Msc bioinstrumentation
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Mar 16, 2021
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About This Presentation
The pdf contain all the information of various technique ,such as chromatography,spectroscopy,centrifugation,electrophoresis special thanks to Dr.Rambir Singh for helping out the topics easily.Contact for help or suggestion @7985214648 whattapp only
Slide Content
Principles and applications of light microscope
The main components of the compound light microscope include a light source that is
focussed at the specimen by a condenser lens. Light that either passes through the
specimen (transmitted light) or is reflected back from the specimen (reflected light) is
focussed by the objective lens into the eyepiece lens. The image is either viewed directly
by eye in the eyepiece or it is most often profected onto a detector, for example
photographic film or, more likely, a digital camera.
Light from the light source passes into the condenser lens, which is mounted beneath
the microscope stage in an upright microscope (and above the stage in an inverted
microscope) in a bracket that can be raised and lowered for focussing.
The objective lens is responsible for producing the magnified image, and can be the
most expensive component of the light microscope.
Numerical aperture (NA) is always marked on the lens. This is a number usually
between 0.04 and 1.4. The NA is a measure of the ability of a lens to collect light from
the specimen.
Lenses with a low NA collect less light than those with a high NA.
Resolution varies inversely with NA, which implies that higher NA objectives yield the
best resolution.
The resolution achieved by a lens is a measure of its ability to distinguish between two
objects in the specimen. The shorter the wavelengths of illuminating light the higher the
resolving power of the microscope.
The limit of resolution for a microscope that uses visible light is about 300nm with a dry
lens (in air) and 200nm with an oil immersion lens.
Darkfield illumination produces images of brightly illuminated objects on a black
background .This technique has traditionally been used for viewing the outlines of
objects in liquid media such as living spermatozoa, microorganisms or cells growing in
tissue culture, or for a quick check of the status of a biochemical preparation. For lower
magnifications, a simple darkfield setting on the condenser will be sufficient. For more
critical darkfield imaging at a higher magnification, a darkfield condenser with a
darkfield objective lens will be required.
Phase contrast is used for viewing unstained cells growing in tissue culture and for
testing cell and organelle preparations for lysis. The method images differences in the
refractive index of cellular structures. Light that passes through thicker parts of the cell
is held up relative to the light that passes through thinner parts of the cytoplasm. It
requires a specialised phase condenser and phase objective lenses (both labelled ‘ph’).
Each phase setting of the condenser lens is matched with the phase setting of the
objective lens. These are usually numbered as Phase 1, Phase 2 and Phase 3, and are
found on both the condenser and the objective lens
The main components of the compound light microscope include a light source that is
focussed at the specimen by a condenser lens. Light that either passes through the
specimen (transmitted light) or is reflected back from the specimen (reflected light) is
focussed by the objective lens into the eyepiece lens. The image is either viewed directly
by eye in the eyepiece or it is most often profected onto a detector, for example
photographic film or, more likely, a digital camera.
Light from the light source passes into the condenser lens, which is mounted beneath
the microscope stage in an upright microscope (and above the stage in an inverted
microscope) in a bracket that can be raised and lowered for focussing.
The objective lens is responsible for producing the magnified image, and can be the
most expensive component of the light microscope.
Numerical aperture (NA) is always marked on the lens. This is a number usually
between 0.04 and 1.4. The NA is a measure of the ability of a lens to collect light from
the specimen.
Lenses with a low NA collect less light than those with a high NA.
Resolution varies inversely with NA, which implies that higher NA objectives yield the
best resolution.
The resolution achieved by a lens is a measure of its ability to distinguish between two
objects in the specimen. The shorter the wavelengths of illuminating light the higher the
resolving power of the microscope.
The limit of resolution for a microscope that uses visible light is about 300nm with a dry
lens (in air) and 200nm with an oil immersion lens.
Darkfield illumination produces images of brightly illuminated objects on a black
background .This technique has traditionally been used for viewing the outlines of
objects in liquid media such as living spermatozoa, microorganisms or cells growing in
tissue culture, or for a quick check of the status of a biochemical preparation. For lower
magnifications, a simple darkfield setting on the condenser will be sufficient. For more
critical darkfield imaging at a higher magnification, a darkfield condenser with a
darkfield objective lens will be required.
Phase contrast is used for viewing unstained cells growing in tissue culture and for
testing cell and organelle preparations for lysis. The method images differences in the
refractive index of cellular structures. Light that passes through thicker parts of the cell
is held up relative to the light that passes through thinner parts of the cytoplasm. It
requires a specialised phase condenser and phase objective lenses (both labelled ‘ph’).
Each phase setting of the condenser lens is matched with the phase setting of the
objective lens. These are usually numbered as Phase 1, Phase 2 and Phase 3, and are
found on both the condenser and the objective lens
Stereomicroscope
A second type of light microscope, the stereomicroscope, is used for the observation of the
surfaces of large specimens .The microscope is used when 3D information is required, for
example for the routine observation of whole organisms, for example for screening
through vials of fruit flies. Stereomicroscopes are useful for micromanipulation and
dissection where the wide field of view and the ability to zoom in and out in magnification
is invaluable. A wide range of objectives and eyepieces are available for different
applications. The light sources can be from above, from below the specimen, encircling the
specimen using a ring light or from the side giving a darkfield effect .
Differential interference contrast (DIC) is a form of interference microscopy that
produces images with a shadow relief . It is used for viewing unstained cells in tissue
culture, eggs and embryos, and in combination with some stains. Here the overall shape
and relief of the structure is viewed using DIC and a subset of the structure is stained with
a coloured dye.
Fluorescence microscopy is currently the most widely used contrast technique since it
gives superior signal-to-noise ratios (typically white on a black background) for many
applications.The most commonly used fluorescence technique is called epifluorescence
light microscopy, where ‘epi’ simply means ‘from above’.
Here the light source comes from above the sample, and the objective lens acts as both
condenser and objective lens.
Fluorescence is popular because of the ability to achieve highly specific labelling of cellular
compartments. The images usually consist of distinct regions of fluorescence (white) over
large regions of no fluorescence (black), which gives excellent signal-to-noise ratios
The light source is usually a high-pressure mercury or xenon vapour lamp, and more
recently lasers and LED sources, which emit from the UV into the red wavelengths . A
specific wavelength of light is used to excite a fluorescent molecule or fluorophore in the
specimen.
Light of longer wavelength from the excitation of the fluorophore is then imaged. This is
achieved in the fluorescence microscope using combinations of filters that are specific for
the excitation and emission characteristics of the fluorophore of interest. There are usually
three main filters: an excitation, a dichromatic mirror (often called a dichroic) and a
barrier filter, mounted in a single housing above the objective lens.
For example, the commonly used fluorophore fluorescein is optimally excited at a
wavelength of 488 nm, and emits maximally at 518nm
A second type of light microscope, the stereomicroscope, is used for the observation of the
surfaces of large specimens .The microscope is used when 3D information is required, for
example for the routine observation of whole organisms, for example for screening
through vials of fruit flies. Stereomicroscopes are useful for micromanipulation and
dissection where the wide field of view and the ability to zoom in and out in magnification
is invaluable. A wide range of objectives and eyepieces are available for different
applications. The light sources can be from above, from below the specimen, encircling the
specimen using a ring light or from the side giving a darkfield effect .
Differential interference contrast (DIC) is a form of interference microscopy that
produces images with a shadow relief . It is used for viewing unstained cells in tissue
culture, eggs and embryos, and in combination with some stains. Here the overall shape
and relief of the structure is viewed using DIC and a subset of the structure is stained with
a coloured dye.
Fluorescence microscopy is currently the most widely used contrast technique since it
gives superior signal-to-noise ratios (typically white on a black background) for many
applications.The most commonly used fluorescence technique is called epifluorescence
light microscopy, where ‘epi’ simply means ‘from above’.
Here the light source comes from above the sample, and the objective lens acts as both
condenser and objective lens.
Fluorescence is popular because of the ability to achieve highly specific labelling of cellular
compartments. The images usually consist of distinct regions of fluorescence (white) over
large regions of no fluorescence (black), which gives excellent signal-to-noise ratios
The light source is usually a high-pressure mercury or xenon vapour lamp, and more
recently lasers and LED sources, which emit from the UV into the red wavelengths . A
specific wavelength of light is used to excite a fluorescent molecule or fluorophore in the
specimen.
Light of longer wavelength from the excitation of the fluorophore is then imaged. This is
achieved in the fluorescence microscope using combinations of filters that are specific for
the excitation and emission characteristics of the fluorophore of interest. There are usually
three main filters: an excitation, a dichromatic mirror (often called a dichroic) and a
barrier filter, mounted in a single housing above the objective lens.
For example, the commonly used fluorophore fluorescein is optimally excited at a
wavelength of 488 nm, and emits maximally at 518nm
Chromatic mirrors and filters can be designed to filter two or three specific wavelengths
for imaging specimens labelled with two or more fluorochromes (multiple labelling).
The fluorescence emitted from the specimen is often too low to be detected by the
human eye or it may be out of the wavelength range of detection of the eye, for example,
in the far-red wavelengths A sensitive digital camera easily detects such signals; for
example a CCD or a PMT.
THE ELECTRON MICROSCOPE (EM)
Electron microscopy is used when the greatest resolution is required, and when the
living state can be ignored. The images produced in an electron microscope reveal the
ultrastructure of cells.
There are two different types of electron microscope – the transmission electron
microscope (TEM) and the scanning electron microscope (SEM). In the TEM, electrons
that pass through the specimen are imaged.
In the SEM electrons that are reflected back from the specimen (secondary electrons)
are collected, and the surfaces of specimens are imaged.
The equivalent of the light source in an electron microscope is the electron gun.
When a high voltage of between 40 000 and 100 000 volts (the accelerating voltage) is
passed between the cathode and the anode, a tungsten filament emits electrons.The
negatively charged electrons pass through a hole in the anode forming an electron
beam. The beam of electrons passes through a stack of electromagnetic lenses (the
column).Focussing of the electron beam is achieved by changing the voltage across the
electromagnetic lenses.The electrons have limited penetration power which means that
specimens must be thin (50–100 nm) to allow them to pass through.Thicker specimens
can be viewed by using a higher accelerating voltage, for example in the high-voltage
electron microscope (HVEM) which uses 1 000 000 V accelerating voltage or in the
intermediate voltage electron microscope (IVEM) which uses an accelerating voltage of
around 400 000 V.
All of the water has to be removed from any biological specimen before it can be imaged
in the EM. This is because the electron beam can only be produced and focussed in a
vacuum.The major drawback of EM observation of biological specimens therefore is the
non-physiological conditions necessary for their observation. specimens have been
traditionally prepared for the TEM by fixation in glutaraldehyde to cross-link proteins
followed by osmium tetroxide to fix and stain lipid membranes. This is followed by
dehydration in a series of alcohols to remove the water, and then embedding in a plastic
such as Epon for thin sectioning.Small pieces of the embedded tissue are mounted and
sectioned on an ultramicrotome using either a glass or a diamond knife.The sections are
for imaging specimens labelled with two or more fluorochromes (multiple labelling).
The fluorescence emitted from the specimen is often too low to be detected by the
human eye or it may be out of the wavelength range of detection of the eye, for example,
in the far-red wavelengths A sensitive digital camera easily detects such signals; for
example a CCD or a PMT.
THE ELECTRON MICROSCOPE (EM)
Electron microscopy is used when the greatest resolution is required, and when the
living state can be ignored. The images produced in an electron microscope reveal the
ultrastructure of cells.
There are two different types of electron microscope – the transmission electron
microscope (TEM) and the scanning electron microscope (SEM). In the TEM, electrons
that pass through the specimen are imaged.
In the SEM electrons that are reflected back from the specimen (secondary electrons)
are collected, and the surfaces of specimens are imaged.
The equivalent of the light source in an electron microscope is the electron gun.
When a high voltage of between 40 000 and 100 000 volts (the accelerating voltage) is
passed between the cathode and the anode, a tungsten filament emits electrons.The
negatively charged electrons pass through a hole in the anode forming an electron
beam. The beam of electrons passes through a stack of electromagnetic lenses (the
column).Focussing of the electron beam is achieved by changing the voltage across the
electromagnetic lenses.The electrons have limited penetration power which means that
specimens must be thin (50–100 nm) to allow them to pass through.Thicker specimens
can be viewed by using a higher accelerating voltage, for example in the high-voltage
electron microscope (HVEM) which uses 1 000 000 V accelerating voltage or in the
intermediate voltage electron microscope (IVEM) which uses an accelerating voltage of
around 400 000 V.
All of the water has to be removed from any biological specimen before it can be imaged
in the EM. This is because the electron beam can only be produced and focussed in a
vacuum.The major drawback of EM observation of biological specimens therefore is the
non-physiological conditions necessary for their observation. specimens have been
traditionally prepared for the TEM by fixation in glutaraldehyde to cross-link proteins
followed by osmium tetroxide to fix and stain lipid membranes. This is followed by
dehydration in a series of alcohols to remove the water, and then embedding in a plastic
such as Epon for thin sectioning.Small pieces of the embedded tissue are mounted and
sectioned on an ultramicrotome using either a glass or a diamond knife.The sections are
then mounted onto copper or gold EM grids, and are subsequently stained with heavy
metals, for example uranyl acetate and lead citrate.
For the SEM, samples are fixed in glutaraldehyde, dehydrated through a series of
solvents and dried completely either in air or by critical point drying. This method
removes all of the water from the specimen instantly and avoids surface tension in the
drying process thereby avoiding artifacts of drying. The specimens are then mounted
onto a special metal holder or stub and coated with a thin layer of gold before viewing
in the SEM.
POLARIZATION
The polarized light microscope is designed to observe and photograph specimens that
are visible primarily due to their optically anisotropic character. In order to accomplish
this task, the microscope must be equipped with both a polarizer, positioned in the light
path somewhere before the specimen, and an analyzer (a second polarizer), placed in
the optical pathway between the objective rear aperture and the observation tubes or
camera port.
Polarized light is a contrast-enhancing technique that improves the quality of the image
obtained with birefringent materials when compared to other techniques such as
darkfield and brightfield illumination, differential interference contrast, phase contrast,
Hoffman modulation contrast, and fluorescence.
Polarized light microscopes have a high degree of sensitivity and can be utilized for both
quantitative and qualitative studies targeted at a wide range of anisotropic specimens.
Qualitative polarizing microscopy is very popular in practice, with numerous volumes
dedicated to the subject.
Inverted
An inverted microscope is a microscope with its light source and condenser on the top,
above the stage pointing down, while the objectives and turret are below the stage
pointing up. Inverted microscopes are useful for observing living cells or organisms at
the bottom of a large container (e.g., a tissue culture flask) under more natural
conditions than on a glass slide, as is the case with a conventional microscope.
An inverted microscope is also used for visualisation of the mycobacterium tuberculosis
bacteria in the technique called Microscopic Observation Drug Susceptibility assay
(MODS).
Inverted microscopes are used in micromanipulation applications where space above
the specimen is required for manipulator mechanisms and the microtools they hold, and
in metallurgical applications where polished samples can be placed on top of the stage
and viewed from underneath using reflecting objectives.
metals, for example uranyl acetate and lead citrate.
For the SEM, samples are fixed in glutaraldehyde, dehydrated through a series of
solvents and dried completely either in air or by critical point drying. This method
removes all of the water from the specimen instantly and avoids surface tension in the
drying process thereby avoiding artifacts of drying. The specimens are then mounted
onto a special metal holder or stub and coated with a thin layer of gold before viewing
in the SEM.
