Analytical tools

1. Centrifuge
 The centrifuge used to separate solids
suspended in a liquid by sedimentation.
 The rotational movements allow forces
much greater than gravity to be generated
in controlled periods of time.
 In the laboratory, centrifuge can used to separate
blood components: red cells, white cells, platelets to
carry out further analysis tests and treatments.
 There is a wide range of centrifuges capable of
serving specific industry and research.

2. Centrifugation is the separation of particles by sedimentation (Relative centrifugal
force and Stoke's law)
A centrifuge works by using the principle of sedimentation: Under the influence of
Gravitational force (g-force), substances separate according to their density.
Centripetal force=object moving in a curved path and that is directed inward
toward the center of rotation
Centrifugal force=object moving in a curved path that acts outwardly away from
the center of rotation
Relative centrifugal force (F); M: mass of
particle r: radius of rotation (cm), ω :Average
angular velocity
Stoke's law

3. Centrifugal Field (G): G=rω2
Depends on the radical distance of the particle from the rotation
axis and the square of the angular velocity
Where;
 S is increased for particle of larger mass (because sedimenting force a
m(1-vr)
 S is increased for particle of larger density (equal volume)
 S is increased for more compact structures (Shape) of equal particle
mass (frictional coefficient is less)
 S is increased with rotational speed
Sedimentation Coefficient (S)= BALANCE between the sedimenting
force and counteracting force
•Sedimentation force, mpω2r where mp = the mass of equal volume
of solvent

4. Theory of operation
 When a body of mass [m] turns around a
central point [O], it is subjected to a
centripetal force [F] directed towards the
rotation axis with a magnitude
F = mω2r.
 where [m] is the mass of the body, [r] is
the radius and ω is the angular speed.
 Denser particles will settle at the bottom of
the tube in shorter periods of time, while
lighter ones require longer periods of time.

6. Components of the centrifuge
 Control section: 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 (optional).
 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. Centrifuge Rotors
1. Fixed angle rotors
Hold tubes at an angle of 45° in relation
to the axis of the rotor
The particles strike the opposite side of
the tube where the particles finally slide
down and are collected at the bottom

8. Swinging bucket rotors/ Horizontal
rotors
Hold the tubes at an angle of 90°
The tubes are suspended in the racks
that allow the tubes to be moved
enough to acquire the horizontal
position
tubes remain horizontal, the
supernatant remains as a flat surface

9. Vertical rotors
The shortest path length, fastest run
time, and the highest resolution of all the
rotors
Particles tend to spread towards the
outer wall of the tubes
Commonly used in isopycnic and
density gradient centrifugation

10. Types of centrifuges
 Benchtop centrifuges
 Refrigerated Centrifuges
 Clinical Benchtop centrifuges
 Microcentrifuges
 Vacuum Centrifuges
 Ultracentrifuge
 Preparative Ultracentrifuges
 Analytical Ultracentrifuges

11. ULTRACENTRIFUGES
 Ultracentrifuge has a maximum speed of 65,000 RPM
(100,000’s x g).
 Intense heat is generated due to high speed thus the
spinning chambers must be refrigerated and kept at a
high vacuum.
 It is used for both preparative work and analytical
work.
 Example
 Preparative Ultracentrifuges
 Analytical Ultracentrifuges

12. High-speed centrifuges
 High-speed centrifuges are used in more sophisticated
biochemical applications, higher speeds and temperature
control of the rotor chamber are essential.
 The high-speed centrifuge has a maximum speed of 15,000 –
20,000 RPM
 The operator of this instrument can carefully control speed and
temperature which is required for sensitive biological samples.
 Three types of rotors are available for high-speed
centrifugation-
 Fixed angle
 Swinging bucket
 Vertical rotors

