Centrifugation uses centrifugal force to separate mixtures based on density. There are several types of centrifuges that differ in maximum speed and other features. Desktop centrifuges have the lowest maximum speed below 3000rpm, while ultracentrifuges can reach speeds over 75,000rpm. Centrifuges consist of a drive motor, temperature control, vacuum, and rotors. Sedimentation velocity separates mixtures in a shallow gradient over a short time, while sedimentation equilibrium separates mixtures to their equilibrium positions in a steep gradient over prolonged high-speed centrifugation.

1. Centrifugation

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

3. 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
75000rpm75000rpm 20000~25000rpm20000~25000rpm 3000rpm3000rpm
Ultra-
centrifuge
High
speed
centrifuge
Desk top
centrifuge

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

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

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

11. Composition
 Centrifuge consist of four parts:
1.Drive and speed control
2.Temperature control
3.Vacuum system
4.Rotors

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

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

16. Rotors
 Two types: angle rotor
swinging bucket rotor
Angle rotor:
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.  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)2
r

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

24. Sedimentation velocity
 v =dr / dt = Φ(ρp - ρm) ω2
r /f
r(cm), the distance from the axis of rotation
to the sedimenting particle or molecule
Φ(cm3
), volume of the particle
ρp(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 / ω2
r)
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.  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. Time
 s = (dr / dt) • (1 / ω2
r)
→ 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. 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

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. 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