1. CENTRIFUGATION
Techniques

2.  A centrifuge is a device that separates particles
from suspensions or even macromolecules from
solutions according to their size, viscosity of the
medium and density by subjecting these dispersed
systems to artificially induced gravitational fields by
rotor length and speed.

3. 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.  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 field

5.  Separation (solid/liquid, solid/liquid/liquid and
solid/solid/liquid separation):
 Centrifugation can be used for solid-liquid separation
provided the solids are heavier than the liquid.
Centrifuge can also be used to separate a heavy phase,
and two lighter liquid phases, with one of the lighter
phases being lighter than the other. Solids can be lighter
than liquid and separation is by flotation of the dispersed
solid phase.

6.  Clarification- minimal solids in liquid product
 Centrifuge can be used to clarify the discharged
separated lighter liquid phase. The objective is to
minimize the discrete suspendedsolids in the light
continuous phase. Usually, only fine submicron biosolids
are left uncaptured by centrifugation and they escape
with the discharged light phase.
 Classification -sort by size and density
 Centrifuge is used to classify solids of different sizes.

7.  Degritting- remove oversized and foreign particles
 Degritting is similar to classification where unwanted
particles, larger or denser, are rejected in the sediment,
with product (smaller or less dense) overflowing in the
lighter liquid phase. Another situation is where smaller
unwanted particles are rejected in the light liquid phase,
and valuable he avier solids are settled with the heavier
phase.

8.  Thickening or concentration- remove liquid,
concentrate solids
 Centrifuge is frequently used to concentrate the solid
phase by sedimentation and compaction, removing the
excess liquid phase in the overflow or concentrate. This
reduces the volume of the product in downstream
processing.

9. THE ANALYTICAL ULTRACENTRIFUGE:
 The sedimentation coefficient of a particle may be
determined experimentally using an instrument
known as an analytical ultracentrifuge.
 The rotor that spins in this ultracentrifuge typically
contains two compartments.
 Into one compartment is placed a “reference cell”
containing sample-free solvent and the other re-
ceives a cell containing the sample to be analyzed.
 The interior of each cell is sector shaped and
bounded above and below by parallel quartz
windows to permit light from below the rotor to pass
through the reference and sample during rotation.

10. THE ANALYTICAL ULTRACENTRIFUGE:
 As the particles sediment, boundaries are formed at the trailing
edges of each particulate species. Changes in the distance
between each boundary and the axis of rotation are measured as
a function of time by the instrument’s optical system.

11. THE ANALYTICAL ULTRACENTRIFUGE:
 These measurements are then used to calculate
the respective sedimentation coefficients of the
particles in the suspension.
 The sedimentation coefficients of many cellular
macromolecules such as proteins and nucleic acids
fall in the range of 1 x 1013 to 200 x 1013 seconds
(i.e., the dimensions of s are seconds).
 For convenience, a unit called the Svedberg unit
(named in honor of T. Svedberg), abbreviated S, is
used to describe sedimentation coefficients and is
equal to the constant 1013 seconds.
 Thus, most cellular proteins have sedimentation
coefficients between 1 and 200 S.

13. PREPARATIVE CENTRIFUGATION:
 The counterpart to analytical centrifugation is pre-
parative centrifugation, which provides for the isola-
tion of cell components for further analysis.

14. PREPARATIVE CENTRIFUGATION:
 Three basic types of
centrifuge rotors are regularly
employed for conventional
preparative centrifugation;
 swinging-bucket rotors
 fixed angle rotors
 vertical rotors
 Each category is designed to
address three key factors:
 type of centrifugation
(differential, rate-zonal, or
isopycnic),
 speed
 volume range

15. SWINGING-BUCKET ROTORS
 swinging-bucket rotors are the most common styles for
benchtop, low-speed, and high-speed floor-model
centrifuge applications.
 The most common benchtop and super speed
centrifuge applications requiring a swinging-bucket
rotor include:
1. high-throughput protocols such as batch harvesting of
whole cells from growth media
2. high-capacity processing of blood collection tubes
3. large-volume tissue culture processing.
 For these applications, the user can start with volumes
as large as 1000 mL and scale down to smaller
volumes of 1.5-mL conical tubes

16. SWINGING-BUCKET ROTORS
 An ultracentrifuge equipped with a swinging-bucket
rotor can support both types of separations: rate-zonal
(i.e., based on mass or size) or isopycnic (i.e., based on
density).
 For rate-zonal separations, a swinging-bucket rotor is
advantageous because the path length of the gradient
(i.e., distance from Rmin [inside top of meniscus] to
Rmax [outside bottom of tube]) is long enough for
separation to occur.
 Since the buckets are positioned at 90° during the run
and returned to a vertical position at the end of the run,
the sample retains its orientation, which minimizes any
pellet or band disturbance.