POLARIZATION
The polarized light microscope is designed to observe and photograph specimens that
are visible primarily due to their optically anisotropic character. In order to accomplish
this task, the microscope must be equipped with both a polarizer, positioned in the light
path somewhere before the specimen, and an analyzer (a second polarizer), placed in
the optical pathway between the objective rear aperture and the observation tubes or
camera port.
Polarized light is a contrast-enhancing technique that improves the quality of the image
obtained with birefringent materials when compared to other techniques such as
darkfield and brightfield illumination, differential interference contrast, phase contrast,
Hoffman modulation contrast, and fluorescence.
Polarized light microscopes have a high degree of sensitivity and can be utilized for both
quantitative and qualitative studies targeted at a wide range of anisotropic specimens.
Qualitative polarizing microscopy is very popular in practice, with numerous volumes
dedicated to the subject.
Inverted
An inverted microscope is a microscope with its light source and condenser on the top,
above the stage pointing down, while the objectives and turret are below the stage
pointing up. Inverted microscopes are useful for observing living cells or organisms at
the bottom of a large container (e.g., a tissue culture flask) under more natural
conditions than on a glass slide, as is the case with a conventional microscope.
An inverted microscope is also used for visualisation of the mycobacterium tuberculosis
bacteria in the technique called Microscopic Observation Drug Susceptibility assay
(MODS).
Inverted microscopes are used in micromanipulation applications where space above
the specimen is required for manipulator mechanisms and the microtools they hold, and
in metallurgical applications where polished samples can be placed on top of the stage
and viewed from underneath using reflecting objectives.
Freeze fracture
Cell membranes consist of phospholipids and attached or embedded proteins.
Membrane proteins play vital roles in the metabolism and life of the cell. You cannot
use ordinary microscopy to visualize or characterize adhesion proteins, transport
proteins and protein channels in the cell membrane. Using electron microscopy and a
technique called "freeze fracture," which splits frozen cell membranes apart, allows
visualization of the membrane structure and the organization of proteins within the sea
of phospholipids. Combining other methods with freeze fracturing not only helps us to
understand the structure of different cell membranes and membrane proteins, but
allows for the visualization and detailed analysis of the function of specific proteins,
bacteria and viruses
Using liquid nitrogen, biological tissue samples or cells are rapidly frozen to immobilize
cell constituents
The frozen sample is cracked or fractured with a microtome, which is a knife-like
instrument for cutting thin tissue slices. This causes the cell membrane to split apart
precisely between the two layers because the attraction between the hydrophobic lipid
tails represents the weakest point
Following fracturing, the sample undergoes a vacuum procedure, called "freeze
etching." The surface of the fractured sample is shadowed with carbon and platinum
vapor to make a stable replica, which follows the contours of the fracture plane.
Freeze etching is the vacuum-drying of an unfixed, frozen and freeze-fractured
biological sample.
Fixation
In the fields of histology, pathology, and cell biology, fixation is the preservation of
biological tissues from decay due to autolysis or putrefaction. It terminates any ongoing
biochemical reactions and may also increase the treated tissues' mechanical strength or
stability.
Fixation is usually the first stage in a multistep process to prepare a sample of biological
material for microscopy or other analysis. Therefore, the choice of fixative and fixation
protocol may depend on the additional processing steps and final analyses that are
planned
Types
Heat fixation:
Immersion
Perfusion:
Cell membranes consist of phospholipids and attached or embedded proteins.
Membrane proteins play vital roles in the metabolism and life of the cell. You cannot
use ordinary microscopy to visualize or characterize adhesion proteins, transport
proteins and protein channels in the cell membrane. Using electron microscopy and a
technique called "freeze fracture," which splits frozen cell membranes apart, allows
visualization of the membrane structure and the organization of proteins within the sea
of phospholipids. Combining other methods with freeze fracturing not only helps us to
understand the structure of different cell membranes and membrane proteins, but
allows for the visualization and detailed analysis of the function of specific proteins,
bacteria and viruses
Using liquid nitrogen, biological tissue samples or cells are rapidly frozen to immobilize
cell constituents
The frozen sample is cracked or fractured with a microtome, which is a knife-like
instrument for cutting thin tissue slices. This causes the cell membrane to split apart
precisely between the two layers because the attraction between the hydrophobic lipid
tails represents the weakest point
Following fracturing, the sample undergoes a vacuum procedure, called "freeze
etching." The surface of the fractured sample is shadowed with carbon and platinum
vapor to make a stable replica, which follows the contours of the fracture plane.
Freeze etching is the vacuum-drying of an unfixed, frozen and freeze-fractured
biological sample.
Fixation
In the fields of histology, pathology, and cell biology, fixation is the preservation of
biological tissues from decay due to autolysis or putrefaction. It terminates any ongoing
biochemical reactions and may also increase the treated tissues' mechanical strength or
stability.
Fixation is usually the first stage in a multistep process to prepare a sample of biological
material for microscopy or other analysis. Therefore, the choice of fixative and fixation
protocol may depend on the additional processing steps and final analyses that are
planned
Types
Heat fixation:
Immersion
Perfusion:
Chemical fixation
In both immersion and perfusion fixation processes, chemical fixatives are used to
preserve structures in a state (both chemically and structurally) as close to living tissue
as possible. This requires a chemical fixative.
Crosslinking fixatives – aldehydes
Crosslinking fixatives act by creating covalent chemical bonds between proteins in
tissue. This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to
the tissue. Preservation of transient or fine cytoskeletal structure such as contractions
during embryonic differentiation waves is best achieved by a pretreatment using
microwaves before the addition of a cross linking fixative. The most commonly used
fixative in histology is formaldehyde
Precipitating fixatives – alcohols
Precipitating (or denaturing) fixatives act by reducing the solubility of protein molecules
and often by disrupting the hydrophobic interactions that give many proteins their
tertiary structure. The precipitation and aggregation of proteins is a very different
process from the crosslinking that occurs with aldehyde fixatives.
The most common precipitating fixatives are ethanol and methanol.
ELECTROPHORESIS
Electrophoresis is the migration of charged particles or molecules in a medium under the
influence of an applied electric field.
The usual purposes for carrying out electrophoretic experiments are (i) to determine the
number. amount. and mobility of components in a given sample or to separate them. and (iO to
obtain information about the electrical double layers surrounding the particles.
The first recorded measurements of electrophoretic phenomenon were performed in 1861 by
Quincke.
Even if two molecules have the same charge, they might not migrate together because if there is
difference in their molecular weights, they will have different charge/mass ratio (this differences
is of more use in electrophoresis on gels).
The extent of the friction, as described by Stoke's equation, will depend upon (i) the size and
shape of the molecule, and (iij on the viscosity of the medium through which the molecule will
migrate. Thus where F is the friction exerted on the spherical molecule, r is the radius ofthis
molecule,n is the viscosity of the solution, and v is the velocity at which the molecule is
migrating
In both immersion and perfusion fixation processes, chemical fixatives are used to
preserve structures in a state (both chemically and structurally) as close to living tissue
as possible. This requires a chemical fixative.
Crosslinking fixatives – aldehydes
Crosslinking fixatives act by creating covalent chemical bonds between proteins in
tissue. This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to
the tissue. Preservation of transient or fine cytoskeletal structure such as contractions
during embryonic differentiation waves is best achieved by a pretreatment using
microwaves before the addition of a cross linking fixative. The most commonly used
fixative in histology is formaldehyde
Precipitating fixatives – alcohols
Precipitating (or denaturing) fixatives act by reducing the solubility of protein molecules
and often by disrupting the hydrophobic interactions that give many proteins their
tertiary structure. The precipitation and aggregation of proteins is a very different
process from the crosslinking that occurs with aldehyde fixatives.
The most common precipitating fixatives are ethanol and methanol.
ELECTROPHORESIS
Electrophoresis is the migration of charged particles or molecules in a medium under the
influence of an applied electric field.
The usual purposes for carrying out electrophoretic experiments are (i) to determine the
number. amount. and mobility of components in a given sample or to separate them. and (iO to
obtain information about the electrical double layers surrounding the particles.
The first recorded measurements of electrophoretic phenomenon were performed in 1861 by
Quincke.
Even if two molecules have the same charge, they might not migrate together because if there is
difference in their molecular weights, they will have different charge/mass ratio (this differences
is of more use in electrophoresis on gels).
The extent of the friction, as described by Stoke's equation, will depend upon (i) the size and
shape of the molecule, and (iij on the viscosity of the medium through which the molecule will
migrate. Thus where F is the friction exerted on the spherical molecule, r is the radius ofthis
molecule,n is the viscosity of the solution, and v is the velocity at which the molecule is
migrating
F=6 π rn v
FACTORS AFFECTING ELECTROPHORETIC MOBILITY
Charge/mass ratio of the sample dictates its electrophoretic mobility. The mass consists of not
only the size (molecular weight) but also the shape of the molecule.
(i) Charge. The higher the charge, greater is the electrophoretic mobility. The charge,
however, is dependent on pH of the medium.
(ii) (ii) Size. The bigger the molecule, greater are the frictional and electrostatic forces
exerted upon it by the medium of suspension. Consequently, larger particles have a
smaller electrophoretic mobility compared to the smaller particles. (
(iii) Shape. Rounded contours elicit lesser frictional and electrostatic retardation
compared to sharp contours. As an example consider the case of globular and
fibrous proteins. Given the same size (molecular 'weight) the globular protein will
migrate faster than the fibrous protein.
The rate of migration under unit potential gradient is referred to as mobility of the ion.
The Medium
An inert supporting medium is chosen for electrophoresis. But even this inert medium can exert
adsorption and/or molecular sieving effects on the particle thereby influencing its rate of
migration. The medium may also give rise to electro-osmosis, which may also influence the rate
of sample migration
(i) Adsorption. Adsorption, here, means retention of a component on the surface of
supporting medium
(ii) Molecular sieving. Supporting media such as polyacrylamide, agar, starch and
Sephadex have cross-linked structures giving rise to pores within the gel beads.
(iii) The Buffer Apart from maintaining the pH of the supporting medium, the buffer
can affect the electrophoretic mobility of the sample in various other ways.
Commonly used buffers are formate, citrate, phosphate, EDTA, acetate pyridine,
Tris, and barbitone etc. The choice of buffer depends upon the type of sample being
electrophoresced.
FACTORS AFFECTING ELECTROPHORETIC MOBILITY
Charge/mass ratio of the sample dictates its electrophoretic mobility. The mass consists of not
only the size (molecular weight) but also the shape of the molecule.
(i) Charge. The higher the charge, greater is the electrophoretic mobility. The charge,
however, is dependent on pH of the medium.
(ii) (ii) Size. The bigger the molecule, greater are the frictional and electrostatic forces
exerted upon it by the medium of suspension. Consequently, larger particles have a
smaller electrophoretic mobility compared to the smaller particles. (
(iii) Shape. Rounded contours elicit lesser frictional and electrostatic retardation
compared to sharp contours. As an example consider the case of globular and
fibrous proteins. Given the same size (molecular 'weight) the globular protein will
migrate faster than the fibrous protein.
The rate of migration under unit potential gradient is referred to as mobility of the ion.
The Medium
An inert supporting medium is chosen for electrophoresis. But even this inert medium can exert
adsorption and/or molecular sieving effects on the particle thereby influencing its rate of
migration. The medium may also give rise to electro-osmosis, which may also influence the rate
of sample migration
(i) Adsorption. Adsorption, here, means retention of a component on the surface of
supporting medium
(ii) Molecular sieving. Supporting media such as polyacrylamide, agar, starch and
Sephadex have cross-linked structures giving rise to pores within the gel beads.
(iii) The Buffer Apart from maintaining the pH of the supporting medium, the buffer
can affect the electrophoretic mobility of the sample in various other ways.
Commonly used buffers are formate, citrate, phosphate, EDTA, acetate pyridine,
Tris, and barbitone etc. The choice of buffer depends upon the type of sample being
electrophoresced.
(iv) Ionic strength. As we have already seen above. increased ionic strength of the
buffer means a larges share of the current being carried by the buffer ions and a
meager proportion carried by the sample ions. This situation gets translated into a
slower migration of the sample components. Since the overall current will also
increase there will be heat production
(v) pH Since pH determines the degree of ionization of organiC compounds. it can also
affect the rate of migration of these compounds. Increase in pH increases ionization
of organiC acids and a decrease in pH increases the ionization of organic bases
TYPES OF ELECTROPHORESIS
Electrophoresis can be divided into two main techniques: free electrophoresis or electrophoresis
without stabilizing media and zone electrophoresis or electrophoresis in stabilizing media.
FREE ELECTROPHORESI - Microelectrophoresis And Moving Boundary Electrophoresis.
Microelectrophoresis -This electrophoretic technique involves the observation of motion of
small particles in an electric field with a microscope (such as R.B.Cs. neutrophils. bacteria etc.).
. In modem days this technique is applied only for measuring the zeta potentials of cells such as
R.B.Cs, neutrophils, bacteria etc.
Moving Boundary Electrophoresis-This is the prototype of all modem methods of
electrophoresis and was first developed by A. Tiselius of Sweden in the 1930s
In moving boundary electrophoresis a buffered solution of macromolecules is placed under a
layer of pure buffer solution in a U-shaped observation cell
The whole cell may then be immersed in a constant temperature bath insulated from vibrations.
The power is switched on generating an electric field between the electrodes. Normally in a
complex sample containing many macromolecules, there will be species, which will bear a net
negative charge and therefore move towards the anode while at the same time the
macromolecules bearing a net positive charge will move towards cathode
This situation of movements in mutually opposite directions will not be good for a satisfactory
resolution. The pH of the buffer is, therefore, so chosen that all the macromolecules bear a net
negative charge
Moving boundary method was very popular for quantitative analysis of complex mixtures of
macromolecules, especially proteins,e.g., those in blood plasma..
buffer means a larges share of the current being carried by the buffer ions and a
meager proportion carried by the sample ions. This situation gets translated into a
slower migration of the sample components. Since the overall current will also
increase there will be heat production
(v) pH Since pH determines the degree of ionization of organiC compounds. it can also
affect the rate of migration of these compounds. Increase in pH increases ionization
of organiC acids and a decrease in pH increases the ionization of organic bases
TYPES OF ELECTROPHORESIS
Electrophoresis can be divided into two main techniques: free electrophoresis or electrophoresis
without stabilizing media and zone electrophoresis or electrophoresis in stabilizing media.
FREE ELECTROPHORESI - Microelectrophoresis And Moving Boundary Electrophoresis.
Microelectrophoresis -This electrophoretic technique involves the observation of motion of
small particles in an electric field with a microscope (such as R.B.Cs. neutrophils. bacteria etc.).
. In modem days this technique is applied only for measuring the zeta potentials of cells such as
R.B.Cs, neutrophils, bacteria etc.
Moving Boundary Electrophoresis-This is the prototype of all modem methods of
electrophoresis and was first developed by A. Tiselius of Sweden in the 1930s
In moving boundary electrophoresis a buffered solution of macromolecules is placed under a
layer of pure buffer solution in a U-shaped observation cell
The whole cell may then be immersed in a constant temperature bath insulated from vibrations.
The power is switched on generating an electric field between the electrodes. Normally in a
complex sample containing many macromolecules, there will be species, which will bear a net
negative charge and therefore move towards the anode while at the same time the
macromolecules bearing a net positive charge will move towards cathode
This situation of movements in mutually opposite directions will not be good for a satisfactory
resolution. The pH of the buffer is, therefore, so chosen that all the macromolecules bear a net
negative charge
Moving boundary method was very popular for quantitative analysis of complex mixtures of
macromolecules, especially proteins,e.g., those in blood plasma..
