Method in biochemistry lecture note

1. SPECTROPHOTOMETRY

2. CONTENT
⚫ Introduction,
⚫ Electromagnetic spectrum,
⚫ Interaction of electromagnetic radiations with the matter,
⚫ Mathematical statement and derivation of Lambert’s Law
and Beer’s Law,
⚫ Terminology involved in spectrophotometric analysis,
⚫ Instrumentation of single beam colorimeter,
⚫ Instrumentation of single and double beam
spectrophotometer,
⚫ Principle of additivity of absorbance and simultaneous
determination,
⚫ Spectrophotometric Titrations,
⚫ Experimental Applications-Structure of organic compounds,
Structure of complexes,

3. INTRODUCTION
⚫Spectrophotometry is a branch of science
that deals with the study of interaction of
electromagnetic radiation with matter.
⚫During such interactions the energy is
either absorbed or emitted by the matter
in descrete amount called quanta.

4. ELECTROMAGNETIC SPECTRUM
Electromagnetic radiation –
⚫ It may be considered as simple harmonic wave
propagated from a source and travelling in
straight lines except when reflected or refracted.
⚫ This radiation will be associated with the
properties of the waves.

5. Wavelength
⚫It is the distance between two successive
maxima on an electromagnetic wave.
Frequency
The number of complete wavelength units
passing through a given point in unit time
is called the frequency of radiation (v)
Wave number
It is the number of waves per centimeter in
vaccum.

6. The concept of travelling wave

7. Relation between frequency, wave
number and wavelength
⚫The product of wavelength and frequency
is equal to the velocity of the wave in the
medium.  x v = velocity
⚫As the velocity of light is represented by
c, which is maximum in vacuum
v = c = 3 x 1010 cm sec-1
Thus 1/  v / c = v

8. INTERACTIONS OF RADIATION WITH MATTER
⚫ The entire range over which electromagnetic radiation
exists is known as electromagnetic spectrum.
⚫ Spectrophotometry is mainly concerned with the ultraviolet
(200-400nm) and visible (400-800nm) regions.
⚫ Main instruments – photometers, colorimeters and
spectrophotometers

9. REGIONS OF THE ELECTROMAGNETIC
SPECTRUM

10. TERMINOLOGY USED IN ABSORPTION MEASUREMENT
⚫ Radiant Energy : It is defined as energy transmitted as
electromagnetic radiation. It has the properties of both
particle and wave motion.
⚫ Electromagnetic radiation possesses a certain amount of
energy. The energy of a unit radiation, called photon, is
related to frequency by –
hc
E = hv = ------

⚫ Where E is the energy of the photon in ergs
⚫ h is Planck’s constant (6.62 x 10 -27 erg sec)

11. ⚫Radiant Power, P : Formerly known as
Intensity (I). Energy per unit time is
called as power. Radiant power is the rate
at which energy is transported in a beam
of radiant energy.

12. ⚫Transmittance, T: It is simply the fraction
of the incident power transmitted by a
sample.
⚫Absorbance, A : It was formerly known as
an optical density (O.D.) or extinction (E).
The absorbance is the logarithm to base
10 of the reciprocal of the transmittance.

13. ⚫Absorptivity, a : Formerly known as the
extinction coefficient or specific extinction.
⚫Absorptivity is defined as the ratio of the
absorbance to the product of the length of
optical path b and the concentration of
the sample C.
⚫Absorptivity is the measure of the ability
of sample to absorb light.

14. ⚫Molar absorptivity, € : Formerly known as
molar extinction coefficient or molar
absorption coefficient. Molar absorptivity
is the product of absorptivity and
molecular weight of the material.
⚫Path Length, b : It was formerly denoted
by l or d. It is the internal thichkness
(diameter) of the cell in which the test
sample is taken.

15. FUNDAMENTAL LAWS OF PHOTOMETRY
⚫ When light is incident upon a homogeneous
medium, a part of the radiant power of the
incident light is reflected, a part is absorbed and
the remainder is transmitted.
Pa
Medium of conc C
P
Transmitted
Radiation
P0
Incident
Radiation
Pr
Reflected
Radiation Length b

16. TWO FUNDAMENTAL LAWS OF PHOTOMETRY
⚫ Lambert’s Law : It states the relationship
between the radiant power of absorbed light
with the thickness of the medium.

