Centrifuge centrifugal force centrifugal working of centrifuge type of centrifuge RPM rotation pre minutes rcf MLT laboratory

1. PRINCIPLES AND APPLICATIONS OF
CENTRIFUGATION
Prakash
B.Sc MLT Ist year
Department of Biochemistry
Post Graduate Institute of Medical Education And Research, Chandigarh
Moderated by:
Respected Dinesh Sir

2. INTRODUCTION
WHAT IS CENTRIFUGE?
Centrifuge is a device for separating particles from a solution according to there size,
shape, density, viscosity of the medium.
WHAT IS CENTRIFUGATION?
Centrifugation is a process which involves the use of the centrifugal force for the
sedimentation of heterogeneous mixtures with a centrifuge.

3.  Centrifugal force, word from Latin Centrum, meaning “centre”, and fugere, means
“to flee”, is the apparent force that draws a rotating body away from the centre of
rotation.
 The term centrifugal force is used to refer to one of two distinct concepts: an
inertial force observed in a non-inertial reference frame, and a reaction force
corresponding to a centripetal force.
 The concept of centrifugal force is applied in rotating devices such as centrifuges,
centrifugal pumps. The two different forces are equal in magnitude, but centrifugal
forces is opposite in direction to the centripetal force.
Centrifugal force

4. Principle
The centrifuge involves principle of centrifugation, where the
acceleration at centripetal force causes denser substances to separate out
along the radial direction at the bottom of the tube.
In a solution, particles whose density is higher than that of the solvent
sink (sediment), and particles that are lighter than it float to the top.
The greater the difference in density, the faster they move.

5. Relative Centrifugal Force (RCF)
 RCF, is the ratio of the centrifugal acceleration at a specified radius and the
speed to the standard acceleration of gravity.
 Relative Centrifugal force is defined as f=Mω2 r
Where,
 F= intensity of centrifugal force
 M= mass of particle
 ω= angular velocity of rotation
 R= distance of migrating particles from central axis of rotation.

6. The RCF value for any centrifuge may be calculated from the
following equation: RCF = 1.18 × r × (rpm) 2 ×10-5
Where,
1.18×10-5 = An empirical factor
r is the radius in cm from the centre of the centrifuge shaft to the
external tip of centrifuge tube.
rpm is the number of revolution per minute of the centrifuge rotor

7.  The time required to separate particles depends on the rotor speed, radius of rotor
and effective path length travelled by sedimented particles.
 The following formula useful for the calculation of the speed required for a rotor
with a radius that differ from the radius with which a prescribed,
 R.P.M. =1000× √RCF
1.18×r
 The length of time of centrifugation also can be calculated so, that running with an
alternative rotor of a different size is equivalent to running with a original rotor.

8. Types of Centrifuge depends on :
 Speed of sedimentation (ultra centrifuge or high speed centrifuge).
 Presence /absence of vacuum (ultra centrifuge or small bench top)
 Temperature control refrigeration.
 Volume of sample and capacity of centrifugation tubes

9.  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.

10.  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
Small Bench top

11. Micro centrifuge (“microfuge”,
“Eppendrof”)
 Sample volume is small in
eppendrof tubes
 Refrigerated with or without
 Centrifuge maximum approx
10000 g
 Take tube of small volume
up to 2ml.
 Commonly used of
concentration protein

12. High speed centrifuge
 Refrigerated
 Use for protein precipitates, large intact
organelles cellular debris from tissue
homogenization and microorganism
 They operate maximal centrifugal force of
approx 50000g
 Use for research applications
 Differential separation of nucleus,
mitochondrial, protein precipitate, etc.

13. Ultra centrifuge
 Refrigerated and evacuated
 The detail biochemistry analysis
of subcellular structures and isolate
biomolecules.
 Operate at upto 90000 g

14. Type of rotor
 Fixed angle rotor
 Swinging bucket rotor

15. Fixed angle rotor
 Ideally tool for pelleting
 Isopycnic banding may form
 Centrifugation tubes are held at at fixed angle of between
14'-40' to vertical axis of rotation.
Start of centrifugation particles are driven outward
horizontally but strike side of the tube so that
sediment pack against the side & bottom of the tube,
with the surface of the sediment parallel to the shaft
of centrifuge.

