Centrifugation is the separation technique commonly used in clinical and research laboratories. It is based on the behavior of particles in an applied centrifugal field. 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.

1. TapeshwarYadav
(Lecturer)
BMLT, DNHE,
M.Sc. Medical Biochemistry
CentrifugationCentrifugation

2. IntroductionIntroduction
Centrifugation is the separation technique commonly
used in clinical and research laboratories.
It is based on the behavior of particles in an applied
centrifugal field.
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.

3. PrinciplePrinciple
Particles which differ in density, size or shape, sediment at
different rates in a centrifugal field.
The particles will tend to sediment under the influence of gravity.
If the particles suspended in a liquid are so small or have a density
so close to that of the liquid, then the force of gravity fails to
sediment the particles into a separate layer.
So the basis of centrifugation techniques is to exert a larger force
than the gravitational force to enhance the effective sedimentation
force for the separating such particles from the liquid.

4. Contd…Contd…
In centrifugation, the particles are normally suspended in a
specific liquid medium, held in tubes, which are located in a
rotor.
The rotor is positioned centrally on the drive shaft of the
centrifuge.
Particles which differ in density, shape or size can be
separated since they sediment at different rates in the
centrifugal field, each particle sedimenting at a rate which is
proportional to the applied centrifugal field.

5. Sedimentation coefficientSedimentation coefficient
The rate at which the sedimentation occurs in centrifugation
is expressed in terms of sedimentation coefficient and is
given by the formula:
Where, V = sedimentation of the molecules
ω = Rotation of the rotor in
radians/sec.
(angular velocity)
r = Distance in cm, from the centre of
the rotor
S =
V/ω2
r

6. Relative centrifugal force (RCF)Relative centrifugal force (RCF)
The usual expression of RPM (revolution per minute) gives only the
centrifugation speed and does not express the centrifugal force.
A better expression of forces generated by centrifuge is RCF which is
calculated by considering the speed of rotation (RPM), and the radius
from the center of rotation.
RCF is expressed as some number times gravity
i.e. some number × g.
RCF = R × (RPM)2
× 118 × 10-7
NOTE: RCF applied to a centrifuge tube in a fixed-angle head is much less than that
applied to the same tube in a horizontal-head rotor, due to the increased ‘R’
(radius) in swing out type of rotation.

7. CentrifugeCentrifuge
The equipment used to perform centrifugation is called
centrifuge.
The basic centrifuge consists of two components:
i. Electric motor: with drive shaft to spin the sample and
ii. Rotor: to hold tubes or other containers of the sample.
• The rotors can be of three types:
1. Fixed angle rotor
2. Vertical tube rotor
3. Swinging bucket rotor.

8. 1.FIXED ANGLE
ROTOR
 Fixed-angle rotors are general-purpose
rotors that are especially useful for
pelleting subcellular particles and in
short column banding of viruses and
subcellular organelles.
 Tubes are held at an angle (usually 20
to 45 degrees) to the axis of rotation in
numbered tube cavities.

9. 2.VERTICAL
ROTOR
 Vertical rotors hold tubes parallel to
the axis of rotation; therefore, bands
separate across the diameter of the
tube rather than down the length of
the tube.
 Vertical rotors are useful for isopycnic
and, in some cases, rate zonal
separations when run time reduction is
important.

10. 3.SWINGING BUCKET
ROTOR
 Swinging-bucket rotor are used for
pelleting, isopycnic studies and rate
zonal studies.
 Tubes are attached to the rotor body by
hinge pins or a crossbar.The buckets
swing out to a horizontal position.
 Isopycnic studies (separation as a
function of density).
 Rate zonal studies (separation as a
function of sedimentation coefficient).

12. Rotor BalanceRotor Balance
 The mass of a properly loaded rotor will be evenly distributed on
the ultracentrifuge drive hub, causing the rotor to turn smoothly
with the drive.
 An improperly loaded rotor will be unbalanced; consistent
running of unbalanced rotors will reduce ultracentrifuge drive
life.
 To balance the rotor load, fill all opposing tubes to the same level
with liquid of the same density.
 Weight of opposing tubes must be distributed equally.
 Place tubes in the rotor symmetrically.

13. Types of CentrifugeTypes of Centrifuge
1. Hand centrifuge
2. Small bench centrifuges (Low speed
centrifuges)
3. High speed centrifuges
4. Ultra-centrifuges
5. Crayo-centrifuges

14. Applications of centrifugeApplications of centrifuge
The centrifuge is used in a laboratory for various purposes such
as-
i. The separation of serum or plasma from red blood cells.
ii. Separation of sediment in urine.
iii.Separation of protein free filtrate.
iv.Washing of red blood cells by normal saline.
v. Separation of antigen bound fraction or antibody bound
fraction from the free fraction in immunoassays.

15. Sign up for it on website and log in on the log book.
Turn on the centrifuge.
Set up the parameters, speed, time, temperature, accelerate
and decelerate speed.
Open the centrifuge door.
Install the rotors with O-ring that contain balanced bottles or
tubes.
Make sure the rotor is properly seated to the drive hub.
Secure the rotor with lid, close the centrifuge door.
Operating ProceduresOperating Procedures

16. Press enter then run/start.
Wait until reaching desired speed.
After completely stopped, open the door and take out the
sample bottles or tubes, check whether spilling was happened
or not, if there is spill, take out the rotor, wash and dry it and
return to its storage place; never leave rotor inside the
centrifuge; also clean the chamber.
Leave the high speed centrifuge door open and ultra speed
centrifuge door close and turn off the machine.
Contd…Contd…

17. Care of Centrifuges and RotorsCare of Centrifuges and Rotors
 Carefully read the operating manual or receive proper instructions
before use any centrifuge.
 Select the proper operating conditions on the instrument.
 Check the rotor chamber for cleanliness and for damage.
 Select the proper rotor. Many sizes and types are available.
 Be sure the rotor is clean and undamaged.
 Filled centrifuge tubes or bottles should be weighed carefully and
balanced before centrifugation.
 Rotor manufactures provide a max. allowable speed limit for each
rotor. Do nor exceed that limit.
 Keep an accurate record of centrifuge and rotor use.
 If an unusual noise or vibration develops during centrifugation,
immediately turn the centrifuge off.
 Carefully clean the rotor chamber and rotor after centrifugation.