17. LAMBERT’S LAW
⚫ When a beam of monochromatic light is
allowed to pass through a transparent
medium, the rate of decrease of radiant
power with the thickness of the medium is
directly proportional to the radiant power of
the incident light.
⚫ dP is change in radiant power, db is small thickness of
sample, k1 is proportionality constant and minus sign
indicate radiant power decrease
-dP P
db
-dP = k1P
db

18. integrating above
AFTER REARRANGING AND
FORMULA WE GET
A = K1*B
A = Absorbance = log10(Po/P)
K1 = Absorption coefficient = k1/2.303
Thickness db
b=0 b=b
P
P0

19. ⚫ Beer’s Law : It states the relationship between
the radiant power of absorbed light with the
concentration of the medium
⚫ When a beam of monochromatic light is allowed
to pass through a transparent medium, the rate
of decrease of radiant power with the
concentration of the medium is directly
proportional to the radiant power of the incident
light.
⚫ Combined formed is called Lambert-Beer’s law.

20. LAMBERT-BEER’S LAW
⚫ The law deals with the relationship between the
radiant power of the incident light and
transmitted light as a function of both the
thickness of the medium and the concentration
of the absorbing species.
⚫ Lambert’s law
Absorbance = Constant x Thickness of the medium
x Concentration of the
⚫ Beer’s Law
Absorbance = Constant
medium

21. ⚫ Lambert-Beer’s Law
THE COMBINED LAW MAY BE GIVEN BY THE RELATION –
x
Absorbance = Constant x [Thickness of the medium]
[Concentration of the medium]
It states that, for the given system and the
thickness of the medium, the absorption of the
medium is directly proportional to the
concentration of an absorbing species.

22. INSTRUMENTATION

23. INSTRUMENTATION

24. INSTRUMENTATION - COMPONENTS
⚫ Radiation source
⚫ Filters and Monochromator – Prism, Diffraction
grating
⚫ Cuvette (sample container)
⚫ Detection of Radiation – Detector – Photovoltaic
cell or Photronic cell.

25. A. PHOTOVOLTAIC CELL
⚫ It is also known as barrier-layer cell or photronic cell
⚫ In this cell radiant energy falling on it generates a current
at the interface of a metal and a semi-conductor.
⚫ It operates without battery.
⚫ It consists of a metal base plate made up of iron or copper,
which acts as one electrode.
⚫ A thin layer of semiconducting material is deposited on the
surface of the metal plate.
⚫ Then the surface of semiconducting material is covered by
a very thin layer of silver or gold which acts as a second
collector electrode.

27. CHARACTERISTICS OF PHOTOVOLTAIC CELL
⚫ It is completely different in design and principle of
the
 photomissive cell and works without the use of battery
⚫ These cells generate their own e.m.f.
⚫ The magnitude of photocurrent is directly
proportional to
 the radiant power of incident radiation striking on it.
⚫ The cells are sensitive over the whole visible region. The typical cell
has maximum sensitivity at about 550nm
⚫ The current output of the cell depends upon the wavelength of
 the incident radiation.

28. DISADVANTAGES OF PHOTOVOLTAIC CELL
readily
⚫ The current produced by the cell cannot be
amplified because of the low internal resistance
⚫ The cells show fatigue effect – its current output decreases
slowly during continued illumination. The fatigue effect can
be minimised by careful selection of the optimum level of
illumination.

29. B. PHTOTUBES
⚫ These cells are also
as
known
Photoemissive
tubes
⚫ In this tube, radiant
energy falling on
photosensitive solid
causes
of
surface
emission
electrons.

30. ⚫ When light (photons) falls on cathode, electrons are
emitted by the cathode due to photoelectric effect.
⚫ The liberated electrons are attracted to the anode,
causing an electric current to flow through an
external circuit.
⚫ The current is amplified and measured by readout
device.
⚫ The current measures the radiant power of light
radiation striking the photosensitive surface.

