Centrifugation

1. Working Principle and Uses

2. Centrifugation is a process in which centrifugal force is
used to separate solid matter from a liquid suspension.
Parts
Head or Rotor
Carriers,or shields
 They are attached to the vertical shaft of a motor and enclosed in a
metal covering.
 Centrifuge always has a lid and an on/off switch;
 Many models include a brake or a built in tachometer, which indicates
speed, and some centrifuges are refrigerated.

3. Depends on three variables:
Mass,
Speed, and
Radius.
The speed is expressed in revolutions per minute (rpm),
The centrifugal force generated is expressed in terms of relative
centrifugal force (RCF) or gravities (g).
Centrifugal Force

4.  Particles which differ in density, molecular weight, size
or shape sediment at different rates.
 The rate of sedimentation depends upon:
 Applied centrifugal field.
 Density and radius of the particle
 Viscosity of the suspending medium.

6. Angular velocity = W radians/sec
= W x 60 rad/min
One Revolution = 3600
= 2π rad (π rad= 180)
Revolution /min (rpm) = W x 60
2π
W = 2 π x(rpm)
60
Centrifugal Field = W2xr = (2π x rpm)2 x r
(60)2
= 4π2 x (rpm)2 x r
3600
* r= Radical distance of particle from axis of rotation.

7.  Relative Centrifugal Force (RCF) measures acceleration
applied to the sample
RCF=(W2 x r)/g
r=distance from center or motor to sample
ω= angular velocity (radians/ second)
g=9.8m/s2
RCF = 1.118 x 10-5 x r x (rpm)2

8.  Centrifugation is used to separate two immiscible liquids.
 More-dense components of the mixture migrate away from
the axis of the centrifuge, while less-dense components of
the mixture migrate towards the axis.
 The remaining solution is properly called the "supernate" or
“supernatant liquid".
 The supernatant liquid is then either quickly decanted from
the tube without disturbing the precipitate.

9. Power
LED
Timer
Rotor

11. Operation
Place tubes in centrifuge.
Always use correct size tubes to prevent tube damage.
Always using a counterbalance:
 By mass NOT volume
 Put the tubes opposite each other in the centrifuge.
If you have more than two tubes, only the ones
opposite each other have to be equal in mass.
If centrifuge has variable speeds, enter RPM.
Close lid.
Turn timer on or press start.
Remove the tubes carefully after the centrifuge has
completely stopped spinning to prevent remixing.

12. Types of Centrifuges
Centrifuges are generally divided into 3 categories based on their
maximum attainable speed:
1. "Low-speed": max ~5 x 103 rpm.
2. "High-speed": max ~2 x 104 rpm.
3. "Ultracentrifuges": max ~105 rpm
 Others
 Variable Temperatures
 Vacuum Capabilities

13. Types of Centrifuges:
1. Small Benchtop – with or without refrigeration
– slow speed (eg up to 4000 RPM)
– common in clinical labs (blood/plasma/serum
separation)
– can take approx (up to) 100 tubes, depending on
diameter
2. Microcentrifuges (“microfuge”, “Eppendorf”)
– take tubes of small vols (up to 2 mL)
– very common in biochemistry/molecular
biology/biological labs
– can generate forces up to ~15,000 x g
– with or without refrigeration.

14. 3. High Speed centrifuges
– 15,000 – 20,000 RPM
– Large sample capacity depending on rotor
– Normally refrigerated
– Research applications
4. Ultracentrifuges → 65,000 RPM (100,000’s x g)
– Limited lifetime
– Expensive
– Require special rotors
– Care in use – balance critical!
– Research applications

15. Types of Rotors
1.Swing-out (swinging bucket)
• Common in low speed centrifuges.
• Also high speed, ultracentrifuges.
• Tubes accommodated in a pivoted bucket which rotates from a
vertical to a horizontal position during acceleration.
• Bucket returns to vertical as centrifuge decelerates.
• Meniscus of sample always remains at right angles to axis of
tube.
• Six-place rotor (6 buckets) most useful – can spin 2,3,4
or 6 samples (or sets of samples)

16. 2. Fixed-Angle Rotors
 Tubes in pocket at fixed angle in rotor.
 Angle 10 to 50 degrees from vertical – at rest and during
spin.
 Use up to 600,000 x g.
3. Vertical Rotors – For high and Ultracentrifuge.

18. Applications
Separate solids from solution
Used during urinalysis to separate disease identifying
components
Separate blood into plasma and cellular components

19.  Used to separate serum or plasma from the blood
 To separate a supernatant from a precipitate during an
analytic reaction
 To separate two immiscible liquids, such as a lipid-laden
sample; or to expel air.
Applications

20. Safety
 Before using, read the owner's manual.
 Do not operate a centrifuge until having been shown
proper use by an experienced operator.
Check that the centrifuge chamber and drive spindle
of the rotor are free of scratches or burrs.
Decontaminate centrifuge of biological hazards
before servicing.

21. Safety
Never repair a cracked or bent rotor.
If rotor has been replaced, tighten knob/screw fastening
rotor to the shaft to prevent hazardous loosening.
Prolonged contact w/ some disinfectants can cause
damage to rotor and other components (e.g. 10% sodium
hypochlorite).
Never attempt to touch or stop a spinning rotor by hand or
with another tool or object.

22.  Daily cleaning of any spills or debris, such as blood or glass.
 Ensure that the centrifuge is properly balanced and free from
any excessive vibrations.
 Balancing the centrifuge load is critical .
 Many newer centrifuges will automatically decrease their
speed if the load is not evenly distributed, but more often,
the centrifuge will shake and vibrate or make more noise
than expected.
Care to be taken..

23. Result of unbalanced
centrifuge

24. Thank You