ZONE ELECTROPHORESIS -Konig published the first experiment on the use of
filter paper as stabilizing medium in electrophoresis. This paved the way for several other
porous stabilizing media, most of which are gels such as agar, starch, and polyacrylamide. Zone
electrophoresis is the name given to the separation technique employing these stabilizing media.
It is also known as electrophoresis in stabilized media.
Upon separation the molecules are immobilized by fIxation in different zones. The molecules
are then detected by staining them on the supporting medium.
Other methods to detect the separated molecules are (i) visualization by ultraviolet light. (ii)
detection by virtue of enzymic reaction. or (iii) detection by radioactivity. if the molecules are
radiolabeled
GENERAL TECHNIQUES OF ZONE ELECTROPHORESIS
PAPER ELECTROPHORESIS -Filter paper as a stabilizing medium is very popular for the
study of normal and abnormal plasma proteins. Paper of good quality should contain atleast
95% of cellulose and should have only a very slight adsorption capacity. Chromatography
paper is suitable for electrophoresis and needs no preparation other than to be cut to size.
The equipment required for electrophoresis consists basically of two items, a power pack and an
electrophoretic cell. The power pack provides a stabilized direct current and has controls for
both voltage and current output. Power packs, which have an output of 0-500 V and 0-150 rnA
are available and can be programmed to give either constant voltage or current.
The electrophoretic cell contains the electrodes, buffer reservoirs, a support for paper and a
transparent insulating cover. The electrodes are usually made of platinum.
Sample Application -This is the single most critical procedure in the whole electrophoresis
process. The sample may be applied as a spot (about 0.5 cm in diameter) or as a narrow
uniform streak. Special devices are available commercially for this purpose. There is no hard
and fast rule for the time of application of the sample to the electrophoretogram
filter paper as stabilizing medium in electrophoresis. This paved the way for several other
porous stabilizing media, most of which are gels such as agar, starch, and polyacrylamide. Zone
electrophoresis is the name given to the separation technique employing these stabilizing media.
It is also known as electrophoresis in stabilized media.
Upon separation the molecules are immobilized by fIxation in different zones. The molecules
are then detected by staining them on the supporting medium.
Other methods to detect the separated molecules are (i) visualization by ultraviolet light. (ii)
detection by virtue of enzymic reaction. or (iii) detection by radioactivity. if the molecules are
radiolabeled
GENERAL TECHNIQUES OF ZONE ELECTROPHORESIS
PAPER ELECTROPHORESIS -Filter paper as a stabilizing medium is very popular for the
study of normal and abnormal plasma proteins. Paper of good quality should contain atleast
95% of cellulose and should have only a very slight adsorption capacity. Chromatography
paper is suitable for electrophoresis and needs no preparation other than to be cut to size.
The equipment required for electrophoresis consists basically of two items, a power pack and an
electrophoretic cell. The power pack provides a stabilized direct current and has controls for
both voltage and current output. Power packs, which have an output of 0-500 V and 0-150 rnA
are available and can be programmed to give either constant voltage or current.
The electrophoretic cell contains the electrodes, buffer reservoirs, a support for paper and a
transparent insulating cover. The electrodes are usually made of platinum.
Sample Application -This is the single most critical procedure in the whole electrophoresis
process. The sample may be applied as a spot (about 0.5 cm in diameter) or as a narrow
uniform streak. Special devices are available commercially for this purpose. There is no hard
and fast rule for the time of application of the sample to the electrophoretogram
The current is switched on after the sample has been applied to the paper and the paper has
been equilibrated with the buffer.
Once removed. the paper is dried in vacuum oven at 11 oDe (if the compounds are not
thermolabile. in which case the paper is allowed to hang and air dried).
Detection and Quantitative Assay-To identify unknown components in the resolved mixture the
electrophoretogram may be compared with another electrophoretogram on which standard
components have been electrophoresced under identical conditions
Quantitative estimation. A rough idea of the quantity of the components of a sample can be had
by visually comparing ("eyeballing") the color of different zones with their standards of a
known quantity. But this is seldom accurate.
Cellulose Acetate Electrophoresis
Cellulose acetate as a medium for electrophoresis was introduced by Kohn in 1958. was
developed from bacteriological cellulose acetate membrane filters and is commercially available
as high purity cellulose acetate strips, which are thin and have a uniform micropore structure.
Although paper electrophoresis is still the choice for routine fast diagnostic analyses, the
resolution of a given protein might suffer because of substantial adsorption on paper. This
disadvantage of paper is completely taken care of if cellulose acetate strips are used instead of
paper.
Additional advantages of cellulose acetate are (i) it is chemically pure; it does not contain
lignins, hemicelluloses or nitrogen, (ii) cellulose strips are translucent and this makes them
suitable for direct photoelectric scanning for separated bands of components, (iii) because of the
very low content of glucose these strips are suitable for electrophoresis of polysaccharides; upon
staining by Schiff's reagent, background staining is negligible
been equilibrated with the buffer.
Once removed. the paper is dried in vacuum oven at 11 oDe (if the compounds are not
thermolabile. in which case the paper is allowed to hang and air dried).
Detection and Quantitative Assay-To identify unknown components in the resolved mixture the
electrophoretogram may be compared with another electrophoretogram on which standard
components have been electrophoresced under identical conditions
Quantitative estimation. A rough idea of the quantity of the components of a sample can be had
by visually comparing ("eyeballing") the color of different zones with their standards of a
known quantity. But this is seldom accurate.
Cellulose Acetate Electrophoresis
Cellulose acetate as a medium for electrophoresis was introduced by Kohn in 1958. was
developed from bacteriological cellulose acetate membrane filters and is commercially available
as high purity cellulose acetate strips, which are thin and have a uniform micropore structure.
Although paper electrophoresis is still the choice for routine fast diagnostic analyses, the
resolution of a given protein might suffer because of substantial adsorption on paper. This
disadvantage of paper is completely taken care of if cellulose acetate strips are used instead of
paper.
Additional advantages of cellulose acetate are (i) it is chemically pure; it does not contain
lignins, hemicelluloses or nitrogen, (ii) cellulose strips are translucent and this makes them
suitable for direct photoelectric scanning for separated bands of components, (iii) because of the
very low content of glucose these strips are suitable for electrophoresis of polysaccharides; upon
staining by Schiff's reagent, background staining is negligible
Cellulose acetate is especially used for clinical investigations such as separation of glycoproteins,
lipoproteins and hemoglobins from blood.
GEL ELECTROPHORESIS
The gels, however, are porous and the size of the pores relative to that ofthe molecule
determines whether the molecule will enter the pore and be retarded or will bypass it. The
separation thus not only depends on the charge on the molecule but also on its size.
Types orGel
Starch gel.- the resolving power of starch gels is very high and can be matched only by
polyacrylamide gels. One of their important applications is the analysis of isoenzyme patterns
(zymograms).
Agar- Agar gel electrophoresis was flrst described by Gordon in 1949 and has been since used
increasingly for biochemical separations. Agar is being used to separate high molecular weight
macromolecules like proteins and nucleic acids. In the latter case its use has become legion.
Polyacrylamide. The components used in the formation of this gel are known to be neurotoxins
and thus care has to be taken while preparing the gel. The most commonly used components to
synthesize the matrix are acrylamide monom~r, N, N'-methylenebisacrylamide (bis) ,
ammonium per sulphate and tetramethylenediamine (TEMED).
This feature makes this gel particularly suitable for resolving mixtures of proteins and nucleic
acids in a very reproducible manner. Other desirable features of these gels are (i) a low
adsorption capacity, (n) lack of electroosmosis, and (iii) suitability for in situ stochemical and
qualitative analysis.
Agarose-Acrylamide. The need to separate very high molecular weight nucleic acids (200 Kd or
more) prompted the development of this complex gel. These gels have been successfully tried for
isolation of 3.5 x 106 daltons DNA.
Other gels. which have been used include pectin. gypsum. Sephadex. polyvinyl chloride and
polyvinyl acetate. However. they are not used often and hence a discussion is avoided
Electrophoretic Mobility in Gels
The pore size. thus the molecular sieving action and therefore the effect on electrophoretic
mobility of a molecule are functions of gel concentration. Rodbard and Chrambach have
developed a set of mathematical relationships to describe the effects of gel concentration upon a
macromolecule's mobility.
log E = log E' – KrG
where E is the electrophoretic mobility. E' is the mobility in a sucrose solution. Kr is the
retardation coefficient and G is the total gel concentration. The retardation coefficient has been
described as follows
Kr= C (R + r)
lipoproteins and hemoglobins from blood.
GEL ELECTROPHORESIS
The gels, however, are porous and the size of the pores relative to that ofthe molecule
determines whether the molecule will enter the pore and be retarded or will bypass it. The
separation thus not only depends on the charge on the molecule but also on its size.
Types orGel
Starch gel.- the resolving power of starch gels is very high and can be matched only by
polyacrylamide gels. One of their important applications is the analysis of isoenzyme patterns
(zymograms).
Agar- Agar gel electrophoresis was flrst described by Gordon in 1949 and has been since used
increasingly for biochemical separations. Agar is being used to separate high molecular weight
macromolecules like proteins and nucleic acids. In the latter case its use has become legion.
Polyacrylamide. The components used in the formation of this gel are known to be neurotoxins
and thus care has to be taken while preparing the gel. The most commonly used components to
synthesize the matrix are acrylamide monom~r, N, N'-methylenebisacrylamide (bis) ,
ammonium per sulphate and tetramethylenediamine (TEMED).
This feature makes this gel particularly suitable for resolving mixtures of proteins and nucleic
acids in a very reproducible manner. Other desirable features of these gels are (i) a low
adsorption capacity, (n) lack of electroosmosis, and (iii) suitability for in situ stochemical and
qualitative analysis.
Agarose-Acrylamide. The need to separate very high molecular weight nucleic acids (200 Kd or
more) prompted the development of this complex gel. These gels have been successfully tried for
isolation of 3.5 x 106 daltons DNA.
Other gels. which have been used include pectin. gypsum. Sephadex. polyvinyl chloride and
polyvinyl acetate. However. they are not used often and hence a discussion is avoided
Electrophoretic Mobility in Gels
The pore size. thus the molecular sieving action and therefore the effect on electrophoretic
mobility of a molecule are functions of gel concentration. Rodbard and Chrambach have
developed a set of mathematical relationships to describe the effects of gel concentration upon a
macromolecule's mobility.
log E = log E' – KrG
where E is the electrophoretic mobility. E' is the mobility in a sucrose solution. Kr is the
retardation coefficient and G is the total gel concentration. The retardation coefficient has been
described as follows
Kr= C (R + r)
where C is constant. R is the mean radius of the macromolecule and r is the radius of the gel
fibers.
Solubilizers
Several proteins of biological importance contain more than one polypeptide chain. These
proteins are referred to as oligomeric proteins. The whole structure is stabilized by hydrogen
bonding. disulphide bridges. or by hydrophobic associations. These proteins migrate as a single
band during gel electrophoresis. If. however. the subunits of these proteins are to be separated
from each other. a class of substance~ known to destabilize the quaternary structure are
employed. These substances are collectively known as solubilizers.
Urea- Urea at concentration from 3-12 M is known to disrupt hydrogen bonds
Sodium dodecyl sulphate. (CH3(CH2) lOCHPSO; Na+). Sodium dodecyl sulphate
(SDS) is an anionic detergent and disrupts macromolecules whose structure has been stabilized
by hydrophobic associations. SDS has been shown to bind to the hydrophobic regions of
proteins and to separate most ofthem into their component subunits unless the subunits are
covalently bound. SDS binding also imparts a large negative charge to the denatured
polypeptides. This charge shadows any other charge previously present on the polypeptide.
Since all the polypeptide chains now have an equal negative charge due to SDS, they will
migrate in gel solely on the basis of their size
B-mercaptoethanol. Many proteins have their plural polypeptide chains linked together by
disulphide bridges. These bonds are broken by heating the protein solution in presence of
mercaptoethanol.
Electrophoretic Procedure-The equipment consists of two components
A Buffer Reservoir
A D.C Power Supply.
The buffer reservoir system has an upper and a lower buffer reservoir connected by the gel.
Save for the gel, there is no other electrical connection between the two reservoirs. Platinum
electrodes are positioned in each reservoir and are connected to terminals extending from the
top of the unit. The whole assembly may be covered by a perspex shield.
The sample, prepared in a high density component such as glycerol or ficoll to prevent its
mixing with the upper reservoir buffer is loaded on top of the gel. A 'tracking dye' (usually
bromophenol blue) is often mixed with the sample.
The extent of migration of the dye gives an index of electrophoretic process. The dye migrates
faster than all macromolecules.
The pH is usually fixed at 9, which gives a net negative charge to most macromolecules. The
anode, to which these negatively charged macromolecules would migrate under an electric field,
is therefore placed in the lower buffer reservoir
fibers.
Solubilizers
Several proteins of biological importance contain more than one polypeptide chain. These
proteins are referred to as oligomeric proteins. The whole structure is stabilized by hydrogen
bonding. disulphide bridges. or by hydrophobic associations. These proteins migrate as a single
band during gel electrophoresis. If. however. the subunits of these proteins are to be separated
from each other. a class of substance~ known to destabilize the quaternary structure are
employed. These substances are collectively known as solubilizers.
Urea- Urea at concentration from 3-12 M is known to disrupt hydrogen bonds
Sodium dodecyl sulphate. (CH3(CH2) lOCHPSO; Na+). Sodium dodecyl sulphate
(SDS) is an anionic detergent and disrupts macromolecules whose structure has been stabilized
by hydrophobic associations. SDS has been shown to bind to the hydrophobic regions of
proteins and to separate most ofthem into their component subunits unless the subunits are
covalently bound. SDS binding also imparts a large negative charge to the denatured
polypeptides. This charge shadows any other charge previously present on the polypeptide.
Since all the polypeptide chains now have an equal negative charge due to SDS, they will
migrate in gel solely on the basis of their size
B-mercaptoethanol. Many proteins have their plural polypeptide chains linked together by
disulphide bridges. These bonds are broken by heating the protein solution in presence of
mercaptoethanol.
Electrophoretic Procedure-The equipment consists of two components
A Buffer Reservoir
A D.C Power Supply.
The buffer reservoir system has an upper and a lower buffer reservoir connected by the gel.
Save for the gel, there is no other electrical connection between the two reservoirs. Platinum
electrodes are positioned in each reservoir and are connected to terminals extending from the
top of the unit. The whole assembly may be covered by a perspex shield.
The sample, prepared in a high density component such as glycerol or ficoll to prevent its
mixing with the upper reservoir buffer is loaded on top of the gel. A 'tracking dye' (usually
bromophenol blue) is often mixed with the sample.
The extent of migration of the dye gives an index of electrophoretic process. The dye migrates
faster than all macromolecules.
The pH is usually fixed at 9, which gives a net negative charge to most macromolecules. The
anode, to which these negatively charged macromolecules would migrate under an electric field,
is therefore placed in the lower buffer reservoir
Gel electrophoresis is usually carried out in anyone of the two modes
(i) column electrophoresis, or (ii) slab gel electrophoresis
Column electrophoresis -The gel is set or polymerized in a column. This column is then fitted
between the upper and the lower buffer reservoirs. An apparatus is commercially available
which has between 8-12 columns fitted into buffer reservoirs
Slab gel electrophoresis. The gel is set or polymerized into a thin slab between two glass plates.
