Centrifugation uses centrifugal force to separate particles in a solution based on their size, shape, density, and other properties. A centrifuge applies this force by rapidly spinning samples placed in tubes or other holders. There are different types of centrifuges and rotors that are suited for different separation purposes like pelleting, density gradient separation, or zonal centrifugation. The relative centrifugal force or RCF is used to quantify and compare the force applied across different centrifuge models and rotor configurations.

1. Centrifugation
Mr. R. K. Lodha

2. Definition of Centrifugation
• Centrifugation is a technique
of separating substances
which involves the
application of centrifugal
force.
• A centrifuge is a device for
separating particles from a
solution according to their
size, shape, density,
viscosity of the medium and
rotor speed

3. Parts of a Centrifuge
▶ Core part is a rotor
▶ Fixed no. or arms radiating from it
a certain angle
▶ Hollow tubes attached for
sample placement
▶ Ball bearings
▶ Aluminium/Titanium graded drum
▶ Motor and brake assembly
▶ Controlling nobs

4.  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.
 If there is no difference in density
(isopyknic conditions), the particles
stay steady.
 To take advantage of even tiny
differences in density to separate
various particles in a solution,
gravity can be replaced with the
much more powerful “centrifugal
force” provided by a centrifuge. 4
Principle of Centrifugation

5. principle, where the centripetal
The centrifuge works using the sedimentation
acceleration
causes denser substances and particles to move
outward in the radial direction.
At the same time, objects that are less dense are
displaced and move to the center.
In a laboratory centrifuge that uses sample tubes, the
radial acceleration causes denser particles to settle
to the bottom of the tube, while low- density
substances rise to the top.
A centrifuge is a piece of equipment that puts an
object in rotation around a fixed axis (spins it in a
circle), applying a potentially strong force
perpendicular to the axis of spin (outward) (g-force).

7.  When centrifugal force applied by the centrifuge,
particles move faster (> g).
 For example, when sand particles added in the
water filled bucket it travels slower but it sediment
faster when bucket is swung around in a circle.
 The biological materials show a drastic increase
sedimentation when they undergo under
acceleration in centrifugal force.
 Relative centrifugal force (RCF) is expressed as a
multiple of the acceleration (G) due to gravity (g).

8. Basic Principle of Sedimentation
 When a biological sample moves in centrifuge, it
experiences an outward centrifugal force.
 Rate of sedimentation of biological sample is depend on the
applied centrifugal field.
 The applied centrifugal force is determined by the radial
distance of the particle from the axis of rotation.
 If the angular velocity of particle ω and the radial distance
of a particle r , applied centrifugal field is G would be
G= ω2 r …………………….(1)
 If the mass of the particle m, centrifugal force F
,then
F = mG = m ω2 r

9. 9
Basic Principle of Sedimentation
Relative centrifugal force F  M2
r
M: mass of particle
r: radius of rotation (cm) (ie
distance of particle from axis
of rotation)
 :Average angular velocity
(radians/sec)
 
2 rev min-1
60
Rev: revolution per minute
(r.p.m.)
1 revolution = 2π radians
=360

10. Angular velocity ω = 2π s
Where s = frequency
 Frequency s can defined as numbers of revolutions (cycles) per
second. We can express the angular velocity in per minute then,
ω = 2π (rev per min)/ 60 ………… (2)
Put value of ω from equation (2) to equation (1)
G= 4 π 2 (rev per min) 2/ 3600 ---------------------(3)
 The centrifugal field is generally expressed in multiples of the
gravitational field g (981cm/s 2).

11. Relative centrifugal force (RCF) is the ratio of the centrifugal
acceleration (G) and gravitational acceleration (g).
RCF= G/g
Putting value of G from previous eq.(3),
RCF= 4 π 2 r (revper min) 2/ 3600*981
RCF=1.12 x 10-5 (r.p.m.)2 x r
RCF unit is dimensionless
So the relative centrifugal force (RCF) applied to the particle in
centrifugation can be calculated.
Relative centrifugal force