31. C. PHOTOMULTIPLIER TUBES
⚫ It contains a photosensitive surface as well as
many other surfaces that emit a cascade of
electrons from the photosensitive area.
⚫ It is more sensitive than a phototube for the
visible and UV regions.
⚫ It is mostly used in spectrophotometer

32. INSTRUMENTATION

33. CONSTRUCTION
⚫ A photomultiplier tube consists of a cathode, an anode and
many additional electrodes that are called dynodes.
⚫ All these are enclosed in an evacuated glass tube.
⚫ A photoemissive cathode is in the form of a half cylinder of
metal.
⚫ The inner surface of cathode is coated with a light sensitive
material such as oxide of cesium or potassium or silver.
⚫ Most photomultiplier tubes have nine dynodes.
⚫ A dynode is an electrode with a coating of cesium which
emits several electrons (2-5) for each striking on its
surface.

34. WORKING
⚫ When the light radiation strikes the cathode surface,
it ejects electrons due to photoelectric effect.
⚫ These primary electrons get accelerated in the
electrostatic field between the cathode and the first
dynode and fall on dynode.
⚫ When electrons strike dynode 1, each electron causes
emission of several electrons. These secondary
electrons in turn are accelerated towards dynode 2
⚫ Likewise the process is repeated nine times, where
each secondary electron releases several electrons
(2-5).
⚫ The resulting current is then amplified and
measured.

35. COLORIMETER
⚫ A colorimeter is a device used in colorimetry.
⚫ In scientific fields the word generally refers to the device that measures the
absorbance of particular wavelengths of light by a specific solution.
⚫ This device is commonly used to determine the concentration of a known
solute in a given solution by the application of the Beer-Lambert law.
⚫ Beer Lambert Law states that the concentration of a solute is proportional
to the absorbance.
⚫ It is invented by Louis Jules Duboscq in 1870.
⚫ It involves quantitative estimation of colour.
⚫ The colour of light is the function of its wavelength.

36. SINGLE BEAM PHOTOELECTRIC COLORIMETER
 The basic components are –
⚫ A source of light such as tungsten filament lamp
with
 concave reflector and collimating lens
⚫ An adjustable diaphragms or slits
⚫ A coloured glass filter for monochromatic light
for holding the
⚫ A cuvette such as glass tube
solution/solvent
⚫ A detector such as photovoltaic cell
⚫ A recorder such as galvanometer
⚫ https://youtu.be/_qddp1fd1Do

37. INSTRUMENTATION

38. FUNCTIONING
⚫ Polychromatic light from a incandescent tungsten filament
lamp reflects by concave mirror in the direction of the filter
and also the light passes through the collimating lens.
⚫ It makes the beam of light parallel
⚫ The light passes further through slit (1) which controls the in-
going light.
⚫ The filter converts the polychromatic light to narrow band
width wavelength light.
⚫ It passes further through absorbing material where the
material absorbs the part of it depending upon the colour
density of the solution and transmits the remaining
intensity through slit (2) towards the detector.
⚫ The detector converts the light energy into electrical
energy and feeds it to the recorder.
⚫ The signal magnitude of the recorder is the measure of
the absorbance or transmittance of the solution.

39. SPECTROPHOTOMETER
⚫ If you pass white light through a colored substance,
some of the light gets absorbed.
⚫ A solution containing hydrated copper(II) ions, for
example, looks pale blue because the solution
absorbs light from the red end of the spectrum.
⚫ The remaining wavelengths in the light combine in
the eye and brain to give the appearance of cyan
(pale blue).
⚫ Some colorless substances also absorb light - but in
the ultra-violet region.
⚫ Since we can't see UV light, we don't notice this
absorption.

40. ⚫ Different substances absorb different wavelengths of light,
and this can be used to help to identify the substance - the
presence of particular metal ions, for example, or of
particular functional groups in organic compounds.
⚫ The amount of absorption is also dependent on the
concentration of the substance if it is in solution.
⚫ Measurement of the amount of absorption can be used to
find concentrations of very dilute solutions.
⚫ An absorption spectrometer measures the way that the
light absorbed by a compound varies across the UV and
visible spectrum.

41. ⚫ Single-Beam spectrophotometers are often sufficient for
making quantitative absorption measurements in the UV-
Vis spectral region. The concentration of an analyte in
solution can be determined by measuring the absorbance
at a single wavelength and applying the Beer-Lambert Law.

42. INSTRUMENTATION
⚫ Single-beam spectrophotometers can utilize a fixed
wavelength light source or a continuous source.
⚫ The simplest instruments use a single-wavelength light
source, such as a light-emitting diode (LED), a sample
container, and a photodiode detector.
⚫ Instruments with a continuous source have a dispersing
element and aperture or slit to select a single wavelength
before the light passes through the sample cell.
⚫ In either type of single-beam instrument, the instrument is
calibrated with a reference cell containing only solvent to
determine the Po value necessary for an absorbance
measurement.