17. Swinging bucket rotors:
 Sample tubes are loaded into individual buckets that hang vertically while
the rotor is at rest.
 When the rotor begins to rotate the buckets swing out to a horizontal
position. Useful when samples are to be resolved in density gradients.
 The longer path length permits better separation of individual particle types
from a mixture.
 This rotor is relatively inefficient for pelleting .

19. Centrifuge Calibration
Purpose
This procedure provides accurate rotation speed, timer
verification and centrifuges that are temperature
controlled in a laboratory environment.

20. Verification of rotation speed:
Centrifuges used in the laboratory are to be considered as contaminated and
should only be handled with gloves and other personal protective equipment
and/or thoroughly disinfected before calibration verification.
Place a small section of black and white reflective tape that comes with the
tachometer on the center spindle of the test centrifuge.
In order to measure the rotation speed, there must be a viewing port in the top
cover that will allow the tachometer line of sight to the reflective tape.
Place a normal well-balanced load using specimen covers into the centrifuge.
Start the centrifuge and allow it come to equilibrium at a normal operating
speed.
Use the tachometer through the viewing port above the reflective tape to take
a rotation rate reading.
Record the rotation speed indicated by the centrifuge either by the dial setting
or by a built in tachometer on the centrifuge .

21. Verification of timer :
Centrifuges used in the laboratory are to be considered as
contaminated and should only be handled with gloves and other
personal protective equipment and/or thoroughly disinfected before
calibration verification.
Set the centrifuge timer at a setting frequently used in procedures, and
start the stopwatch simultaneously.
Stop the stopwatch at the same time as the centrifuge timer ends.
Calculate the difference between the two times .

22. Interpretation of results:
Verification of rotation speed:
If the difference between the test centrifuge and the certified tachometer
is ± 5% of the procedure speed, then the test centrifuge rotation
calibration is verified as acceptable.
Verification of timer:
Acceptable difference between the test timer and the certified timer must
be ±2% of the total test timer setting.

23. SAFETY MEASURES
 The work surface must be level and firm.
 Balance the tubes in the rotor.
 Do not open the lid while the rotor is moving.
 Bucket is properly cushioned.
 If you see it wobbling or shaking, turn it off or pull the plug. A little vibration is normal, but
excessive amounts can mean danger.
 Wear a face shield and / or safety goggles if you have to work anywhere near a centrifuge
that’s in use.
 Examine tubes and bottles for cracks or stress marks before using them.
 Discard any centrifuge tubes that have cracks in them
 Clean the rotor and centrifuge well after each use

24. Maintenance of Centrifuge
Daily maintenance
 Wipe the inside of the bowl with disinfectant solution and rinse thoroughly.
 The centrifuge must not be used if the interior is hot, if unusual vibrations or noises occur, or if
deterioration (corrosion of parts) is detected.
 A qualified service technician should be contacted.
 Most vibrations are due to improper balancing and can be corrected by rebalancing the buckets and
tubes.
Monthly maintenance
 Clean the centrifuge housing, rotor chamber, rotors and rotor accessories with a neutral cleaning
agent.
 Clean plastic and non-metal parts with a fresh solution of 0.5% sodium hypochlorite .

25. Application
 Remove cellular elements from blood to provides cell free serum or
plasma
 Conc. Cellular element for microscopy.
 Remove protein precipitate from analytic sample.
 Isolation of macromolecules such as DNA, RNA, proteins, or lipids
 Use in haematology lab for PCV determination.

26. Reference
 Principles and technique in biochemistry-L WALKER&
WILSON
 Lynch's Medical Laboratory Technology (4th edition)

27. Thank you