12.  2
r g-1
f
RCF  c

fg Mg
M2
r
60
 r g-1
 
RCF  
 2 rmp 
2
RCF value
"No. x g"
(multiples of earth's gravitational force).
Relative Centrifugal Force
(RCF)
RCF =
1.12 x 10-5 x (rpm)2 x r
Because rotors are
different from various
manufactures, we use
RCF to represent the
centrifugation force.
rpm:
revolution per minute
r: radius of rotor RCF (x1000)
RPM
(x1000)
Radius
Min
Radius
Ave
Radius
Max
Radius

13. Relative centrifugal force
RCF =
1.12 x 10-5 x (rpm)2 x r
rmin
rmax

14. Centrifugal Field
G=r2 depends on the radical distance of the particle from the
rotation axis and the square of the angular velocity
4 2
rev min-1
2
r
3600
10
G 

15. 11
Angular Velocity
60
 
2 rev min-1
rev: revolution per minute (r.p.m.)

16. Interacting Forces in Centrifugation
Sedimenting force, mp2r, is opposed by...
mp = the mass of equal volume of solvent
. Frictional Resistance against particle
moving through fluid = f.v
M -M 
2
r - fv
p s
Fcentrifuge
Ffriction + Fbuoyancy
f = frictional coefficient of particle in the solvent
v = particle velocity
. Flotation Force F=ms r  2
BALANCE between the sedmenting force and counteracting force
Net force = (mp –ms)r  2 - fv

17. Sedimentation Coefficient (s)
w 2r(mp-ms) - fv = 0
Theodor Svedberg (1884-1971),
Chemist from Sweden 1926
Nobel prize 1908.
He described a new method (ultracentrifuge) of
producing colloid particles and gave convincing
evidence of the validity of the theory on the
Brownian movements

18. m= particle mass
f = frictional coefficient of the particle in the solvent
= density of solution
v = particle velocity
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
Mild, non-denaturing procedure, useful for protein
purification, and for intact cells and organelles
S Can be considered
“Sedimentation Rate” of a particle
under centrifugation force
=(dr/dt)/(1/ r2)

19. Separation by Sedimentation
100 kg 1
30 kg 10 kg 10 kg
Stone Iron Stone
8
Weight
Material Iron Cotton Iron
100 kg
10 kg
1
30 kg
10 kg
8
 Mass
 Density
 Shape
Sedimentation
Higher
density

20. 6
Centrifugation
A centrifuge is used to separate particles or macromolecules:
-Cells
-Sub-cellular components
-Proteins
-Nucleic acids
Basis of separation:
-Size
-Shape
-Density
Methodology:
-Utilizes density difference between the
particles/macromolecules and the medium in which these are
dispersed
-Dispersed systems are subjected to artificially induced
gravitational fields

21. Subcellular Fractionation
Densities and sedimentation coefficients for
biomolecules, cell organelles, and viruses.
Require high
density media
High concentrated
CsCl

22. Sedimentation
Soluble
protein
DNA
RNA

23. g (RCF) & RPM Conversion
 Relative Centrifugal Force (RCF) or g force is the acceleration
applied to the sample. RCF is relative to the force of Earth’s gravity
and depends on revolutions per minute (RPM) and radius of the
rotor.
 Centrifugation protocols use Relative centrifugal force (RCF) as this
is more precise than RPM because the rotor size might differ, and
RCF will be different while the revolutions per minute stay the same.
Modern centrifuges have an automatic converter, but older ones do
not.
 There are several ways to convert g force (RCF) into revolutions
per minute (RPM) and vice versa:
1. Use online converters
2. Use the formula:
a. RCF = (rpm)2 × 1.118 × 10-5 × r
b. RPM = √[RCF/(r × 1.118)] × 1,000
3. Use a nomogram (nomograph).

24. 21
Radical
distance
(mm)
Relative
centrifugal
field (xg)
Rotor
speed
(r.p.m)
NOMOGRAMS
Conversion
between
relative
centrifugal
force

26. Five types of rotors are available for
centrifugation:
1. Fixed-angle rotor,
2. Swinging-bucket rotor
3. Vertical rotor and
4. Near-vertical rotor
5. Continuous-flow rotor
Centrifuge Rotors types

27. 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.

28. 2.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.