The thickness of the slab of the gel can be adjusted by placing spacers of various.
thickness between the two glass plates. Sample wells are made at one end of the gel by placing a
comb-shaped jig into the gel before it sets or polymerizes. After the gel has set. the comb is
removed leaving the sample wells etched into the gel.
. Since a number of wells can be cast side-by-side. number of samples can be loaded
simultaneously and compared under conditions which remain essentially identical. This is a
great advantage of this technique over the column mode. The technique is becoming extremely
popular. especially in the field of molecular biology.
Applications of Gel Electrophoresis
Apart from separation and isolation of a large number of protein and other macromolecules.
larger number of analytical applications.
Determination of DNA sequences
Southern and Northern blotting
\ Restriction mapping of DNA
(i) column electrophoresis, or (ii) slab gel electrophoresis
Column electrophoresis -The gel is set or polymerized in a column. This column is then fitted
between the upper and the lower buffer reservoirs. An apparatus is commercially available
which has between 8-12 columns fitted into buffer reservoirs
Slab gel electrophoresis. The gel is set or polymerized into a thin slab between two glass plates.
The thickness of the slab of the gel can be adjusted by placing spacers of various.
thickness between the two glass plates. Sample wells are made at one end of the gel by placing a
comb-shaped jig into the gel before it sets or polymerizes. After the gel has set. the comb is
removed leaving the sample wells etched into the gel.
. Since a number of wells can be cast side-by-side. number of samples can be loaded
simultaneously and compared under conditions which remain essentially identical. This is a
great advantage of this technique over the column mode. The technique is becoming extremely
popular. especially in the field of molecular biology.
Applications of Gel Electrophoresis
Apart from separation and isolation of a large number of protein and other macromolecules.
larger number of analytical applications.
Determination of DNA sequences
Southern and Northern blotting
\ Restriction mapping of DNA
Determination of molecular weight of proteins by gel electrophoresis
Discontinuous (Disc) Gel Electrophoresis
Disc gel electrophoresis (so called because of the discontinuous buffer employed and discoid
appearance of the macromolecular zones) is a modification of conventional zone electrophoresis.
which allows the sample to enter the gel as a sharp band. thereby helping further resolution.
Here the macromolecule mixture to be analyzed is subjected to an electric field in a retarding
gel support that is separated into two sections differing in porosity and buffered at different
pHs.
macromolecular mixture migrates from the more porous into the less porous gel. a process
accompanied by a change in pH.
The gel that is preferred for disc gel electrophoresis is polyacrylamide. The Two Different
Porosity Gels Used Are Known As The Stacking Gel (High Porosity) And Separating Or
Running Gel (Low Porosity).
Gradient Gel Electrophoresis
This is performed in a pore-gradient gel. Here the size of the pores goes on continuously
decreasing in the direction in which the macromolecules migrate in an electric field. The gel
used is polyacrylamide decrease in pore size is achieved by increments in the acrylamide
concentration. When the electric field is applied, initially the macromolecules move according to
their electrophoretic mobility
The separation of molecules is essentially a function of their size and not their mobilities. This
method is extremely powerful and is chiefly used for separating native proteins..
High Voltage Electrophoresis (H.V.E.)
Heat generation is the paramount problem in H.V.E. The main component of an H.V.E.
apparatus is therefore its cooling system. When Michl described his first apparatus for H.V.E.,
chlorobenzene or toluene was used to dissipate the heat.
However, these inert organic compounds were found unsuitable for voltages of the order of
10,000 V. Modem equipments use either water or water-glycol mixture as the cooling agent. The
cooling agent is made to flow through the cooling plates.
Isoelectric Focussing
While paper electrophoresis resolves plasma proteins into six bands, isoelectric focussing
resolves it into atleast 40 bands.
In isoelectric focussing (also known as electrofocussing). on the other hand. a stable pH gradient
is arranged; the pH increases gradually from anode to cathode. A protein introduced into this
system at a point where the pH is lower than its isoionic point will possess a net positive charge
and will migrate in the direction of cathode.
Discontinuous (Disc) Gel Electrophoresis
Disc gel electrophoresis (so called because of the discontinuous buffer employed and discoid
appearance of the macromolecular zones) is a modification of conventional zone electrophoresis.
which allows the sample to enter the gel as a sharp band. thereby helping further resolution.
Here the macromolecule mixture to be analyzed is subjected to an electric field in a retarding
gel support that is separated into two sections differing in porosity and buffered at different
pHs.
macromolecular mixture migrates from the more porous into the less porous gel. a process
accompanied by a change in pH.
The gel that is preferred for disc gel electrophoresis is polyacrylamide. The Two Different
Porosity Gels Used Are Known As The Stacking Gel (High Porosity) And Separating Or
Running Gel (Low Porosity).
Gradient Gel Electrophoresis
This is performed in a pore-gradient gel. Here the size of the pores goes on continuously
decreasing in the direction in which the macromolecules migrate in an electric field. The gel
used is polyacrylamide decrease in pore size is achieved by increments in the acrylamide
concentration. When the electric field is applied, initially the macromolecules move according to
their electrophoretic mobility
The separation of molecules is essentially a function of their size and not their mobilities. This
method is extremely powerful and is chiefly used for separating native proteins..
High Voltage Electrophoresis (H.V.E.)
Heat generation is the paramount problem in H.V.E. The main component of an H.V.E.
apparatus is therefore its cooling system. When Michl described his first apparatus for H.V.E.,
chlorobenzene or toluene was used to dissipate the heat.
However, these inert organic compounds were found unsuitable for voltages of the order of
10,000 V. Modem equipments use either water or water-glycol mixture as the cooling agent. The
cooling agent is made to flow through the cooling plates.
Isoelectric Focussing
While paper electrophoresis resolves plasma proteins into six bands, isoelectric focussing
resolves it into atleast 40 bands.
In isoelectric focussing (also known as electrofocussing). on the other hand. a stable pH gradient
is arranged; the pH increases gradually from anode to cathode. A protein introduced into this
system at a point where the pH is lower than its isoionic point will possess a net positive charge
and will migrate in the direction of cathode.
Due to the presence of the pH gradient; the protein will migrate to an environment of
successively higher pH values. which. in turn will influence the ionization and net charge of the
molecule. Finally. the protein will encounter a pH where its net charge is zero and will stop
migrating. . This is the isoelectric point of the protein.
Electrofocusing has been widely used for separation and identification of serum proteins. It is
being used by the food and agricultural industries, forensic and human genetics laboratories,
and for research in enzymology, immunology and membrane . biochemistry etc.
Two Dimensional Gel Electrophoresis
This powerful technique combines the resolving power of isoelectric focussing with SDS gel
electrophoresis. The resolving power of the technique is so acute that it can resolve a mixture
containing 5000 proteins into individual species
Capillary electrophoresis
• Capillary tube is placed between two buffer reservoir, and an electric field is applied,
separation depends on electrophoretic mobility & electro-osmosis .
A buffer filled fused-silica capillary 10-100 µm in internal diameter & 40-100 cm long • Two
electrode(platinum) • High voltage supply (5 to 30 kv) • Sample injector (by pressure or
vacuum) • Detector • Buffer solution (like sodium dihydrogen phosphate,NaH2 PO4)
Detectors similar to those used in GC,HPLC • majority of instruments have UV detectors
available. • Alternative detector modes include commercially available fluorescence, laser
induced fluorescence, conductivity and indirect detection. • The mass spectrometers is
frequently used to give structural information on the resolved peaks. • Sensitive detectors are
needed for small concentrations in CE
PULSED-FIELD GEL ELECTROPHORESIS
As opposed to the continuous unidirectional electric fields applied in conventional gel
electrophoresis, pulsed-field gel electrophoresis uses pulsed, alternating, orthogonal electric
fields.
When such a field is applied to a gel, large DNA molecules become trapped into their reptation
tubes every time the direction of the electric field is changed. These molecules remain immobile
till they reorient. themselves along the direction of the new electric field. It is here that different
DNA molecules adapt a behaviour consonant with their respective sizes.
Factors, which are of extreme importance for determining the limit of resolution of pulsed-field
gel electrophoresis are given below.
(i) The absolute periods of the electric pulses. (ii) The angles at which the two electric fields are
applied to the gel. (iii) The relative field strengths of the two electric fields and the degree of
uniformity of the two electric fields. (iv) The ratio of the periods of the electric pulses employed
to generate the two electric fields.
successively higher pH values. which. in turn will influence the ionization and net charge of the
molecule. Finally. the protein will encounter a pH where its net charge is zero and will stop
migrating. . This is the isoelectric point of the protein.
Electrofocusing has been widely used for separation and identification of serum proteins. It is
being used by the food and agricultural industries, forensic and human genetics laboratories,
and for research in enzymology, immunology and membrane . biochemistry etc.
Two Dimensional Gel Electrophoresis
This powerful technique combines the resolving power of isoelectric focussing with SDS gel
electrophoresis. The resolving power of the technique is so acute that it can resolve a mixture
containing 5000 proteins into individual species
Capillary electrophoresis
• Capillary tube is placed between two buffer reservoir, and an electric field is applied,
separation depends on electrophoretic mobility & electro-osmosis .
A buffer filled fused-silica capillary 10-100 µm in internal diameter & 40-100 cm long • Two
electrode(platinum) • High voltage supply (5 to 30 kv) • Sample injector (by pressure or
vacuum) • Detector • Buffer solution (like sodium dihydrogen phosphate,NaH2 PO4)
Detectors similar to those used in GC,HPLC • majority of instruments have UV detectors
available. • Alternative detector modes include commercially available fluorescence, laser
induced fluorescence, conductivity and indirect detection. • The mass spectrometers is
frequently used to give structural information on the resolved peaks. • Sensitive detectors are
needed for small concentrations in CE
PULSED-FIELD GEL ELECTROPHORESIS
As opposed to the continuous unidirectional electric fields applied in conventional gel
electrophoresis, pulsed-field gel electrophoresis uses pulsed, alternating, orthogonal electric
fields.
When such a field is applied to a gel, large DNA molecules become trapped into their reptation
tubes every time the direction of the electric field is changed. These molecules remain immobile
till they reorient. themselves along the direction of the new electric field. It is here that different
DNA molecules adapt a behaviour consonant with their respective sizes.
Factors, which are of extreme importance for determining the limit of resolution of pulsed-field
gel electrophoresis are given below.
(i) The absolute periods of the electric pulses. (ii) The angles at which the two electric fields are
applied to the gel. (iii) The relative field strengths of the two electric fields and the degree of
uniformity of the two electric fields. (iv) The ratio of the periods of the electric pulses employed
to generate the two electric fields.
The original apparatus used alternately pulsed electric fields or perpendicular orientations and
linear electrodes. The apparatus of this type, however, suffers from certain drawbacks due to
which the resolution is not so good. The electric field generated in such apparatus is never
uniform. This affects the speed and direction of the DNA which depend more and more on the
position at which they are loaded into the gel. It is typical of such apparatus that towards the
edge of the gel, the DNA path becomes skewed leading to a not-so-good resolution.
SPECTROPHOTOMETRY
BASIC PRINCIPLES-The term electromagnetic is a precise description of the radiation in that
the radiation is made up of an electrical and a magnetic wave which are in phase and
perpendicular to each other and to the direction of propagation.
v = c/λ
Sometimes radiation, mostly in the infrared region , is characterized by another term known as
the wave number and denoted by the symbol v. Wave number means the number of complete
cycles occurring per centimetre,
The energy E, of a photon can be related to its wave:length and frequency with the help of
Planck's constant, h
E=hv=hc/ λ
A beam of radiation from an electric bulb consists of several wavelengths and is known as
polychromatic, A beam in which all the rays have the same wavelength is known as
monochromatic.
THE LAWS OF ABSORPTION -
The first of these laws is known as the Lambert law. It states that the amount of light absorbed
is proportional to the thickness of the absorbing material and is independent of the intensity of
the incident light. I*b
The second law of absorption is known as the Beer's law. This law states that the amount of
light absorbed by a material is proportional to the number of absorbing molecules i.e., the
concentration of absorbing solution.I*C
Log10 IO/I=abC
linear electrodes. The apparatus of this type, however, suffers from certain drawbacks due to
which the resolution is not so good. The electric field generated in such apparatus is never
uniform. This affects the speed and direction of the DNA which depend more and more on the
position at which they are loaded into the gel. It is typical of such apparatus that towards the
edge of the gel, the DNA path becomes skewed leading to a not-so-good resolution.
SPECTROPHOTOMETRY
BASIC PRINCIPLES-The term electromagnetic is a precise description of the radiation in that
the radiation is made up of an electrical and a magnetic wave which are in phase and
perpendicular to each other and to the direction of propagation.
v = c/λ
Sometimes radiation, mostly in the infrared region , is characterized by another term known as
the wave number and denoted by the symbol v. Wave number means the number of complete
cycles occurring per centimetre,
The energy E, of a photon can be related to its wave:length and frequency with the help of
Planck's constant, h
E=hv=hc/ λ
A beam of radiation from an electric bulb consists of several wavelengths and is known as
polychromatic, A beam in which all the rays have the same wavelength is known as
monochromatic.
THE LAWS OF ABSORPTION -
The first of these laws is known as the Lambert law. It states that the amount of light absorbed
is proportional to the thickness of the absorbing material and is independent of the intensity of
the incident light. I*b
The second law of absorption is known as the Beer's law. This law states that the amount of
light absorbed by a material is proportional to the number of absorbing molecules i.e., the
concentration of absorbing solution.I*C
Log10 IO/I=abC
This combined law states that the amount of light absorbed (absorbance or extinction) is
proportional to the concentration of the absorbing substance and to the thickness of the
absorbing material (path-length). The quantity loll is known as the absorbance or the optical
density (O.D.). The reverse, 1110 is known as the transmittance, T (the amount of light which
escapes absorption and is transmitted).
A (absorbance) = log I0 - log I, but 10 is always set at 100% and log 100 = 2, A = 2 -log I
ABSORPTION SPECTRUM - The pattern of energy absorption by a substance when light of
varying wavelength passes through it is uniquely characteristic of the substance. This pattern is
known as an absorption spectrum.
The data so obtained is plotted in the form of a curve relating transmittance or optical density
(the latter is preferred) to wavelength. The application of absorption spectra is not limited to the
visible region of the spectrum but may be applied equally well to characterization of the
ultraviolet or infrared absorption of many substances
The instruments that are used to study the absorption or emission of electromagnetic radiation
as a function of wavelength are called spectrometers or spectrophotometers.
The essential components of a spectrophotometer include: (i) a stable and cheap radiant energy
source, (ii) a monochromator to break the polychromatic radiation into component wavelengths
or "bands" of wavelengths, (iii) transparent vessels (cuvettes) to hold the sample, and (iv) a
photosensitive detector and an associated readout system (meter or recorder). Instruments
available commercially involve quite a bit of complex arrangements,
Sources of ultraviolet radiation: Most commonly used sources of ultraviolet radiation are the
hydrogen lamp and the deuterium lamp
Sources of visible radiation : Tungsten filament lamp is the most commonly used source for
visible radiation. It is inexpensive and emits continuous radiation in the region between 350 and
2500 nm.
Sources of infrared radiation: Nernst Glower and Globar are the most satisfactory sources of
infrared radiation.
Wavelength selectors are of two types
Filters
Monochromators.
Filters- Filters resolve polychromatic light into a relatively wide bandwidth of about 40 nm and
are used only in colorimeters. One disadvantage of glass filters is their low transmittance (5-
20%).