17. SWINGING-BUCKET ROTORS

18. FIXED-ANGLE ROTORS
 Fixed-angle rotors are the most ubiquitous rotors used
in centrifugation.
 The majority are used for basic pelleting applications
(differential separations), either to pellet particles from a
suspension and discard the excess debris, or to collect
the pellet.
 The cavities in these rotors range in volume from 0.2
mL to 1 L, with speeds ranging from single digits to
1,000,000×g (relative centrifugal force, RCF).

19. FIXED-ANGLE ROTORS
 Two factors determine the type of fixed-angle rotor
required:
1. desired g-force (RCF)
2. desired volume
 Generally speaking, the size of the rotor is inversely
proportional to its maximum speed capability (i.e., the
larger the rotor, the lower the maximum speed).

20. FIXED-ANGLE ROTORS
 An important specification when selecting a fixed-angle
rotor is the K factor, which indicates the pelleting
efficiency of the rotor at top speed, taking into account
the maximum and minimum radius (path length) of the
rotor cavity.
 A low K factor indicates a higher pelleting efficiency;
therefore, the K factor can be a useful metric for
comparing the speed at which particles will pellet
across a range of rotors

21. VERTICAL ROTORS
 Vertical rotors are fairly specialized—their most common use
is during ultracentrifugation for isopycnic separations,
specifically for the banding of DNA in cesium chloride.
 In this type of separation, the density range of the solution
contains the same density as the particle of interest; thus the
particles will orient within this portion of the gradient.
 Isopycnic separations are not dependent on the path-length
of the gradient but rather on run time, which must be
sufficient for the particles to orient at the proper position
within the gradient.
 Vertical rotors have very low K factors (typically in the range
of 5–25), indicating that the particle must only travel a short
distance to pellet (or in this case form a band); therefore run
time is minimized.

22. DIFFERENTIAL CENTRIFUGATION
 During centrifugation, particles sediment through the
medium in which they are suspended at rates related
to their size, shape, and density.
 Differences in the sedimentation coefficients of the
various subcellular particles provide the means for
their effective separation.

23. DIFFERENTIAL CENTRIFUGATION
 The material to be fractionated is subjected first to low-
speed centrifugation to sediment the largest (or
densest) particles present. Following this, the
unsedimented material (called the supernatant) is
transferred to another tube and centrifuged at a higher
speed (and usually also for a longer period of time) to
sediment particles of somewhat smaller size (and/or
lower density).
 The sequence is repeated several times until all
particles have been sedimented; each sediment is
then used for further experimentation and analysis.

24. DIFFERENTIAL CENTRIFUGATION
 The procedure that is routinely used for the differential fractionation of liver
tissue serves as a convenient example of this method.

25. DIFFERENTIAL CENTRIFUGATION
 Differential
centrifugation has
several major
disadvantages.
Because the
homogenate is
initially distributed
uniformly
throughout the
centrifuge tube, the
first particles
sedimented will
necessarily be
contaminated with
small amounts of all
other constituents
of the homogenate.

26. DENSITY GRADIENT CENTRIFUGATION:
 The resolution achieved during centrifugation can be
greatly improved if the mixture of particles is confined
at the outset to a narrow zone at the top of the
centrifuge tube and the particles then permitted to
sediment from this position.
 Initial stability under these conditions can be obtained
only if the particles are layered onto a density
gradient, that is, a column of fluid of increasing
density.
 If a particle reaches a position in the gradient where
the particle’s density and the gradient’s density are the
same then s becomes zero, and no further
sedimentation of the particle occurs.
 This technique, known as density gradient
centrifugation.

27. DENSITY GRADIENT CENTRIFUGATION:
 Density gradients may be stepwise (discontinuous) or
continuous.
 A step gradient is prepared by successively layering
solutions of decreasing density in the centrifuge tube.
 Continuous density gradients are prepared by mixing
dense and light solutions in varying proportions at a
controlled rate and delivering the mixture to a
centrifuge tube in a continuous stream.

