Centrifugation

1. Centrifugation

2. WHAT IS CENTRIFUGE?
Centrifuge is device for separating particles from a
solution according to there size, shape, density,
viscosity of the medium.
WHAT IS CENTRIFUGATION?
Centrifuge uses centrifugal force to separate
phases of different densities.

3. Centrifugation is a procedure that involves the use of centrifugal
force for the sedimentation of mixture with a centrifuge used in
industry and in laboratory settings. More dense components of the
mixture move away from the axis of the centrifuge while less
dense components of the mixture move towards the axis.

4. A particle whether it is a precipitate a macromolecule or a cell organelle is
subjected to a centrifugal force when it is rotated at a high rate of speed. The
centrifugal force F is denoted by equation
F=mω2 r
Where
F= intensity of the centrifugal force
m= effective mass of the sedimenting particle
ω= angular velocity of rotation
r= distance of the migrating particles from the central axis of
rotation
A more common measurement of F in terms of the earths gravitation force , g,
is relative centrifugal force, RCF its defined by
RCF = 1.119 x 10-5 (rpm)2 (r)
This equation relates RCF to revolutions per minute of the sample . Equation
dictates that the RCF on a sample will vary with r, the distance of the
sedimenting particles from the axis of rotation . The RCF value is reported as “ a
number times gravity ,g .”

5. On and off control, operation time control (timer),
rotation speed control, temperature control (in
refrigerated centrifuges), vibration control (safety
mechanism) and brake system
Refrigeration system (in refrigerated centrifuges).
Base
Lid/cover
Casing
Electric motor
Rotor.
There are different types of rotors. The most common are
the fixed angle, the swinging buckets, the vertical tube
and the almost vertical tube types.

7. 1. Low –speed centrifuges .
Its also called as clinical centrifuge.
The common centrifuge has a maximum speed in the range of
4000 to 5000 rpm with RCF value approx. up to 3000Xg. These
instruments usually operate at low temperature with no means
of temperature control of the samples.
Two types of rotors fixed angle and swinging bucket may be
used in this instruments.

10. a)Preparative
1. Differential centrifugation
2. Density gradient centrifugation
3. Rate-zonal density gradient
centrifugation.
b) Analytical
4. Isopycnic
centrifugation

11. based on the size of the particles
used for simple pelleting, for the separation of
subcellular organelles and macromolecules
first, sample must be homogenised
ultra centrifugation
sedimentation depends on mass, shape and
partial specific volume of a macromolecule, as
well as solvent density, rotor size, rate of
rotation.
Usually uses a fixed angle rotor

13. method to purify subcellular organelles and
macromolecules.
Density gradients generated by placing layer
after layer of gradient media
Particles are placed on top of gradient and
centrifuge .
Particles travel through gradient until
density matches with sucrose.
Now fraction is removed and analyzed.

15. use of continuous density gradient of solvent
such as sucrose.
density increases towards the bottom of the
tube
sample layered on the top
molecules form discrete bands after
centrifugation
separation based on size of the molecules
Swinging bucket rotors
2.Rate- zonal Density
Gradient centrifugation

17. based on the density of the molecules
Mix gradient material with the sample molecule (CsCl)
molecules move to the position where their density is
same as the gradient material (isopycnic position)
inorder to generate a gradient, we select a CsCl
concentration that will give a range of densities that
includes the range of molecules that have to be
separated.
used for the separation of DNA
Swinging bucket or fixed angle rotor
3. Isopycnic
Centrifugation

19. USES SMALL SIZE
SAMPLES BUILT-IN
OPTICAL SYSTEM
USES RELATIVELY PURE
SAMPLE

20. Types centrifuges depends upon
Maximum speed of sedimentation
Presence /absence of vacuum
Temperature control refrigeration.
Volume of sample and capacity of
centrifugation tubes
Depending on the particular application,
centrifuges differ in their overall design and size.
a common feature in all centrifuges is the
central motor that spins a rotor containing the
samples to be separated

22. With or without refrigeration
Slow speed (eg up to 4000 RPM)
Common in clinical lab (blood/plasma/serum
separation)
can take approx (up to) 100 tubes, depending
on diameter

24. (“MICROFUGE”,
“EPPENDORF”)
Sample volume is small in tubes
Refrigerated with or without
can generate forces up to ~15,000 x g
Take tube of small volume up to 2ml.
Commonly used of concentration protein
very common in biochemistry/molecular
biology/biological labs

26. Refrigerated
use for protein precipitates, large intact
organelles cellular debris from tissue
homogenisation and micoorganism
They operate maximal centrifugal force of
approx 10000g
Use for research applications
differntial sepration of nucleus, mitrochondrial,
protein precipitate,etc.
15000-20000
RPM

28. Refrigerated and evacuated
The detail biochmestry analysis of subcellular
structures and isolate biomolecules.
Operate at upto 90000 g
limited lifetime
expensive
require special rotors
care in use – balance critical!
research applications

30. FIXED ANGLE
SWINGING BUCKET ROTORS
(VERTICAL/ HORIZONTAL)

31. THE ROTOR (MAINLY MADE OF ALUMINIUM) IS
VERY COMPACT. THERE ARE BOREHOLES WITH A
SPECIFIC ANGLE (LIKE 45°) WITHIN THE ROTOR.
THESE BOREHOLES ARE USED FOR THE SAMPLE
TUBES.

32. a. Vertical rotors
Vertical rotors Sample tubes are held in vertical position during rotation
This type of rotor is not suitable for pelleting applications but is most
efficient for isopycnic (density) separations due to the short pathlength .
Applications include plasmid DNA, RNA, and lipoprotein isolations.
b. horizontal rotors
The rotor looks like a cross with bucket.
Within these buckets, different tubes can be centrifuged.
For a safe centrifugation, a specific adadpter for every tube shape is
mandatory.

33. Centrifugal field from the center of rotation may be
calculated using the equation
G=ω2 r
Angular velocity ω may be calculated by using
this equation
ω= 2 π rev min-1
60

34. Lubricate and clean motor.
Clean case.
Inspect power cords and plugs.
Inspect controls and switches.
Ensure appropriate menu settings for proper use.
Ensure tightness of rotor.
Check lights and indicators.
Verify that alarms are operating properly.
Ensure safety switch is functioning.
If refrigerated, ensure temperature reading is working.
Replace/repair gaskets, seals, and vacuum pump (if
applicable).

35. Application
s
• To separate two miscicible
substances
• To analyze hydrodynamic
properties of macrmolecules .
• Purification of mammalian
cell.
• Fractionation of subcellular
organelles and membrane
vesical.
• Separation of chalk from water.
• To separate fat from milk .
• Particulate matter from air flow
using cyclonic precipitator.

36. Thankyou

38. Thankyo
u