrifugation before the first species reaches the bottom of the tube This method works well for species that differ in size but not in density

Sums to be prepare

Sedimentation equilibrium Allowing the sedimenting species to move through the gradient until they reach a point no further sedimentation occurs because they are floating on a “cushion” of material that has a density greater than their own Maximum gradient density > the most dense sedimenting species prolonged periods and at relatively higher speeds This technique is used to separate particles similar in size but of differing densities

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