13. LOW-SPEED CENTRIFUGE
 Most laboratories have a standard low-speed centrifuge used
for routine sedimentation of heavy particles
 The low-speed centrifuge has a maximum speed of 4000-
5000rpm
 These instruments usually operate at room temperatures with
no means of temperature control.
 Two types of rotors are used in it,
 Fixed angle
 Swinging bucket.
 It is used for sedimentation of red blood cells until the particles
are tightly packed into a pellet and supernatant is separated by
decantation
Example: Clinical Benchtop centrifuges

14. Types of centrifugation
 Analytical centrifugation
 Preparative ultracentrifuges
 Density gradient centrifugation
 Rote-zonal centrifugation
 Isopycnic centrifugation

15. Analytical centrifugation
 Technique for determining the shape, molecular weight, and purity of
pure chemicals and macromolecules.
 This method is employed in two different types of experiments
 Sedimentation velocity and sedimentation equilibrium studies, both
of which are important in macromolecular characterisation.
Preparative ultracentrifuges
 Biological materials are processed in preparative ultracentrifuges for
subsequent examination. Preparative ultracentrifugation is most
commonly used in tissue and subcellular fractionation to extract
increasingly smaller components of biological samples. i.e
Differential Centrifugation, Rote-zonal centrifugation,
Density gradient centrifugation, Isopycnic centrifugation

18. Differential Pelleting (differential
centrifugation)
 Tissue such as the liver is
homogenized at 32 degrees
in a sucrose solution that
contains buffer.
 The homogenate is then
placed in a centrifuge and
spun at constant centrifugal
force at a constant
temperature
 Differential centrifugation is
commonly used for the
separation of cell organelles
and membranes found in the
cell.
 It can also be used for low-
resolution separation of the
nucleus.
 As this technique separates
particles based on their sizes,
this can be used for the
identification and
comparison of particles of
different sizes.

19. Density Gradient Centrifugation
 Important technique for purifying proteins and
particularly nucleic acids.
 Two different types of density gradient centrifugation,
for two different purposes are:
 Zonal (or Rate Zonal) Centrifugation (Sucrose density
gradient centrifugation)
 Iso-density (Isopycnic) Centrifugation (Caesium
chloride density gradient centrifugation)

20. Moving Zone (differential) Centrifugation
Under centrifugal force, the particles will begin sedimenting through
the gradient in separate zones according to their size shape and
density

21. Iso-density (Isopyncic) Centrifugation
 Molecules separated on equilibrium position, NOT by
rates of sedimentation.

23. General Application of cetntrifugation
 To separate two miscible substances
 To analyze the hydrodynamic properties of macromolecules
 Purification of mammalian cells
 Fractionation of subcellular organelles (including
membranes/membrane fractions) Fractionation of membrane vesicles
 Separating chalk powder from water
 Removing fat from milk to produce skimmed milk
 Separating particles from an air-flow using cyclonic separation
 The clarification and stabilization of wine
 Separation of urine components and blood components in forensic
and research laboratories
 Aids in the separation of proteins using purification techniques such
as salting out, e.g. ammonium sulfate precipitation.

24. There is a vibration.
•The rotor is unbalanced.
•The speed selected is near the
rotor’s critical speed range
•The rotor is incorrectly
mounted.
•There is a lack of lubrication in
the rotor’s supports.
 Balance the rotor’s load. Fill all the opposite tubes with
the same level of liquid of same density.
 Distribute the weight of the opposite tubes
symmetrically.
 Load fixed angle or vertical tube rotors symmetrically
 Select a rotation outside of the critical speed range.
 Select a rotation outside of the critical speed range.
 Verify the rotor’s assembly. Test that it is well adjusted
 Lubricate the pivoting axis according to the
manufacturer’s recommendation.

25. The tubes are cracked or broken.
The tubes can be broken
or become fragile if they
are used below the
recommended
temperature.
The tubes become fragile
with age and use.
 If the sample is frozen, warm to 2 °C before
centrifuging. Evaluate how the tubes behave
at low temperatures before centrifuging
 Discard expired tubes, use new ones.

26. Thank You.