Monochromators- As the name suggests, a monochromator resolves polychromatic radiation
into its individual wavelengths and isolates these wavelengths into very narrow bands
proportional to the concentration of the absorbing substance and to the thickness of the
absorbing material (path-length). The quantity loll is known as the absorbance or the optical
density (O.D.). The reverse, 1110 is known as the transmittance, T (the amount of light which
escapes absorption and is transmitted).
A (absorbance) = log I0 - log I, but 10 is always set at 100% and log 100 = 2, A = 2 -log I
ABSORPTION SPECTRUM - The pattern of energy absorption by a substance when light of
varying wavelength passes through it is uniquely characteristic of the substance. This pattern is
known as an absorption spectrum.
The data so obtained is plotted in the form of a curve relating transmittance or optical density
(the latter is preferred) to wavelength. The application of absorption spectra is not limited to the
visible region of the spectrum but may be applied equally well to characterization of the
ultraviolet or infrared absorption of many substances
The instruments that are used to study the absorption or emission of electromagnetic radiation
as a function of wavelength are called spectrometers or spectrophotometers.
The essential components of a spectrophotometer include: (i) a stable and cheap radiant energy
source, (ii) a monochromator to break the polychromatic radiation into component wavelengths
or "bands" of wavelengths, (iii) transparent vessels (cuvettes) to hold the sample, and (iv) a
photosensitive detector and an associated readout system (meter or recorder). Instruments
available commercially involve quite a bit of complex arrangements,
Sources of ultraviolet radiation: Most commonly used sources of ultraviolet radiation are the
hydrogen lamp and the deuterium lamp
Sources of visible radiation : Tungsten filament lamp is the most commonly used source for
visible radiation. It is inexpensive and emits continuous radiation in the region between 350 and
2500 nm.
Sources of infrared radiation: Nernst Glower and Globar are the most satisfactory sources of
infrared radiation.
Wavelength selectors are of two types
Filters
Monochromators.
Filters- Filters resolve polychromatic light into a relatively wide bandwidth of about 40 nm and
are used only in colorimeters. One disadvantage of glass filters is their low transmittance (5-
20%).
Monochromators- As the name suggests, a monochromator resolves polychromatic radiation
into its individual wavelengths and isolates these wavelengths into very narrow bands
Essential components of a monochromator are:
An Entrance Slit Which Admits Polychromatic Light From The Source,
A Collimating Device Such As A Lens Or A Mirror Which Collimates The
Polychromatic Light On To The Dispersion Device,
A Wavelength Resolving Device Like A Prism Or A Grating Which Breaks The
Radiation Into Component Wavelengths,
A Focussing Lens Or A Mirror,
An Exit Slit Which Allows The Monochromatic Beam To Escape.
Prism: A prism disperses polychromatic light from the source into its constituent wavelengths
by virtue of its ability to refract different wavelengths to a different extent; the shorter
wavelengths are diffracted most
Gratings: Gratings (Figure 8.14) are often used in the monochromators of spectrophotometers
operating in ultraviolet. visible. and infrared regions. The grating possesses a highly aluminized
surface etched with a large number of parallel grooves which are equally spaced. These grooves
are also known as lines
Samples to be studied in the ultraviolet or visible region are usually gases or solutions and are
put in cells known as cuvettes. Cuvettes meant for the visible region are made up of either
ordinary glass or sometimes quartz. Since glass absorbs in the ultraviolet region. quartz or
fused silica .::ells are used in this region.
. The solvents which can be used in the UV and visible region are water. methyl-. ethyl-.
isopropyl-alcohols. chloroform. hexane etc
Ultraviolet and visible radiation detectors- Photocells. phototubes. and photomultiplier tubes
Photocell - A typical photocell consists of a thin coating of selenium over a thin transparent
silver film on a steel base. This arrangement ensures that electrons pass easily from selenium to
An Entrance Slit Which Admits Polychromatic Light From The Source,
A Collimating Device Such As A Lens Or A Mirror Which Collimates The
Polychromatic Light On To The Dispersion Device,
A Wavelength Resolving Device Like A Prism Or A Grating Which Breaks The
Radiation Into Component Wavelengths,
A Focussing Lens Or A Mirror,
An Exit Slit Which Allows The Monochromatic Beam To Escape.
Prism: A prism disperses polychromatic light from the source into its constituent wavelengths
by virtue of its ability to refract different wavelengths to a different extent; the shorter
wavelengths are diffracted most
Gratings: Gratings (Figure 8.14) are often used in the monochromators of spectrophotometers
operating in ultraviolet. visible. and infrared regions. The grating possesses a highly aluminized
surface etched with a large number of parallel grooves which are equally spaced. These grooves
are also known as lines
Samples to be studied in the ultraviolet or visible region are usually gases or solutions and are
put in cells known as cuvettes. Cuvettes meant for the visible region are made up of either
ordinary glass or sometimes quartz. Since glass absorbs in the ultraviolet region. quartz or
fused silica .::ells are used in this region.
. The solvents which can be used in the UV and visible region are water. methyl-. ethyl-.
isopropyl-alcohols. chloroform. hexane etc
Ultraviolet and visible radiation detectors- Photocells. phototubes. and photomultiplier tubes
Photocell - A typical photocell consists of a thin coating of selenium over a thin transparent
silver film on a steel base. This arrangement ensures that electrons pass easily from selenium to
silver but not in the reverse direction. Due to the inability of electrons to move away from the
silver film, the silver acts as the collecting electrode for electrons liberated from selenium by the
incident radiation. The steel plate functions as the other electrode. The current flowing between
the two electrodes is then measured by a microammeter
Photomultipliers: These detectors are designed to amplify the initial photoelectric effect and are
suitable for use at very low light intensities. A photomultiplier consists of (A) an evacuated glass
tube into which are sealed the cathode and the anode additional intervening electrodes known
as dynodes
Photodiodes: Photodiodes are semiconductors that change their charged voltage (usually 5 V)
upon being struck by light. The voltage change is converted to current and is measured.
Double Beam Operation Voltage fluctuations inducing fluctuations in the source intensity can
cause large scale errors in spectrophotometer operation. To obviate this situation, double beam
spectrophotometers have been designed,
Double beam instruments employ some type of beam splitter prior to the sample containers.
One of the split beams passes through the "blank" or reference cell while the other passes
through the sample cell. The two transmitted beams are then compared either continuously or
alternately several times in a second
silver film, the silver acts as the collecting electrode for electrons liberated from selenium by the
incident radiation. The steel plate functions as the other electrode. The current flowing between
the two electrodes is then measured by a microammeter
Photomultipliers: These detectors are designed to amplify the initial photoelectric effect and are
suitable for use at very low light intensities. A photomultiplier consists of (A) an evacuated glass
tube into which are sealed the cathode and the anode additional intervening electrodes known
as dynodes
Photodiodes: Photodiodes are semiconductors that change their charged voltage (usually 5 V)
upon being struck by light. The voltage change is converted to current and is measured.
Double Beam Operation Voltage fluctuations inducing fluctuations in the source intensity can
cause large scale errors in spectrophotometer operation. To obviate this situation, double beam
spectrophotometers have been designed,
Double beam instruments employ some type of beam splitter prior to the sample containers.
One of the split beams passes through the "blank" or reference cell while the other passes
through the sample cell. The two transmitted beams are then compared either continuously or
alternately several times in a second
Dual wavelength spectrophotometry refers to the photometric measurement of a material by
passing radiation of two different wavelengths through the same sample before reaching the
detector. Light from two different sources is allowed to be resolved into two different
wavelength with the help of a pair of diffraction gratings.
Applications Of Uv-Vis Spectrophotometry
• Qualitative & Quantitative Analysis:
– It is used for characterizing aromatic compounds and conjugated olefins.
– It can be used to find out molar concentration of the solute under study.
• Detection of impurities:
– It is one of the important method to detect impurities in organic solvents.
• Detection of isomers are possible.
• Detection of unknown compound.
• Enzyme Assay
INFRARED SPECTROSCOPY
Infrared radiation is of much higher wavelength as compared to the ultraviolet and the Visible
region. Infrared radiation is, therefore, not associated with electronic tranSitions; rather, it is
associated with vibrational transitions of molecules as we will see below.
passing radiation of two different wavelengths through the same sample before reaching the
detector. Light from two different sources is allowed to be resolved into two different
wavelength with the help of a pair of diffraction gratings.
Applications Of Uv-Vis Spectrophotometry
• Qualitative & Quantitative Analysis:
– It is used for characterizing aromatic compounds and conjugated olefins.
– It can be used to find out molar concentration of the solute under study.
• Detection of impurities:
– It is one of the important method to detect impurities in organic solvents.
• Detection of isomers are possible.
• Detection of unknown compound.
• Enzyme Assay
INFRARED SPECTROSCOPY
Infrared radiation is of much higher wavelength as compared to the ultraviolet and the Visible
region. Infrared radiation is, therefore, not associated with electronic tranSitions; rather, it is
associated with vibrational transitions of molecules as we will see below.
This increase or decrease in bond length is occurring, the atoms remain in the same bond axis.
These vibrations are known as stretching vibrations
. The other type of molecular vibration, known as bending vibration (Bll involves changes in
the positions of the atoms with respect to the original bond axis.
Vibrational transitions are low energy transitions and these energy levels correspond to the
energies of electromagnetic radiation in the infrared region of the spectrum.
INFRARED SPECTROPHOTOMETER: MODE OF OPERATION
The spectrometer consists of a source of infrared light, emitting radiation throughout the whole
frequency range of the instrument. Light from the source is split into two beams of equal
intensity. One beam is made to pass through the sample while the other is allowed to behave as
the reference beam.
It is the function of the detector to convert infrared thermal energy to electrical energy. At the
wavelength where the sample has absorbed, the detector will receive a weak beam from the
sample while the reference beam will retain full intensity. This will lead to a pulsating, or
alternating current to flow from the detector to the amplifier.
The sampling techniques depend on whether the sample is in vapor phase, a liquid, or a solid.
Applications of Infrared Spectroscopy
identification of compounds, assaying the rate of reactions, studying the conformation of
molecules, and understanding interactions between molecules.
Disadvantages - It is very difficult to obtain quantitative data with infrared spectrophotometry.
This is again a problem related to the solvent.
Fluorescense spectroscopy(Emission)
The phenomenon whereby a molecule. after absorbing radiations. emits radiation of a longer
wavelength is known as fluorescence. Therefore. fluorescence spectra are band spectra. They
are usually independent of the wavelength of the radiation absorbed.
These vibrations are known as stretching vibrations
. The other type of molecular vibration, known as bending vibration (Bll involves changes in
the positions of the atoms with respect to the original bond axis.
Vibrational transitions are low energy transitions and these energy levels correspond to the
energies of electromagnetic radiation in the infrared region of the spectrum.
INFRARED SPECTROPHOTOMETER: MODE OF OPERATION
The spectrometer consists of a source of infrared light, emitting radiation throughout the whole
frequency range of the instrument. Light from the source is split into two beams of equal
intensity. One beam is made to pass through the sample while the other is allowed to behave as
the reference beam.
It is the function of the detector to convert infrared thermal energy to electrical energy. At the
wavelength where the sample has absorbed, the detector will receive a weak beam from the
sample while the reference beam will retain full intensity. This will lead to a pulsating, or
alternating current to flow from the detector to the amplifier.
The sampling techniques depend on whether the sample is in vapor phase, a liquid, or a solid.
Applications of Infrared Spectroscopy
identification of compounds, assaying the rate of reactions, studying the conformation of
molecules, and understanding interactions between molecules.
Disadvantages - It is very difficult to obtain quantitative data with infrared spectrophotometry.
This is again a problem related to the solvent.
Fluorescense spectroscopy(Emission)
The phenomenon whereby a molecule. after absorbing radiations. emits radiation of a longer
wavelength is known as fluorescence. Therefore. fluorescence spectra are band spectra. They
are usually independent of the wavelength of the radiation absorbed.
The higher the number of 1t-electrons. the higher is the fluorescence.
The instrumentation of a spectrofluorimeter differs from that of the spectrophotometer in two
important respects besides other minor variations.(i) There are two monochromators instead of
one as in a spectrophotometer; one monochromator is placed before the sample holder and one
after it. and (ii) As fluorescence is maximum between 25-30oC. the sample hoider has a device to
maintain the temperature.
The main components of a spectrofluorimeter
(i) A continuous source of radiant energy (mercury lamp or xenon arc);
(ii) A monochromator usually a prism (P1)' to choose the wavelength with which the sample is
to be irradiated;
(iii) A second monochromator (P2) which. placed after the sample. enables the determination of
the fluorescent spectrum of the sample;
(iV) A detector. usually a photomultiplier suited for wavelengths greater than 500 nm; and
(v) An amplifier.
Disadvantages
A drawback of spectrofluorlmetty is a high degree of absorption of fluorescent radiation by the
emitting sample itself. This is known as quenching. Quenching also occurs due to impurities.
Detergents, filter paper and many laborat of tissues cause interference in fluorlmetric assays
because they can release strong fluorescing materials
Application
Studies On Protein Structure
assay of riboflavin, thiamine, hormones such as cortisol, oestrogen. serotonin and dopamine,
organophosphorus pesticides. tobacco smoke carcinogens. drugs such as lysergic acid and
barbiturates. porphyrins. cholesterol, and even some metal ions);Fluorescent microscopy
The instrumentation of a spectrofluorimeter differs from that of the spectrophotometer in two
important respects besides other minor variations.(i) There are two monochromators instead of
one as in a spectrophotometer; one monochromator is placed before the sample holder and one
after it. and (ii) As fluorescence is maximum between 25-30oC. the sample hoider has a device to
maintain the temperature.
The main components of a spectrofluorimeter
(i) A continuous source of radiant energy (mercury lamp or xenon arc);
(ii) A monochromator usually a prism (P1)' to choose the wavelength with which the sample is
to be irradiated;
(iii) A second monochromator (P2) which. placed after the sample. enables the determination of
the fluorescent spectrum of the sample;
(iV) A detector. usually a photomultiplier suited for wavelengths greater than 500 nm; and
(v) An amplifier.
Disadvantages
A drawback of spectrofluorlmetty is a high degree of absorption of fluorescent radiation by the
emitting sample itself. This is known as quenching. Quenching also occurs due to impurities.
Detergents, filter paper and many laborat of tissues cause interference in fluorlmetric assays
because they can release strong fluorescing materials
Application
Studies On Protein Structure
assay of riboflavin, thiamine, hormones such as cortisol, oestrogen. serotonin and dopamine,
organophosphorus pesticides. tobacco smoke carcinogens. drugs such as lysergic acid and
barbiturates. porphyrins. cholesterol, and even some metal ions);Fluorescent microscopy
Nuclear Magnetic Resonance Spectrometry
Nuclear magnetic resonance spectrometry addresses itself towards detecting the minute amount
of energy absorbed or emitted as the nuclei jump from one energy level to another.
Nuclei possessing an odd mass number (either the number of neutrons or the number of protons
should be odd. but never both odd) are assigned half-integral spin quantum numbers.
Nuclei possessing even numbers of both protons and neutrons (l2C. 160. 32S. etc.) are not
measured in n.m.r. experiments because they do not possess an angular momentum (I = 0) and
do not exhibit magnetic properties. Any given nucleus can have 21 + 1 possible levels or
orientations
In order to change from low energy state to high energy state. such nuclei must absorb the
appropriate quantum of energy. In a magnetic field of several hundred tesla (several thousand
gauss) such nuclei absorb radiation in the radiowave region of the electromagnetic spectrum.