43. INSTRUMENTATION

44. DOUBLE BEAM SPECTROPHOTOMETER
It has components as –
The light source
⚫ You need a light source which gives the entire visible spectrum plus
the near ultra-violet so that you are covering the range from about
200 nm to about 800 nm.
⚫ You can't get this range of wavelengths from a single lamp, and so a
combination of two is used - a deuterium lamp for the UV part of the
spectrum, and a tungsten / halogen lamp for the visible part.
⚫ The combined output of these two bulbs is focused on to a diffraction
grating.

45. The diffraction grating
and the slit
⚫ You are probably familiar
with the way that a prism
splits light into its
component colors.
⚫ A diffraction grating does
the same job, but more
efficiently.

46. show the way the
light are sent off in
various
different
⚫ The blue arrows
wavelengths of the
directions.
⚫ The slit only allows light of a very narrow range of
wavelengths through into the rest of the
spectrometer.
⚫ By gradually rotating the diffraction grating, you can
allow light from the whole spectrum (a tiny part of
the range at a time) through into the rest of the
instrument.

47. THE ROTATING DISKS
⚫ Each disk is made up of a
number of different
segments.
⚫ Those in the machine we
are describing have three
different sections - other
designs may have a
different number.

48. The sample and reference cells
⚫ These are small rectangular glass or quartz containers.
⚫ They are often designed so that the light beam travels a
distance of 1 cm through the contents.
⚫ The sample cell contains a solution of the substance you
are testing - usually very dilute.
⚫ The solvent is chosen so that it doesn't absorb any
significant amount of light in the wavelength range we are
interested in (200 - 800 nm).
⚫ The reference cell just contains the pure solvent.

49. The detector and computer
⚫ The detector converts the incoming light into a
current.
⚫ The higher the current, the greater the intensity
of the light.
⚫ For each wavelength of light passing through the
spectrometer, the intensity of the light passing
through the reference cell is measured.

50. INSTRUMENTATION

51. ⚫ The absorption
spectrum is a plot
of absorbance
against
wavelength.

52. COLORIMETER VS SPECTROPHOTOMETER

53. SPECTROPHOTOMETRIC TITRATIONS
⚫ Definition: The process of determining the quantity
of a sample by adding measured increments of a
titrant until the end-point, at which essentially all of
the sample has reacted, is reached.
⚫ The titration is followed by measuring the absorbance
of radiation in the range ultraviolet to near-infrared
(0.1--2.5 mum) by the sample.
⚫ The titration in which absorbance of a solution is used
to determine the end point are called
spectrophotometric titrations.

54. INSTRUMENTATION

55. TECHNIQUE
⚫ The absorbance measurements are made at a fixed
wavelength.
⚫ First an optimum wavelength is selected and zero
adjustment is made.
⚫ The solution to be titrated (tirand) is taken in the cell
⚫ Then the cell is kept in the beam of light in the
instrument.
⚫ A known volume of titrant is added to the stirred
solution in the cell.
⚫ Thus the absorbance of the titrand solution is
measured after each addition of the titrant and the
end point of the titration is detected.
⚫ The absorbance is plotted against the volume of the
titrant added.

56. TITRATION CURVE

57. ADVANTAGES OF PHOTOMETRIC TITRATIONS
⚫ Useful to solutions with lower or higher ionic
strength or non-aqueous solvents
⚫ Used for highly coloured solutions which cannot
be determined the visual indicators.
⚫ The slight changes in colour are readily detcted
by the spectrophotometer and hence end point
determination is sharp and accurate.
⚫ Other absorbing species do not interfere with the
actual titration, because only the changes in
absorbance are taken into account and not the
absolute values of absorbance.

58. ⚫ These titrations can be applied to a large number
of non-absorbing constituents, since only one
absorber is necessary among the reactant, the
titrant or the reaction products.
⚫ These titrations provide more accurate results
tan a routine analysis because the data fro many
measurements are brought together in
determining the end point.
⚫ It is particularly suitable to titration reactions
where a relatively large degree of reaction
incompletion exists at the equivalence point.