29. 3.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.
 Not suitable for pelleting

30. 4.NEAR VERTICAL
ROTOR
 Near-vertical rotors are
designed for gradient
centrifugation when there are
components in a sample
mixture that do not participate
in the gradient.
 Tubes are held at an angle
(typically 7 to 10 degrees) to the
axis of rotation in numbered
tube cavities.
 Used in isolation of
molecules like plasmid DNA,
RNA, Lipoprotein.

31. 5. CONTINUOUS FLOW ROTOR
 Used industrially and for few
lab separations like recovery of
bacteria from litres of culture
solution.
 The sample is ejected
through the centre and
separated at a specific speed
 Sample is extracted by
injecting high density liquid
from outside wall while the
rotor is running

33. Fixed Angle Rotor
Swinging
Bucket Rotor
Vertical Tube Rotor
Centrifuge Rotors types

36. Fixed Angle Rotor  Swinging Bucket Rotor
Sedimenting particles have only
short distance to travel before
pelleting. Shorter run time.
The most widely used rotor type.
Longer distance of travel may allow
better separation, such as in density
gradient centrifugation. Easier to
withdraw supernatant without
disturbing pellet.

37. Types of Centrifuge
Types based on-
 Maximum speed of sedimentation
 Presence /absence of vacuum
 Temperature control refrigeration
 Volume of sample and capacity of
centrifugation tubes

38. Types of Centrifuge
Large-capacity low-speed preparative
centrifuges
Refrigerated high-speed preparative centrifuges
Analytical ultracentrifuges
Preparative ultracentrifuges
Large-scale clinical centrifuges
Small-scale laboratory microfuges

40. 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.

41. Microcentrifuge
s⚫Microcentrifuges are used to
process small volumes of biological
molecules, cells, or nuclei.
⚫Microcentrifuge tubes generally
hold 0.5 - 2 ml of liquid, and are
spun at maximum angular speeds
of 12000–13000 rpm.
⚫Microcentrifuges are small enough
to fit on a table-top and have rotors
that can quickly change speeds.
⚫They may or may not have
a refrigeration function.

42. Small Bench Centrifuge
⚫Simplest centrifuges that are used to
separate erythrocytes, Blood samples,
coarse precipitates and cells are known
an bench or laboratory centrifuges.
⚫They have a speed ranging from 4000 –
6000 RPM and a relative centrifugal
force of 3000 – 7000 g.
⚫Small samples are sedimented now a
days with microfuge that after a speed
of 8000- 13000 RPM and relative RCF
of approximately 10000 g.
⚫They sediment small volume (250 mm3
to1.5 cm3 ) of material in 1 or 2 min.

43. High-speed centrifuges
⚫High-speed
centrifuges
or super
can handle
speed
larger
sample volumes, from a few tens of
millilitres toseveral litres.
⚫Additionally, larger centrifuges can
also reach higher angular velocities
(around 20000 rpm).
⚫The rotors may come with different
adapters to hold various sizes
of test tubes, bottles, or microliter
plates.
High-speed centrifuges

44. 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 rotor

46. Ultracentrifuges
⚫Ultracentrifuges can also be used in
the studyof membrane fractionation.
⚫Can reach maximum angular
velocities in excess of 70000 rpm.
⚫Ultracentrifuges
molecules in batch
f low systems.
⚫During the run, the
can separate
or continuous
particles or
molecules will migrate through the
test tube
depending
at different
on their
speeds
physical
properties and the properties of the
solution.

47.  Microfuge
0.5-1.5 cm3, 10,000 g
Concentration of protein samples
 Large-capacity preparative
centrifuge
5-250 cm3, 3,000-7,000 g

48. 24
 High-speed refrigerated centrifuge
5-250 cm3, 100,000 g
Differentiation separation of nucleus,
mitochondrial, protein precipitate, large
intact organelle, cellular debris
 Ultracentrifugation
5-250 cm3, 600,000 g
Microsomal vesicles, ribosome
Has to reduce excessive rotor temperature
generated by frictional resistance
→ sealed chamber, evacuated, cooling

49. ULTRACENTRIFUGES
It is the most sophisticated instrument.
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

52. Types of Ultracentrifugation

53. Ultracentrifugation
Preparative
ultracentrifugation
Analytical
ultracentrifugation
Differential
centrifugatio
n
Density gradient
centrifugation
Rate zonal
centrifugation
Isopycnic
centrifugation