This phenomenon is known as nuclear resonance or nuclear magnetic resonance
The frequency of the radiowave absorbed during n.m.r. is dictated by (i) the isotope being
studied. and (ii) the intensity of the magnetic field.
n.m.r. spectra are thus a plot of energy absorbed against the magnetic field strength applied.
Instrumentation
It would not be wrong to say that the magnets are the heart of an n.m.r. spectrometer. Since the
nuclei absorb in the radiowave region. the source of radiation is a radio frequency transmitter.
The sample must be dissolved to a relatively high concentration in a solvent which is proton-
poor 020. or CDCI3).
Sample is usually placed n a high precision diameter tube (this minimizes the variations in the
magnetic field) and is rotated at high speed by an air turbine. The absorption signal is then
detected by a radio receiver. The signal is amplified and recorded.
Applcations
Structural diagnosis
Nuclear magnetic resonance spectrometry addresses itself towards detecting the minute amount
of energy absorbed or emitted as the nuclei jump from one energy level to another.
Nuclei possessing an odd mass number (either the number of neutrons or the number of protons
should be odd. but never both odd) are assigned half-integral spin quantum numbers.
Nuclei possessing even numbers of both protons and neutrons (l2C. 160. 32S. etc.) are not
measured in n.m.r. experiments because they do not possess an angular momentum (I = 0) and
do not exhibit magnetic properties. Any given nucleus can have 21 + 1 possible levels or
orientations
In order to change from low energy state to high energy state. such nuclei must absorb the
appropriate quantum of energy. In a magnetic field of several hundred tesla (several thousand
gauss) such nuclei absorb radiation in the radiowave region of the electromagnetic spectrum.
This phenomenon is known as nuclear resonance or nuclear magnetic resonance
The frequency of the radiowave absorbed during n.m.r. is dictated by (i) the isotope being
studied. and (ii) the intensity of the magnetic field.
n.m.r. spectra are thus a plot of energy absorbed against the magnetic field strength applied.
Instrumentation
It would not be wrong to say that the magnets are the heart of an n.m.r. spectrometer. Since the
nuclei absorb in the radiowave region. the source of radiation is a radio frequency transmitter.
The sample must be dissolved to a relatively high concentration in a solvent which is proton-
poor 020. or CDCI3).
Sample is usually placed n a high precision diameter tube (this minimizes the variations in the
magnetic field) and is rotated at high speed by an air turbine. The absorption signal is then
detected by a radio receiver. The signal is amplified and recorded.
Applcations
Structural diagnosis
Study of dynamic characteristics of protein structure
Quantitative studies
Intact organ studies with n.m.r
ELECTRON SPIN RESONANCE SPECTROMETRY
A chemical species with an odd number of electrons exhibits characteristic magnetic properties
much like the nucleus. In a manner Similar to the atomic nucleus. an electron in a magnetic
field is able to absorb energy of the proper frequency, ~E = hv which will catapult it from the
lower to a higher energy level. This phenomenon is known as electron resonance. and the
technique which is employed to study this type of behavior is called electron spin resonance
(e.s.r.) spectrometer
As been seen with respect to n.m.r., it is common practice to subject the sample to differing
magnetic intensities keeping the microwave frequency constant.
The area of an e.s.r. peak is directly proportional to the number of unpaired electrons in the
sample investigated and thus to the concentration of the sample.
Instrumentation
Basic components of an e.s.r. spectrometer. Fields of 50-500 millitesla required for accurate
work, are generated by electromagnets.
Monochromatic microwave radiation might be readily obtained by using a klystron oscillator
Samples for e.s.r. must be solids. Biological samples which contain a large amount of water are
therefore frozen in liquid nitrogen before e.s.r. experiment
Electron spin resonance spectrometry has proven most helpful in studying mechanisms of
reactions which proceed through free radical intermediates
Electron spin resonance spectrometry is one of the main methods to study transition metal
containing metalloproteins
Quantitative studies
Intact organ studies with n.m.r
ELECTRON SPIN RESONANCE SPECTROMETRY
A chemical species with an odd number of electrons exhibits characteristic magnetic properties
much like the nucleus. In a manner Similar to the atomic nucleus. an electron in a magnetic
field is able to absorb energy of the proper frequency, ~E = hv which will catapult it from the
lower to a higher energy level. This phenomenon is known as electron resonance. and the
technique which is employed to study this type of behavior is called electron spin resonance
(e.s.r.) spectrometer
As been seen with respect to n.m.r., it is common practice to subject the sample to differing
magnetic intensities keeping the microwave frequency constant.
The area of an e.s.r. peak is directly proportional to the number of unpaired electrons in the
sample investigated and thus to the concentration of the sample.
Instrumentation
Basic components of an e.s.r. spectrometer. Fields of 50-500 millitesla required for accurate
work, are generated by electromagnets.
Monochromatic microwave radiation might be readily obtained by using a klystron oscillator
Samples for e.s.r. must be solids. Biological samples which contain a large amount of water are
therefore frozen in liquid nitrogen before e.s.r. experiment
Electron spin resonance spectrometry has proven most helpful in studying mechanisms of
reactions which proceed through free radical intermediates
Electron spin resonance spectrometry is one of the main methods to study transition metal
containing metalloproteins
Atomic Absorption Spectroscopy
Atomic Absorption Spectroscopy is a very common Atomic Absorption Spectroscopy is a very
common technique for detecting metals and metalloids in technique for detecting metals and
metalloids in samples.
It can analyze over 62 elements. It also measures the concentration of metals in the sample.
PRINCIPLE:
The technique uses basically the principle that free atoms (gas) generated in an atomizer can
absorb atoms (gas) generated in an atomizer can absorb radiation at specific frequency.
Atomic-absorption spectroscopy quantifies the absorption of ground state atoms in the
gaseous state.
The atoms absorb ultraviolet or visible light and make transitions to higher electronic energy
levels. . The analyte concentration is determined from the amount of concentration is
determined from the amount of absorption.
LIGHT SOURCE: LIGHT SOURCE:
common radiation source in AAS. source in AAS.
hollow cylindrical cathode made of the element to be determined.
NEBULIZER:
introduction into flame.
Atomic Absorption Spectroscopy is a very common Atomic Absorption Spectroscopy is a very
common technique for detecting metals and metalloids in technique for detecting metals and
metalloids in samples.
It can analyze over 62 elements. It also measures the concentration of metals in the sample.
PRINCIPLE:
The technique uses basically the principle that free atoms (gas) generated in an atomizer can
absorb atoms (gas) generated in an atomizer can absorb radiation at specific frequency.
Atomic-absorption spectroscopy quantifies the absorption of ground state atoms in the
gaseous state.
The atoms absorb ultraviolet or visible light and make transitions to higher electronic energy
levels. . The analyte concentration is determined from the amount of concentration is
determined from the amount of absorption.
LIGHT SOURCE: LIGHT SOURCE:
common radiation source in AAS. source in AAS.
hollow cylindrical cathode made of the element to be determined.
NEBULIZER:
introduction into flame.
Mix the aerosol and fuel and oxidant thoroughly for introduction into flame
Atomizer Atomizer
atomic sate.
into individual molecules and breaking molecules into atoms. This is molecules and breaking
molecules into atoms. This is done by exposing the analyte to high temperatures in a done by
exposing the analyte to high temperatures in a flame or graphite furnace .
Atomizer- FLAME ATOMIZERS, GRAPHITE TUBE ATOMIZERS
Flame Atomizer
eed to mix an oxidant gas and a. fuel gas.
-acetylene flame or nitrous oxidein most of the cases air-acetylene
flame or nitrous oxideacetylene flame is used.
Graphite Tube Atomizer
ln GFAAS sample, samples are deposited in a small graphite coated tube which can then be
heated to graphite coated tube which can then be heated to vaporize and atomize the analyte
MONOCHROMATOR:
It is used to separate out all of the
thousands of lines.
A monochromator is used to select the specific wavelength of light which is absorbed by the
sample, and wavelength of light which is absorbed by the sample, and to exclude other
wavelengths.
The light selected by the monochromator is directed onto a detector that is typically a
photomultiplier tube , whose function is to convert the light signal into an electrical function is
proportional to the light intensity.
A calibration curve is used to determine the unknown concentration of an element in a
solution. . The instrument is calibrated using several solutions of known
, Determination of even small amounts of metals (lead, mercury, calcium, magnesium, etc) as
follows:
X-ray crystallography
X-ray crystallography is a powerful technique for visualizing the structure of protein. In
crystallography the crystalline atoms cause a beam of incident X-rays to diffract into many
specific directions.
Atomizer Atomizer
atomic sate.
into individual molecules and breaking molecules into atoms. This is molecules and breaking
molecules into atoms. This is done by exposing the analyte to high temperatures in a done by
exposing the analyte to high temperatures in a flame or graphite furnace .
Atomizer- FLAME ATOMIZERS, GRAPHITE TUBE ATOMIZERS
Flame Atomizer
eed to mix an oxidant gas and a. fuel gas.
-acetylene flame or nitrous oxidein most of the cases air-acetylene
flame or nitrous oxideacetylene flame is used.
Graphite Tube Atomizer
ln GFAAS sample, samples are deposited in a small graphite coated tube which can then be
heated to graphite coated tube which can then be heated to vaporize and atomize the analyte
MONOCHROMATOR:
It is used to separate out all of the
thousands of lines.
A monochromator is used to select the specific wavelength of light which is absorbed by the
sample, and wavelength of light which is absorbed by the sample, and to exclude other
wavelengths.
The light selected by the monochromator is directed onto a detector that is typically a
photomultiplier tube , whose function is to convert the light signal into an electrical function is
proportional to the light intensity.
A calibration curve is used to determine the unknown concentration of an element in a
solution. . The instrument is calibrated using several solutions of known
, Determination of even small amounts of metals (lead, mercury, calcium, magnesium, etc) as
follows:
X-ray crystallography
X-ray crystallography is a powerful technique for visualizing the structure of protein. In
crystallography the crystalline atoms cause a beam of incident X-rays to diffract into many
specific directions.
Then crystallographer can produce a three-dimensional picture of the density of electrons
within the crystal, From this electron density, the mean positions of the atoms in the crystal can
be determined.
Ray diffraction by crystals is a reflection of the periodicity of crystal architecture, so that
imperfection in the crystal lattice usually results in poor diffraction properties.
X-ray crystallography uses the uniformity of light diffraction of crytals to determine the
structure of molecule or atom.
Then X-ray beam is used to hit the crystallized molecule. The electron surrounding the
molecule diffract as the X-rays hit them. This forms a pattern. This type of pattern is known as
X-ray diffraction pattern
Bragg’s Law
nλ = 2d sinƟ
Here d is the spacing between diffracting planes, Ɵ is the incident angle, n is any integar, and λ
is the wavelength of the beam.
Generally a typical x-ray diffraction contain below parts:
Detector 2. X-ray source 3. Crystal on the end of mounting needle 4. Liquid nitrogen steam to
keep crystal cold 5. Movable mount to rotate crystal
Knowing this, protein crystallographers use high intensity x-ray sources such as a rotating
anode tube or a strong synchrotron x-ray source for analyzing the protein crystals.
The process begins by crystallizing a protein of interest. 4 critical steps are taken to achieve
protein crystallization:
X-rays are generated and directed toward the crystallized protein, The crystal is rotated so that
the x-rays are able to hit the protein from all sides and angles
An electron density map is created based on the measured intensities of the diffraction pattern
on the film, The mapping gives a three-dimensional representation of the electron densities
observed through the x-ray crystallography
When interpreting the electron density map, resolution needs to be taken into account
Application
X-ray crystallography technique has been a widely used tool for elucidation of compounds
present in milk
Differentiation of Sugar
Medical
In case of new material
Chromatography
within the crystal, From this electron density, the mean positions of the atoms in the crystal can
be determined.
Ray diffraction by crystals is a reflection of the periodicity of crystal architecture, so that
imperfection in the crystal lattice usually results in poor diffraction properties.
X-ray crystallography uses the uniformity of light diffraction of crytals to determine the
structure of molecule or atom.
Then X-ray beam is used to hit the crystallized molecule. The electron surrounding the
molecule diffract as the X-rays hit them. This forms a pattern. This type of pattern is known as
X-ray diffraction pattern
Bragg’s Law
nλ = 2d sinƟ
Here d is the spacing between diffracting planes, Ɵ is the incident angle, n is any integar, and λ
is the wavelength of the beam.
Generally a typical x-ray diffraction contain below parts:
Detector 2. X-ray source 3. Crystal on the end of mounting needle 4. Liquid nitrogen steam to
keep crystal cold 5. Movable mount to rotate crystal
Knowing this, protein crystallographers use high intensity x-ray sources such as a rotating
anode tube or a strong synchrotron x-ray source for analyzing the protein crystals.
The process begins by crystallizing a protein of interest. 4 critical steps are taken to achieve
protein crystallization:
X-rays are generated and directed toward the crystallized protein, The crystal is rotated so that
the x-rays are able to hit the protein from all sides and angles
An electron density map is created based on the measured intensities of the diffraction pattern
on the film, The mapping gives a three-dimensional representation of the electron densities
observed through the x-ray crystallography
When interpreting the electron density map, resolution needs to be taken into account
Application
X-ray crystallography technique has been a widely used tool for elucidation of compounds
present in milk
Differentiation of Sugar
Medical
In case of new material
Chromatography
The basis of all forms of chromatography is the distribution or partition coefficient (Kd), which
describes the way in which a compound (the analyte) distributes between two immiscible
phases.
All chromatographic systems consist of the stationary phase, which may be a solid, gel, liquid or
a solid/liquid mixture that is immobilised, and the mobile phase, which may be liquid or
gaseous, and which is passed over or through the stationary phase after the mixture of analytes
to be separated has been applied to the stationary phase.
it is very easy to separate one from another. But as the individual components of a mixture get
more and more similar in physical and chemical properties. it becomes increasingly difficult to
separate them from one another.
The first detailed description of chromatography is generally credited to Michael Tswett. a
Russian biochemist. who separated chlorophyll from a mixture of plant pigments tn 1906.
the process was coined from the Greek words for color (chromo) and .. to write" (graphy).
The compound which interacts more with the mobile phase and least with the stationary phase
migrates fast.
Partition Coefficient
Partition coeffiCient (also known as distribution codficient) is a definitive term normally used to
describe the way in which a given compound distributes or partitions itself between two
immiscible phases. the stationary and the mobile phase
the concentration of the compound in each of the phases is described by the partition coefficient,
K. which is expressed as follows
K=CS/Cm
whete C. and em are the concentrations of the compound in the stationary and the mobUe
phases respectivelywhete C. and em are the concentrations of the compound in the stationary
and the mobUe phases respectively
Partition Chromatography
The principle of partition is exploited in gas/liquid chromatography (GLC) technique also.
Separations depend upor the partition of the solute molecules between a liqUid, supported on a
suitable solid, and the gas flowing through the system.
In true partition chromatography, the only factor which influences the movement of a
compound as the solvent travels along the stationary phase is the relative solubility of that
compound in the two phases.
Substances more soluble in the mobile phase will migrate greater distances as compared with
the substances more soluble in the stationary phase. Other compounds of intermediate solubility
between the two phases will migrate to intermediate distances depending upon their partition
coefficient
Adsorption Chromatography
describes the way in which a compound (the analyte) distributes between two immiscible
phases.
All chromatographic systems consist of the stationary phase, which may be a solid, gel, liquid or
a solid/liquid mixture that is immobilised, and the mobile phase, which may be liquid or
gaseous, and which is passed over or through the stationary phase after the mixture of analytes
to be separated has been applied to the stationary phase.
it is very easy to separate one from another. But as the individual components of a mixture get
more and more similar in physical and chemical properties. it becomes increasingly difficult to
separate them from one another.