28. DENSITY GRADIENT CENTRIFUGATION:
 A linear gradient can be
made using a simple
equipment called gradient
maker, containing two
cylinder. One of the
cylinder contain lighter
solution while other filled
with denser one.
 The lighter solution mixed
with the denser solution
while moving to the
centrifuge tube.

29. DENSITY GRADIENT CENTRIFUGATION:
 Using this method, a
density gradient is formed
that decreases linearly as
a function of volume
between the limiting
densities originally
present in the two
cylinders.

30. DENSITY GRADIENT CENTRIFUGATION:
 Additives used to provide solutions for density
gradients are selected on the basis of their solubility in
water and their compatibility with cells and subcellular
organelles.
 the additive must form a solution within the required
density range
 the additive must not interfere with, or damage, the
sample
 the solvent must be compatible with the sample
the solution must have a refractive index within the
practical range, as well as a low viscosity
 The additive must be easily removable from the
sample.

31. DENSITY GRADIENT CENTRIFUGATION:
 Additives used to provide solutions for density
gradients are selected on the basis of their solubility in
water and their compatibility with cells and subcellular
organelles.
 the additive must form a solution within the required
density range
 the additive must not interfere with, or damage, the
sample
 the solvent must be compatible with the sample
the solution must have a refractive index within the
practical range, as well as a low viscosity
 The additive must be easily removable from the
sample.

32. DENSITY GRADIENT CENTRIFUGATION:
 The additives for density gradient centrifugation can
be divided into four main categories:
 Salts of Alkali Metals
 Neutral Water Soluble Molecules
 Hydrophilic Macromolecules
 Synthetic Molecules

33. DENSITY GRADIENT CENTRIFUGATION:
 These solutions fulfill most of the above requirements.
However, due to the high ionic strength, hydrogen bonding
within biological macromolecules (protein, nucleic acid -
protein complexes) is impaired by a chaotropic effect.
Therefore these salts are mainly used for DNA and RNA
separations.
 Cesium chloride is used most frequently. Other useful salts
include sodium iodide, sodium bromide, cesium sulfate and
cesium acetate. Potassium tartrate has been used to
separate viruses from host cells.
 It should be kept in mind that the density of the sample is
highly dependent on the hydration of the macromolecule,
which in turn depends to a large extent on the dehydration
power of the salt solution.
SALTS OF ALKALI METALS

34. DENSITY GRADIENT CENTRIFUGATION:
 In this class of compounds sucrose is most widely
used. It has a useful density range of up to 1.29. This
range can be increased to 1.37 by addition of glucose
or by dissolving sucrose in D2O.
 Sucrose has very little effect on macromolecules, but
affects enzyme activity. Due to its high osmotic
pressure, sucrose solution dehydrates cells and their
organellae very efficiently.
 Glycerol solutions are the preferred media for the
separation of enzymes because they do not affect
enzyme activity. They exhibit a high viscosity, requiring
prolonged centrifugation times. More importantly
however, glycerol penetrates biological membranes.
NEUTRAL, WATER-SOLUBLE MOLECULES

35. DENSITY GRADIENT CENTRIFUGATION:
 Dextran gradients have been used for the separation
of microsomes.
 Separations achieved with dextrans show similar
results to those obtained by using synthetic sucrose/
epichlorohydrin co-polymers.
 In some cases bovine serum albumin has been
applied, but the preparation of an appropriate solution
is very difficult.
HYDROPHILIC MACROMOLECULES

36. DENSITY GRADIENT CENTRIFUGATION:
 Sucrose, Ficoll (a copolymer of sucrose and
epicholorhydrin), Metrizamide, Nycodenz, and Percoll
(a colloidal form of silica) are the most popular, as they
have minimal detrimental effects on organelles and
provide densities up to about 1.3 g/cm.
 Cesium chloride, cesium trifluoroacetate, and other
dense salts are used when the limiting density must
be considerably higher (i.e., up to 1.9 g/cm).