54. Type 1– Analytical Ultracentrifugation (AUC)
http://www.cgmh.org.tw/chldhos/intr/c4a90/new_page_50.htm
1.肌膜
肌膜
Determine the mass, shape and stoichiometry ratio of non-
covalent association of macromolecules (protein-protein,
small molecule-protein, quaternary structure)
1. Rotates at high speeds e.g.
30000 rpm
2. The high speeds used in
such devices generate
considerable amounts of
heat
3. Therefore cooling
arrangements are required
in ultracentrifuges

55. Analytical Ultracentrifugation
An analytical ultracentrifuge spins a rotor at an accurately
controlled speed and temperature. The concentration
distribution of the sample is determined at known times
using absorbance measurements.
It can determine:
 Purity of macromole
 Relative molecular mass of solute (within 5% SD)
Change in relative molecular mass of supermolecular
complexes
 Conformational change of protein structure
 Ligand-binding study
Continuously monitor the sedimentation process

56. This figure displays a schematic
diagram of the Beckman
Optima XL-A absorbance
system. A high intensity xenon
flask lamp allows the use of
wavelengths between 190 and
800nm. The lamp is fired briefly
as a selected sector passes the
detector.
(Beckman Optima XL-A):
ck to top
Optical System of an Analytical
Ultracentrifugation

57. Type 2– Preparative Centrifugation
 Collect (isolation) material:
cell, subcellular structure, membrane vesicles
1. Handle larger liquid volumes (i.e.
1 to several thousand litres)
2. Range of designs
3. Typical rotating speed: 500 - 2000
rpm
Immunofluorescent imaging of
human cells (U2OS) with pan
Cadherin antibody

59. It is the most common type of centrifugation employed. 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. After some time a sediment
forms at the bottom of a centrifuge called pellet and an overlying solution
called supernatant. The overlying solution is then placed in another
centrifuge tube which is then rotated at higher speeds in progressing steps.

60. Differential Centrifugation
• Based on the differences in the
sedimentation rate of the biological particles
of different size, shape and density

62. Moving Boundary (differential velocity) Centrifugation
1) The entire tube is filled with sample and centrifuged
2) Through centrifugation, one obtains a separation of two
particles but any particle in the mixture may end up in the
supernatant or in the pellet or it may be distributed in both
fractions, depending upon its size, shape, density, and
conditions of centrifugation
3) Repeat sedimentation at different speed
1) 3)
2)

63. 31
Medium: same density
The sedimentation speed is determined mainly on the
size, shape of particle.
Application: low resolution separation such as
preparation of nucleus
Differential Velocity Centrifugation cont.

67. Density Gradient Centrifugation
• This type of centrifugation is mainly used to purify
viruses, ribosomes, membranes, etc.
• A sucrose density gradient is created by gently
overlaying lower concentrations of sucrose on higher
concentrations in centrifuge tubes
• The particles of interest are placed on top of the
gradient and centrifuge in ultracentrifuges.
• The particles travel through the gradient until they
reach a point at which their density matches the
density of surrounding sucrose.
• The fraction is removed and analyzed.

69. Rate-Zonal Density-Gradient
Centrifugation
• Zonal centrifugation is also known as band or gradient
centrifugation
• It relies on the concept of sedimentation coefficient (i.e.
movement of sediment through the liquid medium)
• In this technique, a density gradient is created in a test tube
with sucrose and high density at the bottom.
• The sample of protein is placed on the top of the gradient and
then centrifuged.
• With centrifugation, faster-sedimenting particles in sample
move ahead of slower ones i.e. sample separated as zones in
the gradient.
• The protein sediment according to their sedimentation
coefficient and the fractions are collected by creating a hole at
the bottom of the tube.

72. Moving Zone Centrifugation
1.Preparation of gradient sucrose density for
centrifugation medium
Density1 < Density2 < Density 3 < Density 4 < DensityAnalyte
2.Sample is applied in a thin zone at the top of the
centrifuge tube on a density gradient
1 2 3 4

73. 3. Under centrifugal force, the particles will begin
sedimenting through the gradient in separate zones
according to their size shape and density
Insufficient time--------- Incomplete separation
Overtime--------------------co precipitation of all analytes35
Moving Zone (differential) Centrifugation –cont.