The first detailed description of chromatography is generally credited to Michael Tswett. a
Russian biochemist. who separated chlorophyll from a mixture of plant pigments tn 1906.
the process was coined from the Greek words for color (chromo) and .. to write" (graphy).
The compound which interacts more with the mobile phase and least with the stationary phase
migrates fast.
Partition Coefficient
Partition coeffiCient (also known as distribution codficient) is a definitive term normally used to
describe the way in which a given compound distributes or partitions itself between two
immiscible phases. the stationary and the mobile phase
the concentration of the compound in each of the phases is described by the partition coefficient,
K. which is expressed as follows
K=CS/Cm
whete C. and em are the concentrations of the compound in the stationary and the mobUe
phases respectivelywhete C. and em are the concentrations of the compound in the stationary
and the mobUe phases respectively
Partition Chromatography
The principle of partition is exploited in gas/liquid chromatography (GLC) technique also.
Separations depend upor the partition of the solute molecules between a liqUid, supported on a
suitable solid, and the gas flowing through the system.
In true partition chromatography, the only factor which influences the movement of a
compound as the solvent travels along the stationary phase is the relative solubility of that
compound in the two phases.
Substances more soluble in the mobile phase will migrate greater distances as compared with
the substances more soluble in the stationary phase. Other compounds of intermediate solubility
between the two phases will migrate to intermediate distances depending upon their partition
coefficient
Adsorption Chromatography
Substances differ in their adsorption-desorption behaviour between a moving solvent (a liquid
or a gas) and a stationary solid phase. This behaviour of a substance can be exploited to achieve
its separation
The solute molecule which interacts more with the adsorbent, which is also the stationary phase,
is retarded more while less interacting solute molecules are retarded less. In this way a
separation of sample components is achieved.
Ion-Exchange Chromatograph
The ion exchanger consists of an inert support medium coupled covalently to positive (anion
exchanger) or negative (cation exchanger) functional groups
To these covalently bound functional groups are bound, through electrostatic attraction,
oppositely charged ions which will be exchanged with like charged ions in the sample.
if anion exchange chromatography is performed, negatively charged sample components will
interact more with the stationary phase and will be exchanged for like charged ions already
bound to the matrix. These sample ions will be retarded whereas other uncharged or positively
charged ions will not be retarded to the same degree and will be eluted out fast.
Molecular Size: Gel-Filtration Chromatography
This technique exploits the molecular size as the basiS of separation. The support medium, a
gel, consists of porous beads where pore size is strictly controlled.
Macromolecules smaller than the pores get entrapped in the pores (and move slowly), while
those bigger than the pores travel unhindered through the column (and elute out faster than the
smaller molecules)
Thus the main interaction between the solute and the stationary phase is with respect to the size
and this is ultimately the basis of separation also.
Affinity Chromatography
The technique utilizes the specificity of an enzyme for its substrate (also receptor for its agonist,
antibody for antigen) or substrate analogue for the enzyme's (other proteins with biological
specificity) separation
A substrate analogue is coupled to the gel matrix and the cellular suspension is allowed to
percolate through. The enzyme which is specific for the substrate analogue binds to the gel
becoming immobile while all other components move down and out. The technique has a very
high resolution power.
or a gas) and a stationary solid phase. This behaviour of a substance can be exploited to achieve
its separation
The solute molecule which interacts more with the adsorbent, which is also the stationary phase,
is retarded more while less interacting solute molecules are retarded less. In this way a
separation of sample components is achieved.
Ion-Exchange Chromatograph
The ion exchanger consists of an inert support medium coupled covalently to positive (anion
exchanger) or negative (cation exchanger) functional groups
To these covalently bound functional groups are bound, through electrostatic attraction,
oppositely charged ions which will be exchanged with like charged ions in the sample.
if anion exchange chromatography is performed, negatively charged sample components will
interact more with the stationary phase and will be exchanged for like charged ions already
bound to the matrix. These sample ions will be retarded whereas other uncharged or positively
charged ions will not be retarded to the same degree and will be eluted out fast.
Molecular Size: Gel-Filtration Chromatography
This technique exploits the molecular size as the basiS of separation. The support medium, a
gel, consists of porous beads where pore size is strictly controlled.
Macromolecules smaller than the pores get entrapped in the pores (and move slowly), while
those bigger than the pores travel unhindered through the column (and elute out faster than the
smaller molecules)
Thus the main interaction between the solute and the stationary phase is with respect to the size
and this is ultimately the basis of separation also.
Affinity Chromatography
The technique utilizes the specificity of an enzyme for its substrate (also receptor for its agonist,
antibody for antigen) or substrate analogue for the enzyme's (other proteins with biological
specificity) separation
A substrate analogue is coupled to the gel matrix and the cellular suspension is allowed to
percolate through. The enzyme which is specific for the substrate analogue binds to the gel
becoming immobile while all other components move down and out. The technique has a very
high resolution power.
There are two basic techniques of chromatography: plane chromatography and column
chromatography.
In plane chromatography the stationary phase is coated onto a plane surface. There are two
variations of plane chromatography: paper chromatography and thin layer chromatography. In
paper chromatography the stationary phase is supported by cellulose fibres of the paper sheet.
In thin layer chromatography the stationary phase is coated onto a glass or plastic surface.
As opposed to plane chromatography, the stationary phase in column chromatography is
packed into a glass or plastic column.
. PLANE CHROMATOGRAPHY
A drop of liqUid spotted on to a piece of paper or cloth will spread in a circular pattern. If the
liquid possesses color, concentric rings of colors will be observed, paper partition
chromatography in which mOisture clustered around cellulose fibres served as the stationary
phase
PAPER CHROMATOGRAPHY
The paper commonly used consists of highly purified cellulose. Cellulose, a homopolysaccharide
of glucose, contains several thousand anhydro-glucose units linked through oxygen atoms.
Many of the hydroxyl groups of glucose, however, become partially Oxidized during
manufacture
The paper also contains impurities through inorganic substances, which gets deposited on the
paper while it is being processed. These impurities may be removed by washing the paper with
0.1 N HCI and drying it before chromatography is carried out
The paper exhibits weak ion exchange and adsorptive properties
The apparatus required for paper chromatography consists of a support for paper. a solvent
trough. and an airtight chamber in which the chromatogram is developed.
chromatography.
In plane chromatography the stationary phase is coated onto a plane surface. There are two
variations of plane chromatography: paper chromatography and thin layer chromatography. In
paper chromatography the stationary phase is supported by cellulose fibres of the paper sheet.
In thin layer chromatography the stationary phase is coated onto a glass or plastic surface.
As opposed to plane chromatography, the stationary phase in column chromatography is
packed into a glass or plastic column.
. PLANE CHROMATOGRAPHY
A drop of liqUid spotted on to a piece of paper or cloth will spread in a circular pattern. If the
liquid possesses color, concentric rings of colors will be observed, paper partition
chromatography in which mOisture clustered around cellulose fibres served as the stationary
phase
PAPER CHROMATOGRAPHY
The paper commonly used consists of highly purified cellulose. Cellulose, a homopolysaccharide
of glucose, contains several thousand anhydro-glucose units linked through oxygen atoms.
Many of the hydroxyl groups of glucose, however, become partially Oxidized during
manufacture
The paper also contains impurities through inorganic substances, which gets deposited on the
paper while it is being processed. These impurities may be removed by washing the paper with
0.1 N HCI and drying it before chromatography is carried out
The paper exhibits weak ion exchange and adsorptive properties
The apparatus required for paper chromatography consists of a support for paper. a solvent
trough. and an airtight chamber in which the chromatogram is developed.
The sample is applied to the paper as a small spot. This is done before dipping the paper into
the eluting solvent.
There are two main techniques. which may be employed for the development of paper
chromatograms - ascending or descending teclmiques
Ascending technique has two advantages. Firstly. the set up required for it is very simple. This
is so because in ascending chromatography. two forces are acting on the solute: the capillary
force. which makes it move up. and the gravitational force which opposes this movement.
Usually in paper chromatography the stationary phase is water since it is very well adsorbed by
cellulose
The mobile phase, which is less polar flows over the polar stationary phase.
Detection There are various methods of detection aVailable. If the sample components are
colored, the analysis becomes simple as the distinctive color itself identifies the component.
When the components are colorless (usually they are), they can be imparted color by spraying
the paper with color producing reagents. A case in point is the detection of amino acids.
Ninhydrin reagent spread on the paper reacts with amines and amino acids to fonn a blue or
purple color
Other methods of detection are (i) ultraviolet and infrared absorption, (ii) fluorescence, and (iii)
radioactivity.
The identification of a given compound may be made on the basis of the distance traversed by
the solute relative to the distance moved by the solvent front.
This ratio, which reflects the distribution coefficient of the given solute, is known as the
retardation factor.
Application
the eluting solvent.
There are two main techniques. which may be employed for the development of paper
chromatograms - ascending or descending teclmiques
Ascending technique has two advantages. Firstly. the set up required for it is very simple. This
is so because in ascending chromatography. two forces are acting on the solute: the capillary
force. which makes it move up. and the gravitational force which opposes this movement.
Usually in paper chromatography the stationary phase is water since it is very well adsorbed by
cellulose
The mobile phase, which is less polar flows over the polar stationary phase.
Detection There are various methods of detection aVailable. If the sample components are
colored, the analysis becomes simple as the distinctive color itself identifies the component.
When the components are colorless (usually they are), they can be imparted color by spraying
the paper with color producing reagents. A case in point is the detection of amino acids.
Ninhydrin reagent spread on the paper reacts with amines and amino acids to fonn a blue or
purple color
Other methods of detection are (i) ultraviolet and infrared absorption, (ii) fluorescence, and (iii)
radioactivity.
The identification of a given compound may be made on the basis of the distance traversed by
the solute relative to the distance moved by the solvent front.
This ratio, which reflects the distribution coefficient of the given solute, is known as the
retardation factor.
Application
The control of purity of pharmaceuticals, the detection of adulterants and contaminants in
foods and drinks, the study of ripening and fermentatioll, the detection of drugs and dopes in
animals and humans.
THIN LAYER CHROMATOGRAPHY
The sample to be separated is spotted at one end. The plate is dipped into the solvent in a glass
jar and the development carried out by the ascending technique. After the development, the
layer can be dried and the components detected by various methods available.
Thin layer chromatography may be either carried out by the adsorption principle (if the thin
layer is prepared by an adsorbent such as Kieselguhr, or alumina) or by the partition principle
(if the layer is prepared by a substance such as silica gel which holds water like the paper).
Preparation of the Layer The glass plate on which the thin layer is prepared should be even and
is thoroughly washed and dried before layer application
The material of which the thin layer is to be made (silica gel, Kieselguhr, etc is usually mixed
with water in such a proportion that a thick suspension, known as slurry results
This slurry is applied to a plate surface as a uniform thin layer by means of a plate" spreader'
starting at one end of the plate and moving to the other in an unbroken uniform motion.
While preparing stationary phase for adsorption chromatography a binder such as calcium
sulphate is mixed with the slurry. The binder helps in better adhesion of the stationary phase to
the glass or foil plate
Detection
Many of these have already been named in the section on paper chromatography (for example.
ultraviolet absorption. fluorescence. autoradiography. if the components are radio-labeled. or
production of colors by chemical treatment).
Those specific for TLC are: (i) spraying the plate with 25-50% sulphuric acid in ethanol and
heating. This results in charring of most of the compounds. which show up as brown f'pots. and
(ii) iodine vapour is used extensively as a universal reagent for organiC compounds
On plate quantification of the separated components might be achieved by employing a
densitometer. which not only measures the ultraviolet or visible absorption of the separated
components but also gives a complete absorption spectrum of the compound for identification
purposes
Application
Thin layer technique has often been used to identify drugs. contaminants and adulterants. It has
also been widely used to resolve plant extracts and many other biochemical preparations.
COLUMN CHROMATOGRAPH
Column chromatography is an often used and routinely carried out technique which is
adaptable to all the major types of chromatography. Although such diverse operations as
foods and drinks, the study of ripening and fermentatioll, the detection of drugs and dopes in
animals and humans.
THIN LAYER CHROMATOGRAPHY
The sample to be separated is spotted at one end. The plate is dipped into the solvent in a glass
jar and the development carried out by the ascending technique. After the development, the
layer can be dried and the components detected by various methods available.
Thin layer chromatography may be either carried out by the adsorption principle (if the thin
layer is prepared by an adsorbent such as Kieselguhr, or alumina) or by the partition principle
(if the layer is prepared by a substance such as silica gel which holds water like the paper).
Preparation of the Layer The glass plate on which the thin layer is prepared should be even and
is thoroughly washed and dried before layer application
The material of which the thin layer is to be made (silica gel, Kieselguhr, etc is usually mixed
with water in such a proportion that a thick suspension, known as slurry results
This slurry is applied to a plate surface as a uniform thin layer by means of a plate" spreader'
starting at one end of the plate and moving to the other in an unbroken uniform motion.
While preparing stationary phase for adsorption chromatography a binder such as calcium
sulphate is mixed with the slurry. The binder helps in better adhesion of the stationary phase to
the glass or foil plate
Detection
Many of these have already been named in the section on paper chromatography (for example.
ultraviolet absorption. fluorescence. autoradiography. if the components are radio-labeled. or
production of colors by chemical treatment).
Those specific for TLC are: (i) spraying the plate with 25-50% sulphuric acid in ethanol and
heating. This results in charring of most of the compounds. which show up as brown f'pots. and
(ii) iodine vapour is used extensively as a universal reagent for organiC compounds
On plate quantification of the separated components might be achieved by employing a
densitometer. which not only measures the ultraviolet or visible absorption of the separated
components but also gives a complete absorption spectrum of the compound for identification
purposes
Application
Thin layer technique has often been used to identify drugs. contaminants and adulterants. It has
also been widely used to resolve plant extracts and many other biochemical preparations.
COLUMN CHROMATOGRAPH
Column chromatography is an often used and routinely carried out technique which is
adaptable to all the major types of chromatography. Although such diverse operations as
column adsorption. partition. ion exchange. exclusion and affinity chromatography are carried
out in a column. the apparatus and general techniques used share a lot in common
The columns are usually made up of glass or polyacrylate plastic. Different columns differ in
their dimensions, The commonly used glass columns have a sintered glass disc at the bottom to
support the stationary phase
Temperature fluctuations may be harmful for certain chromatographic separations. For such
experiments. columns with a thermostat jacket are used
The columns are provided with an inlet and an outlet. The inlet. which may be simple or fitted
with a ground glass adaptor provides for the eluting solvent to enter the column
The column is fitted in the upright position and its bottom is sealed with glass wool or such
other supports. The column is now fIlled to about one third its height with the mobile phase. A
thick suspension. called slurry. of the degassed stationary phase (gel. adsorbent. or resin) is
gently poured into the column with its outlet closed
The slurry is usually added till 3/4th of the column is full. The outlet is now opened and the
column is stabilized by washing it with mobile phase
Introduction of Sample It is necessary that the sample to be applied reachs the surface of the
column below the top layer of the solvent. This can be achieved by sucking the top layer of the
solvent out and then carefully pipetting the sample on to the column surface
The sample is allowed to just run into the column. Solvent is then added to the column to a
height of 5-10 cm. The column is then connected to a suitable reservoir which contains more
solvent. so Ulat the height of the solvent in the column can be maintained to a height of 5-10 cm.