37. DENSITY GRADIENT MEDIA TYPES AND THEIR PRINCIPLE USES

38. DENSITY GRADIENT CENTRIFUGATION:
 Density gradient centrifugation methods are of two
types, the rate zonal technique and the isopycnic
(isodensity or equal density) technique.
 Rate Zonal Technique.
 Isopycnic Technique

39. DENSITY GRADIENT CENTRIFUGATION:
 When the densest region of a density gradient (i.e.,
the liquid at the bottom of the centrifuge tube) is less
dense than the particles being sedimented, all
particles will eventually reach the bottom of the tube (if
centrifugation is continued long enough).
 However, if the duration of centrifugation is carefully
limited, this will not occur; instead, the particles will be
distributed through the density gradient in the order of
their sedimentation coefficient that depends on their
sizes and shapes.
 Fractionations carried out in this manner are called
rate separations and are based on the combined
contributions of particle size and particle density.
RATE ZONAL TECHNIQUE

40. DENSITY GRADIENT CENTRIFUGATION:
 A second density gradient technique, called equilibrium
density-gradient centrifugation is used to separate cellular
components on the basis of their density.
 In this case the cellular mixture is centrifuged through a
steep density gradient that contains a high concentration of
sucrose, or more often, cesium chloride (CsCl).
 In these gradients, the molecules being studied have a
density somewhere in between the highest and lowest
densities of sucrose or CsCl generated in the gradient.
 The components of a sample begin to move down this
gradient in the same way as they do in a rate-zonal density
gradient. When a component of the mixture reaches a point
where the density of the solution is equal to its own density
(isopycnic), it stops moving further and forms a distinct
band.
ISOPYCNIC TECHNIQUE

42. MOTOR
 In general, centrifuge motors are high-torque, series-wound
DC motors, the rotation of which increases as the voltage is
increased. The rotor shaft is driven directly or through a gyro,
although occasionally a pulley system is used. Electrical
contact to the commutator is provided by graphite brushes,
which gradually wear down as they press against the
commutator turning at high speed, and thus should be
replaced at specified intervals.

43. MOTOR
 Modern centrifuges have induction drive motors that have no
brushes to change. The shaft of the motor turns through
sleeve bearings located at the top and bottom of the motor.
Most instruments contain sealed bearings that are
permanently lubricated, while others require periodic
application of oil or grease.
 The speed of the centrifuge
is controlled by a
potentiometer that raises
and lowers the voltage
supplied to the motor. The
calibrations on the speed
control are often only
relative voltage increments
and should never be taken
as accurate indicators of
speed. Therefore, periodic
recalibration is required.

44. IMBALANCE DETECTOR
 Some instruments have an internal imbalance detector that
monitors the rotor during operation, causing automatic shutdown
if rotor loads are severely out of balance.

45. TACHOMETER
 A tachometer indicates the speed in rpm. Most modern centrifuges use
electronic tachometers, in which a magnet rotates around a coil to produce a
current that can be measured.
 The digital handheld tachometer is a gauge for determining speed, velocity
and distance. This handheld tachometer measures in two ways: either
optically and therefore without contact, or mechanically via various adapters.
 In the optical measurement, a focused light beam is directed onto the
object andreflected back by a thin, reflective foil glued on the object with the
frequency of rotation. The result is displayed on the 5-digit LCD.
 The distance between the handheld
tachometer and the measured object can
reach up to 600 mm. The mechanical
measurement of speed is performed with an
adapter tip that is placed on the axis of the
moving parts. For normal speed
or length measurement, a measuring wheel
is place on the adapter.

46. SAFETY LID
 Modern centrifuges must have a door-locking mechanism to prevent the lid
from being opened while the instrument is running. If there is a power failure
or the safety latch fails for some reason it may be necessary to trip the door-
locking mechanism manually to retrieve the samples. Manufacturers’
instructions should be checked for the exact procedure required.

47. BRAKING SYSTEM
 B raking devices are incorporated to provide rapid rotor deceleration. Modern
instruments have an electrical braking system that functions by reversing the
polarity of the electrical current to the motor. Other machines may have a
mechanical brake.

48. REFRIGERATOR
 A centrifuge generates heat as it rotates and if samples are
temperature labile then a refrigerated centrifuge should be used.
Some centrifuges enable the rotor and chamber to be precooled
before a run

49. CENTRIFUGE TUBES
 It is advisable to use a conical-bottomed tube in a swing-out bucket
rotor for the sedimentation of cells. This tube type will retain the pellet
of cells more effectively as the supernatant is removed. All tubes for
use with high-speed rotors are round-bottomed. Pyrex glass tubes
can withstand forces of around 2000 xg , while Corex tubes can be
used up to 12,000 xg.
 Polycarbonate or polyallomer are the most common plastic tubes in
use but great care must be taken when using organic solvents.
Manufactur ers usually provide extensive information about solvent,
salt and pH resistance, as well as sterilisation procedures.