74. Iso-density (Isopyncic) Centrifugation
Isopycnic = Equal density
Molecules separated on equilibrium position, NOT by rates
of sedimentation.
After centrifugation, each molecule floats or sinks (=re-
distribution) to position where density equals density of CsC
(or sucrose)l solution. Then no net sedimenting force on
molecules and separation is on basis of different densities of
the particles.

75. • The sample is loaded into the tube with the gradient-forming
solution (on top of or below pre-formed gradient, or mixed in with
self-forming gradient)
• The solution of the biological sample and cesium salt is uniformly
distributed in a centrifuge tube and rotated in an ultracentrifuge.
• Under the influence of centrifugal force, the cesium salts
redistribute to form a density gradient from top to bottom.
• Particles move to point where their buoyant density equals that
part of gradient and form bands. This is to say the sample
molecules move to the region where their density equals the
density of gradient.
• It is a “true” equilibrium procedure since depends on bouyant
densities, not velocities
• Eg: CsCl, NaI gradients for macromolecules and nucleotides –
“self-forming” gradients under centrifugal force.
Isopycnic Centrifugation

76. Iso-density (Isopyncic) Centrifugation
Preparation of gradient sucrose density for
centrifugation medium
The gradient density has to cover the range of
different densities of analytes

79. 38
Comparison of Two Methods
Isopynic
centrifugation
Moving Zone
Centrifugation
but different in MW)
Sedimentation equilibrium
Similar MW,
different density
Protein (similar density,
Sample:
Sedimentation Rate
Similar density,
different MW
Nucleic acid /
cell organelle
Centrifugation: Lower speed, not
complete sedimented,
stop at proper time
Completely sediment to where
the density is equilibrated, high
speed, long running time

80. Precautions
A centrifuge user should strictly observe the following
precautions :
1. Manufacturer’s manual should be strictly followed.
2. Rotorshould be stored in propercontainers.
3. Attention should be given to imbalance detectors.
4. Rotorspeed should notexceed the assigned speed.
5. Lid of the rotor chamber should remain locked during
operation.
6. To avoid the rotor failure, manufactures instructions
regarding rotor care and use should always be followed.

81. Centrifuge Its Use and Safety
On December 16, 1998, milk samples were running in a
Beckman L2-65B ultracentrifuge using a large aluminum
rotor . The rotor failed due to excessive mechanical stress.

82. Mechanical stress
Always ensure that loads are evenly balanced before
a run.
Always observe the manufacturers maximum
speed and sample density ratings.
Always observe speed reductions when running high
density solutions, plastic adapters, or stainless steel
tubes.

83. Corrosion
⚫Many rotors are made from either titanium or aluminium
alloy, chosen for their advantageous mechanical
properties.
⚫While titanium alloys are quite corrosion-resistant,
aluminium alloys are not.
⚫When corrosion occurs, the metal is weakened and less
able to bear the stress from the centrifugal force exerted
during operation.
⚫The combination of stress and corrosion causes the rotor
to fail more quickly and at lower stress levels than an
uncorroded rotor.

84. Applications in Biological Sciences
⚫ To separatecellularand subcellularcomponents
⚫ Separating onecell type fromanother.
⚫ Removing cells or other suspended particles from their
surrounding milieu on eithera batch oracontinuous-flow basis.
⚫ Isolating viruses and macromolecules, including DNA, RNA,
proteins, and lipids or establishing physical parameters of these
particles from theirobserved behaviourduring centrifugation.
⚫ To study the effects of centrifugal forces on cells, developing
embryos, and protozoa.
⚫ These techniques have allowed scientists to determine certain
viscosity of
properties about cells, including surface tension, relative
the cytoplasm, and the spatial and functional
interrelationship of cell organelles when redistributed in intact
cells.

85.  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
Applications of Centrifugation

86. Conclusion
⚫The centrifugation is a modern & easy technique of
separation and sedimentation on the basis of shape, size
and densityof macromolecules and otherparticles.
⚫In the centrifugation there are different types of forces are
applied like as centrifugal force, gravitational force and
centripedal force etc. and also different types of rotors are
to be used that is; Swinging Bucket Rotor and fixed angle
rotors atdifferent RPM/RCF.