1. Centrifugation

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

3. 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

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

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

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

7. Continuous flow centrifuge
Relatively simple
High capacity
Separating mixed liquids^

8. 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

9. Refrigerated high-speed centrifuge

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

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

12. 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

13. 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

14. 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

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

16. 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°

17. Swinging bucket rotor:
Hang three to six free moving buckets

18. 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

19. 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

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

21. 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

22. 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

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

24. 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

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

26. 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

27. 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℃

28. → 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

29. 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

30. 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

31. Testube

32. 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

33. 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

34. Sums to be prepare

35. 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

36. SUN WEI Pharmacy of woosuk university sunwei880709@hotmail.com
Thank You !
SUN WEI
Pharmacy of woosuk university