50. RELATIVE CENTRIFUGAL FORCE (F)
 The force acting on a particle during centrifugation is given by
the equation
F = Mω2r
 M = mass of particle
 ω (omega) = Angular velocity of the spinning rotor in radians
per second (one unit of circumference contains 2π radians),
 r = radius of rotation (distance of particles from axis of rotation
in cm)
 𝜔 =
2𝜋 𝑟𝑒𝑣 𝑚𝑖𝑛−1
60
 𝜔 =
2(3.1416) 𝑟𝑒𝑣 𝑚𝑖𝑛−1
60
 𝜔 = 0.10472 × 𝑟𝑒𝑣 𝑚𝑖𝑛−1

51. RELATIVE CENTRIFUGAL FORCE (RCF)
 The magnitude of the induced gravitational field is measured in terms
of the G value which is also referred to as the RCF (relative
centrifugal force) value depends on the rotation speed as well as the
manner in which the centrifuge tubes are held by the rotor.
𝐺 (𝑅𝐶𝐹) =
𝑟𝜔2
𝑔
 r = distance from the axis of rotation (mm)
 ω = angular velocity (radians/s)
 g = acceleration due to gravity (m/s2)
 𝐺 (𝑅𝐶𝐹) =
𝑟(0.10472 𝑥 𝑟𝑒𝑣 𝑚𝑖𝑛−1 )2
980
 𝐺 (𝑅𝐶𝐹) =
𝑟(0.10472)2 𝑥 (𝑟𝑒𝑣 𝑚𝑖𝑛−1 )2
980
 𝐺 (𝑅𝐶𝐹) = (𝑟 1.119𝑥10−5 𝑥 𝑟𝑒𝑣 𝑚𝑖𝑛−1 2)

52. RELATIVE CENTRIFUGAL FORCE (RCF)
 What is the maximum relative centrifugal force applied when
red blood cells are sedimented at 1000 rpm in a rotor of
maximum sample radius equal to 10 cm?
 RCFmax = 1.119 x 10-5 (1000)2(10)
 = 1.119 x 102 or 112g

53. RELATIVE CENTRIFUGAL FORCE (RCF)
The RCF value can also be obtained using a nomogram. Using a straight-edged ruler,
line up the known rotating radius (distance from the center of the rotor to the bottom of
the centrifuge bucket) on the left with the known rpm on the far right and read the RCF
value where the line crosses the graph in the center.
nomogram..

54. RELATIVE CENTRIFUGAL FORCE (RCF)

55. RELATIVE CENTRIFUGAL FORCE (RCF)
 Most manufacturers include a nomogram in the instruction manual;
however, most modern centrifuges now have the facility to swap the
figure displayed on the control panel between rpm and RCF,
making manual calculation unnecessary.

56. RELATIVE CENTRIFUGAL FORCE (RCF)
 The sedimentation of particles by centrifugation is, in effect, a
method for concentrating them; therefore, one of the major
physical forces opposing such concentration is diffusion.
 In the case of the sedimentation of cells or sub-cellular organelles
(nuclei, mitochondria, etc.), the effects of diffusion are essentially
nil.
 However, when centrifugation is used to sediment much smaller
particles (such as cellular proteins, nucleic acids, or
polysaccharides), the effects of diffusion become significant.

57. SEDIMENTATION COEFFICIENT
 The RCF or “g force” applied to particles during centrifugation is
independent of the physical properties of the particles being
sedimented. However, a particle’s sedimentation rate at a
specified RCF depends on the properties of the particle itself and
medium.
 The instantaneous sedimentation rate of a particle during
centrifugation is determined by three forces.
 FC, the centrifugal force,
 FB, the buoyant force of the medium
 FF, the frictional resistance to the particle’s movement
 Two particles having different sizes or different densities can have
similar sedimentation coefficients. On the other hand, two particles
with either similar size or similar density can have markedly
different sedimentation coefficients.

58. SEDIMENTATION COEFFICIENT
 The sedimentation coefficient s of a particle is used to
characterize its behavior in sedimentation processes.
 The sedimentation coefficient has the dimention of a unit of time
and is expressed in svedbergs.
 It is equal to 10-13 seconds and is written with no space between
the number and the symbol (e.g. 70S).
 It is named after Theodor Svedberg (1884–1971), the Swedish
chemist who developed the technique of centrifugation for the
study of colloids and macromolecules, for which he was awarded
a Nobel Prize in 1926