59. CENTRIFUGATION AND
CENTRIFUGE
Separation of suspended particles
from solution

60. CENTRIFUGATION
• Centrifugation is the technique of separating components
where the centrifugal force/ acceleration causes the
denser molecules to move towards the periphery while
the less dense particles move to the center
• The process of centrifugation relies on the perpendicular force
created when a sample is rotated about a fixed point.
• The rate of centrifugation is dependent on the size and
density of the particles present in the solution
• Centrifugation is a technique of separating substances
which
involves the application of centrifugal force
• The particles are separated from a solution according to
their size, shape, density, the viscosity of the medium
and rotor speed

61. PRINCIPLE
• In a solution, particles whose density is higher than that of
the solvent sink (sediment), and particles that are lighter
than it floats to the top
• The greater the difference in density, the faster they move
• If there is no difference in density (isopycnic conditions),
the particles stay steady
• 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)
• The centrifuge works using the sedimentation principle,
where the centripetal 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
Centrifugal force

62. PRINCIPLE
Relative Centrifugal Force (RCF)
• Relative centrifugal force is the measure of the strength of rotors of different
types and sizes
• This is the force exerted on the contents of the rotor as a result of the rotation.
• RCF is the perpendicular force acting on the sample that is always relative to the
gravity of the earth
• The RCF of the different centrifuge can be used for the comparison of rotors,
allowing the selection of the best centrifuge for a particular function
The formula to calculate the relative centrifugal force (RCF) can be written as:
• RCF (g Force)= 1.118 × 10-5 × r × (RPM)2
• where r is the radius of the rotor (in centimeters), and RPM is the speed of the
rotor in rotation per minute

63. G= W2
R
G= CENTRIFUGAL FIELD W=
ANGULAR VELOCITY
R= radial distance of particle from axis of rotation
W= 𝟐𝝅𝒓𝒆𝒗
𝒎𝒊𝒏
G= 4𝝅𝒓𝟐
3600

64. CENTRIFUGE
• A centrifuge is a device used to separate components of a
mixture on the basis of their size, density, the viscosity of
the medium, and the rotor speed
• The centrifuge is commonly used in laboratories for the
separation of biological molecules from a crude extract.
• In a centrifuge, the sample is kept in a rotor that is
rotated about a fixed point (axis), resulting in strong force
perpendicular to the axis
• There are different types of centrifuge used for the
separation of different molecules, but they all work on the
principle of sedimentation

66. ROTORS:
in centrifuges are the motor devices that house the tubes with the
samples
Centrifuge rotors are designed to generate rotation speed that can bring
about the separation of components in a sample.
There are three main types of rotors used in a centrifuge, which are:
1. Fixed angle rotors
• These rotors hold the sample tubes at an angle of 45° in relation to the
axis of the rotor
• In this type of rotor, the particles strike the opposite side of the tube
where the particles finally slide down and are collected at the bottom
• These are faster than other types of rotors as the pathlength of the
tubes increases
• However, as the direction of the force is different from the position of
the tube, some particles might remain at the sides of the tubes

67. 1. FIXED ANGLE ROTORS

68. 2. SWINGING BUCKET ROTORS/ HORIZONTAL ROTORS
• Swinging bucket rotors hold the tubes at an angle of 90° as
the rotor swings as the process is started
• In this rotor, the tubes are suspended in the racks that
allow the tubes to be moved enough to acquire the
horizontal position
• In this type of rotors, the particles are present along the
direction or the path of the force that allows the particles to
be moved away from the rotor towards the bottom of the
tubes
• Because the tubes remain horizontal, the supernatant
remains as a flat surface allowing the deposited particles to
be separated from the supernatant

69. 2. SWINGING BUCKET ROTORS/
HORIZONTAL ROTORS

70. 3. VERTICAL ROTORS
• Vertical rotors provide the shortest pathlength, fastest run
time, and the highest resolution of all the rotors
• In vertical rotors, the tubes are vertical during the
operation of the centrifuge
• The yield of the rotor is not as ideal as the position of the
tube doesn’t align with the direction of the centrifugal
force
• As a result, instead of settling down, particles tend o
spread towards the outer wall of the tubes
• These are commonly used in isopycnic and density
gradient centrifugation

