centrifugation
DESCRIPTION
centrifugationTRANSCRIPT
Centrifugation
Contents
1. Definition
2. Classification
3. Composition
4. Relative force & application
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
75000rpm75000rpm 20000~25000rpm20000~25000rpm 3000rpm3000rpm
Ultra-centrifuge
High speed
centrifuge
Desk topcentrifuge
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
Refrigerated high-speed centrifuge
Continuous flow centrifuge
High speed centrifuges
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
The ultracentrifuge
Attain the speed of 75000rpm Isolate viruse
DNA
RNA
protein
Composition
Centrifuge consist of four parts:
1.Drive and speed control
2.Temperature control
3.Vacuum system
4.Rotors
Drive & Speed control
Drive: water-cooled electric motor
Speed control:
1.selected by rheostat
2.monitored with a tachometer
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
Temperature control
highspeed centrifuge:placing a thermocouple in the rotor chambermonitoring only the rotor chamber temperature
Ultracentrifuge:an infrared radiometric sensor placed beneath
the rotor continuously monitors the rotor temperature
Vacuum system
The speed of centrifuge < 15000 to 20000rp Not required
The speed of centrifuge > 4000rpm
Required
Rotors
Two types: angle rotor
swinging bucket rotor
Angle rotor: Consist of a solid piece of metal with 6 to 12
holesAt 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
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 rThe problem is illustrated in the
following example
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
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
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℃
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
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:
1.Zone centrifugation
2.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
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
