Centrifugation

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 = ω
2
r
F is expressed in terms of the earth’s
gravitational force, referred to as the
relative centrifugal force , RCF (× g)
RCF = ω
2
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 = ω
2
r
→ RCF = (π (rpm) /30)
2
× r/ 30
2
/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 , r
min
and r
max
, are 4.8 and 8.0cm
, spinning at a speed of 12000rpm.
Calculate RCF
top
and RCF
bottom

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(ρ
p
- ρ
m
) ω
2
r /f
r(cm), the distance from the axis of rotation
to the sedimenting particle or molecule
Φ(cm
3
), volume of the particle
ρ
p
(g/cm
3
), the density of the particle
ρ
m
(g/cm
3
), 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 / ω
2
r)
Or s = Φ (ρ
p
-ρ
m
) f
S(s), unit:10
-13
seconds
18 ×10
-13
seconds = 18s

26. Frictional coefficient
f = 6 πηr
m
r
m
(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
℃
S
20，w
= s
t,m
• η
t,m
(ρ
p
- ρ
20,w
)/ η
20,w
(ρ
p
- ρ
t,m
)
s
t,m
, the uncorrected sedimentation coefficient determined in medium m,
and temperature t
η
t,m
, the viscosity of the medium at the temperature of centrifugation
η
20,w
,the viscosity of water at 20
℃
ρ
p
,the density of the particle or molecule in solution
ρ
t,m
, the density of the medium at the temperature of centrifugation
ρ
20,w
, the density of water at 20
℃

28. Time
s = (d
r
/ d
t
) • (1 / ω
2
r)
→ s = (ln
rt
–ln
ro
) / (ω
2
(t
t
–t
0
))
→ t
t –t
0 = 1/s • (ln
rt –ln
ro) / ω
2
=Δt
r
t
, the radii at the top of the spinning centrifuge tube
r
0
, 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
centrifugationIncomplete 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
[email protected]