71. 3. VERTICAL ROTORS

72. TYPES OF CENTRIFUGE
1. Benchtop centrifuge
• Benchtop centrifuge is a compact centrifuge that is
commonly used in clinical and research laboratories
• It is driven by an electric motor where the tubes are rotated
about a fixed axis, resulting in force perpendicular to the
tubes
• Because these are very compact, they are useful in smaller
laboratories with smaller spaces
• Different variations of benchtop centrifuges are available in the
market for various purposes
• A benchtop centrifuge has a rotor with racks for the sample
tubes and a lid that closes the working unit of the centrifuge

73. 2. LOW-SPEED CENTRIFUGE
• Low-speed centrifuges are the traditional centrifuges that are
commonly used in laboratories for the routine separation of
particles.
• These centrifuges operate at the maximum speed of 4000-5000 rpm.
• These are usually operated under room temperature as they are not
provided with a system for controlling the speed or temperature of the
operation.
• Two types of rotors are used,Fixed angle and 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
• These are easy and compact centrifuges that are ideal for the
analysis of blood samples and other biological samples

74. 3. HIGH-SPEED CENTRIFUGE
• High-speed centrifuge, as the name suggests, is the centrifuge that can be
operated at somewhat larger speeds
• The speed of the high-speed centrifuge can range from 15,000 to 30,000 rpm.
• The high-speed centrifuge is commonly used in more sophisticated
laboratories with the biochemical application and requires a high speed of
operations
• High-speed centrifuges are provided with a system for controlling the speed
and temperature of the process, which is necessary for the analysis of sensitive
biological molecules
• High-speed centrifuges are used in more sophisticated biochemical
applications, higher speeds and temperature control of the rotor chamber are
essential
• The operator of this instrument can carefully control speed and temperature
which is required for sensitive biological samples
All three types of rotors are available for high-speed centrifugation- Fixed angle,
Swinging bucket and Vertical rotors

75. 4. MICROCENTRIFUGE
• Microcentrifuges are the centrifuges used for the separation of
samples with smaller volumes ranging from 0.5 to 2 µl.
• Microcentrifuges are usually operated at a speed of about
12,000-13,000 rpm
• This is used for the molecular separation of cell organelles
like nuclei and DNAand phenol extraction
• Microcentrifuges, also termed, microfuge, use sample tubes
that are smaller in size when compared to the standard test
tubes used in larger centrifuges
• Some microcentrifuges come with adapters that facilitate the use
of larger tubes along with the smaller ones
• Microcentrifuges with temperature controls are available for the
operation of temperature-sensitive samples

76. 5. REFRIGERATED CENTRIFUGES
• Refrigerated centrifuges are the centrifuges that are provided with
temperature control ranging from -20°C to -30°C
• A different variation of centrifuges is available that has the system of
temperature control which is essential for various processes requiring
lower temperatures
• Refrigerated centrifuges have a temperature control unit in addition to the
rotors and racks for the sample tubes
• These centrifuges provide the RCF of up to 60,000 xg that is ideal for
the separation of various biological molecules
• These are typically used for collecting substances that separate rapidly like
yeast cells, chloroplasts, and erythrocytes
• The chamber of refrigerated centrifuge is sealed off from the outside to
meet the conditions of the operations

77. 6. ULTRACENTRIFUGES
• Ultracentrifuge is the most sophisticated instrument that operate at extremely
high speeds that allow the separation of much smaller molecules like
ribosomes, proteins, and viruses
• It is the most sophisticated type of centrifuge that allows the separation of
molecules that cannot be separated with other centrifuges
• Refrigeration systems are present in such centrifuges that help to balance the
heat produced due to the intense spinning
• The speed of these centrifuges can reach as high as 150,000 rpm with
maximum speed of 65,000 RPM (100,000’s x g)
• It can be used for both preparative and analytical works
• Ultracentrifuges can separate molecules in large batches and in a continuous
flow system
• In addition to separation, ultracentrifuges can also be used for the
determination of properties of macromolecules like the size, shape, and
density.

78. TYPES OF CENTRIFUGATION
1. Differential Pelleting (differential centrifugation)
• 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.

79. 2. DENSITY GRADIENT CENTRIFUGATION
• This type of centrifugation is mainly used to purify viruses, ribosomes,
membranes, etc.
• Asucrose 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.

80. 3. 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.

81. 4. ISOPYNIC CENTRIFUGATION
• 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.

82. APPLICATIONS OF CENTRIFUGATION
• 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.