Continuous passage of a suitable eluant (mobile phase) through the packed column separates
the components of the sample applied to the column.
This process is known as column development. There are two main techniques of elution: (i)
isocratic elution. and (ii) gradient elution.
When a single solvent is used as an eluant during development. the process is known as isocratic
separation
This process where the composition of the mobile phase is changed giving rise to a gradient is
known as gradient elution
ADSORPTION CHROMATOGRAPHY
A solid, which has the property of holding molecules at its surface. can be described as an
adsorbent Adsorption can be fairly specific so that one solute may be adsorbed selectively from
a mixture.
One is the different degree of adsorption of various components on the adsorbent surface and
the other is the varying solubility of different components in the solvent used (mobile phase).
out in a column. the apparatus and general techniques used share a lot in common
The columns are usually made up of glass or polyacrylate plastic. Different columns differ in
their dimensions, The commonly used glass columns have a sintered glass disc at the bottom to
support the stationary phase
Temperature fluctuations may be harmful for certain chromatographic separations. For such
experiments. columns with a thermostat jacket are used
The columns are provided with an inlet and an outlet. The inlet. which may be simple or fitted
with a ground glass adaptor provides for the eluting solvent to enter the column
The column is fitted in the upright position and its bottom is sealed with glass wool or such
other supports. The column is now fIlled to about one third its height with the mobile phase. A
thick suspension. called slurry. of the degassed stationary phase (gel. adsorbent. or resin) is
gently poured into the column with its outlet closed
The slurry is usually added till 3/4th of the column is full. The outlet is now opened and the
column is stabilized by washing it with mobile phase
Introduction of Sample It is necessary that the sample to be applied reachs the surface of the
column below the top layer of the solvent. This can be achieved by sucking the top layer of the
solvent out and then carefully pipetting the sample on to the column surface
The sample is allowed to just run into the column. Solvent is then added to the column to a
height of 5-10 cm. The column is then connected to a suitable reservoir which contains more
solvent. so Ulat the height of the solvent in the column can be maintained to a height of 5-10 cm.
Continuous passage of a suitable eluant (mobile phase) through the packed column separates
the components of the sample applied to the column.
This process is known as column development. There are two main techniques of elution: (i)
isocratic elution. and (ii) gradient elution.
When a single solvent is used as an eluant during development. the process is known as isocratic
separation
This process where the composition of the mobile phase is changed giving rise to a gradient is
known as gradient elution
ADSORPTION CHROMATOGRAPHY
A solid, which has the property of holding molecules at its surface. can be described as an
adsorbent Adsorption can be fairly specific so that one solute may be adsorbed selectively from
a mixture.
One is the different degree of adsorption of various components on the adsorbent surface and
the other is the varying solubility of different components in the solvent used (mobile phase).
Certain adsorbents might not be inert but react and degrade components to be separated
during experiment. Certain other adsorbents might imbibe water from the atmosphere and
become useless
It is necessary to consider the competition between the solutes and the solvent for the
adsorption sites on the surface of the stationary phase. Thus. a solvent which elutes the solutes
too fast will give a poor separation. while the solvent eluting the solutes very slowly will lead to
uncomfortably long retention times which will result into excessive band broadening and
sample dilution
Adsorption chromatography can be carried out using column. TLC. or paper chromatographic
techniques
Adsorption chromatography has been extensively used for biochemical separations. The long
list includes amino acids. mono-. and disaccharides. neutral lipids. cholesterol easters.
carotenoids. phospholipids. etc. The disadvantage with adsorption chromatography. however.
lies in the fact that it is highly empirical and non-reproducible because of variations in the
nature of adsorbents
Liquid-Liquid Chromatography
The stationary phase there was a polar solvent. water. tightly held by the paper on which
migrated the mobile phase which was another solvent of lesser polarity. Similarly when thin
layer chromatography is performed by silica gel layer. the stationary phase is the water
adsorbed by silica gel particles.
the stationary phase is polar. the solutes will not have much solubility in it and will therefore
migrate fast with the mobile phase in which they are more soluble (since it is relatively non-
polar
non-polar organic solvent as the stationary phase and aqueous solvent as the mobile phase. In
other words. the nature of the phases is reversed. The technique is aptly known as reverse-phase
chromatography
Gas-Liquid Chromatography (GLC)
The basis for the separation of the compounds in gas-liquid chromatography is the difference in
the partition coefficients of volatilized compounds in the liquid stationary phase.
Gas-liquid chromatography is a form of column chromatography where the stationary phase is
a non-volatile liqUid. The stationary phase here is known as the liquid phase. This phase is
dispersed over a surface of an inert solid support
. A gas stream termed as the carrier gas flows continuously through this column at a flow rate
which is controlledWhm a small quantity of the volatile sample is introduced into the gas. the
gas promptly carries it on to the column (hence the name "carrier gas").
In the column, the sample components become distributed between the liqUid and the gas
phase. These components therefore travel more slowly than the carrier gas because they are
being retarded by virtue of their interaction with the liquid phase. The retarding effect is
during experiment. Certain other adsorbents might imbibe water from the atmosphere and
become useless
It is necessary to consider the competition between the solutes and the solvent for the
adsorption sites on the surface of the stationary phase. Thus. a solvent which elutes the solutes
too fast will give a poor separation. while the solvent eluting the solutes very slowly will lead to
uncomfortably long retention times which will result into excessive band broadening and
sample dilution
Adsorption chromatography can be carried out using column. TLC. or paper chromatographic
techniques
Adsorption chromatography has been extensively used for biochemical separations. The long
list includes amino acids. mono-. and disaccharides. neutral lipids. cholesterol easters.
carotenoids. phospholipids. etc. The disadvantage with adsorption chromatography. however.
lies in the fact that it is highly empirical and non-reproducible because of variations in the
nature of adsorbents
Liquid-Liquid Chromatography
The stationary phase there was a polar solvent. water. tightly held by the paper on which
migrated the mobile phase which was another solvent of lesser polarity. Similarly when thin
layer chromatography is performed by silica gel layer. the stationary phase is the water
adsorbed by silica gel particles.
the stationary phase is polar. the solutes will not have much solubility in it and will therefore
migrate fast with the mobile phase in which they are more soluble (since it is relatively non-
polar
non-polar organic solvent as the stationary phase and aqueous solvent as the mobile phase. In
other words. the nature of the phases is reversed. The technique is aptly known as reverse-phase
chromatography
Gas-Liquid Chromatography (GLC)
The basis for the separation of the compounds in gas-liquid chromatography is the difference in
the partition coefficients of volatilized compounds in the liquid stationary phase.
Gas-liquid chromatography is a form of column chromatography where the stationary phase is
a non-volatile liqUid. The stationary phase here is known as the liquid phase. This phase is
dispersed over a surface of an inert solid support
. A gas stream termed as the carrier gas flows continuously through this column at a flow rate
which is controlledWhm a small quantity of the volatile sample is introduced into the gas. the
gas promptly carries it on to the column (hence the name "carrier gas").
In the column, the sample components become distributed between the liqUid and the gas
phase. These components therefore travel more slowly than the carrier gas because they are
being retarded by virtue of their interaction with the liquid phase. The retarding effect is
different for different components; the component distributing more in the liqUid phase is
retarded more and the component prefering the gas phase is retarded less.
the long column, which is present in the GLC apparatus, these sample components become
separated from each other on the basis of differences in the retarding effect. These separated
components eventually elute out of the column and reach the detector,which reads the
concentration of a given component present in the carrier gas and converts it to an quivalent
electrical signal
There is a variation of gas liqUid chromatography which is knon as gas-solid chromatography
(GSC). Here, the liqUid phase is absent. The solid phase, which is coated on to the interior of
the column, is not inert in this technique; it interacts with the sample components carried by the
gas by exerting adsorption fOlces. The sample components therefore become distributed
between the gas and the solid surface on the basis of differences in the adsorption-desorption
behaviour on the solid surface. This eventually leads to their separation from each other.
Carrier Gas
In GLC, the carrier gas constitutes the mobile phase and provides transportation for the sample
components through the apparatus. The gas must be chemically inert and pure. Gas used at
high density gives a better separation but takes a long time to achieve
Most commonly used gasses are nitrogen and argon, but, helium, hydrogen and carbon dioxide
are also used. Even steam is used for special purposes.
Columns
Two distinct types of columns are commonly used, packed and open tubular. The open tubular
columns are also known as capillary columns. Packed columns are stainless steel, copper or
glass tubing
Liquid Phase A good rule to follow when selecting liquid phases is "like dissolves like". A good
separation will occur only when the sample dissolves well in the liquid stationary phase. This is
so since the gas phase is inert;
The requirements for a good liquid phase are: (i) it must be non-volatile at the temperature it is
to be used; (ii) it should be thermally stable; (iii) it should provide appropriate partition
coefficient values for the components of interest. and (iv) it should be completely inert towards
the solutes.
Detectors
Located at the exit of the separation column. the detector detects the presence of the individual
components as they leave the column. The detector output is then suitably amplified and is
traced on a strip chart recorde
retarded more and the component prefering the gas phase is retarded less.
the long column, which is present in the GLC apparatus, these sample components become
separated from each other on the basis of differences in the retarding effect. These separated
components eventually elute out of the column and reach the detector,which reads the
concentration of a given component present in the carrier gas and converts it to an quivalent
electrical signal
There is a variation of gas liqUid chromatography which is knon as gas-solid chromatography
(GSC). Here, the liqUid phase is absent. The solid phase, which is coated on to the interior of
the column, is not inert in this technique; it interacts with the sample components carried by the
gas by exerting adsorption fOlces. The sample components therefore become distributed
between the gas and the solid surface on the basis of differences in the adsorption-desorption
behaviour on the solid surface. This eventually leads to their separation from each other.
Carrier Gas
In GLC, the carrier gas constitutes the mobile phase and provides transportation for the sample
components through the apparatus. The gas must be chemically inert and pure. Gas used at
high density gives a better separation but takes a long time to achieve
Most commonly used gasses are nitrogen and argon, but, helium, hydrogen and carbon dioxide
are also used. Even steam is used for special purposes.
Columns
Two distinct types of columns are commonly used, packed and open tubular. The open tubular
columns are also known as capillary columns. Packed columns are stainless steel, copper or
glass tubing
Liquid Phase A good rule to follow when selecting liquid phases is "like dissolves like". A good
separation will occur only when the sample dissolves well in the liquid stationary phase. This is
so since the gas phase is inert;
The requirements for a good liquid phase are: (i) it must be non-volatile at the temperature it is
to be used; (ii) it should be thermally stable; (iii) it should provide appropriate partition
coefficient values for the components of interest. and (iv) it should be completely inert towards
the solutes.
Detectors
Located at the exit of the separation column. the detector detects the presence of the individual
components as they leave the column. The detector output is then suitably amplified and is
traced on a strip chart recorde
Three most commonly used detectors are described below.
Flame ionization detector. This is by far the most widely used detector. It measures all organiC
compounds and it can detect as low as one nanograrn of any given compound. Hydrogen. either
used as carrier or introduced into the detector through elsewhere. is burnt to give a nearly
colorless flame. the jet of which forms one electrode
Electron capture detector. This detector has radioactive source (63Ni) which ionizes the carrier
gas coming out of the column. The electrons produced give rise to a current across the
electrodes to which a suitable voltage is applied
Thermionic emission detector. This detector employs fuel-poor hydrogen plasma. This low
temperature source suppresses the normal flame ionization response of the compounds not
containing nitrogen or phosphorous.
Much in the same manner as the retardation factor. Rf (see section on paper chromatography).
retention time aids in qualitative analysis in gas chromatograph
Under standard conditions of temperature. gas flow. gas compressibility etc .. the time taken for
a compound to emerge from a column is constant and is known as the retention time.
retention times are inconveniently long in GSC. However, retention times are considerably
smaller when working with such gases as hydrogen, nitrogen, argon, and oxygen (these gases
give too short a retention time in GLC).
upper temperature limit in GLC is limited by the necessity of retaining the liquid stationary
phase. However, since there is no liqUid phase in GSC, considerably higher temperatures may
be used
Application of Gas Chromatography
Apart from the separation of components of tobacco smoke, atmospheriC pollutants, solvents,
plant extracts, essential oils, volatile vegetable oils and organiC acids etc., for which it is
routinely used, gas chromatography is being increasingly used as an analytical tool to study
Flame ionization detector. This is by far the most widely used detector. It measures all organiC
compounds and it can detect as low as one nanograrn of any given compound. Hydrogen. either
used as carrier or introduced into the detector through elsewhere. is burnt to give a nearly
colorless flame. the jet of which forms one electrode
Electron capture detector. This detector has radioactive source (63Ni) which ionizes the carrier
gas coming out of the column. The electrons produced give rise to a current across the
electrodes to which a suitable voltage is applied
Thermionic emission detector. This detector employs fuel-poor hydrogen plasma. This low
temperature source suppresses the normal flame ionization response of the compounds not
containing nitrogen or phosphorous.
Much in the same manner as the retardation factor. Rf (see section on paper chromatography).
retention time aids in qualitative analysis in gas chromatograph
Under standard conditions of temperature. gas flow. gas compressibility etc .. the time taken for
a compound to emerge from a column is constant and is known as the retention time.
retention times are inconveniently long in GSC. However, retention times are considerably
smaller when working with such gases as hydrogen, nitrogen, argon, and oxygen (these gases
give too short a retention time in GLC).
upper temperature limit in GLC is limited by the necessity of retaining the liquid stationary
phase. However, since there is no liqUid phase in GSC, considerably higher temperatures may
be used
Application of Gas Chromatography
Apart from the separation of components of tobacco smoke, atmospheriC pollutants, solvents,
plant extracts, essential oils, volatile vegetable oils and organiC acids etc., for which it is
routinely used, gas chromatography is being increasingly used as an analytical tool to study
GEL PERMEATION CHROMATOGRAPHY
Gel permeation chromatography is a separation method dependent upon molecular size. The
method is also known as molecular sieve, gel filtration, or molecular exclusion chromatography
The advantages are: (i) gentleness of the technique permitting separation of a labile molecular
species, (iO almost 100% solute recovery, (iii) excellent reproducibility, and (iv) comparatively
short time and relatively inexpensive equipment needed for its operation
Gel permeation chromatography is based on a very simple principle. A column of gel beads or
porous glass granules is allowed to attain equilibrium with a solvent suitabfe forthe molecules to
be separated. If the mixture of molecules of different size is placed on the top of such an
equilibrated column, the larger molecules pass through the interstitial spaces between the beads
A gel filtration medium should possess the following characteristics:
(i) The gel material should be chemically inert.
(ii) It should preferably contain vanishingly small number of ionic groups.
(iii) Gel material should provide a wide choice of pore and particle sizes.
(iv) A given gel should have uniform particle and pore sizes.
(v) The gel matrix should have high mechanical rigidity.
Types of Gels
Sephadex.
Polyacrylamide
Agarose
Styragel
Molecular Weight Determination by Gel Filtration
Analytical Uses of Gel Permeation
ION-EXCHANGE CHROMATOGRAPHY
Ion exchange may be defined as the reversible exchange of ions in solution with ions
electrostatically bound to inert support medium. The governing factor in ion exchange reactions
is the electrostatic force of attraction, which in turn depends mainly on the relative charge. the
radius of the hydrated ions. and the degree of non-bonding interactions
Ion exchange separations are carried out usually in columns packed with an ion exchanger. Ion
exchangers can be divided into two groups: anion exchangers and cation exchangers.
This technique is extremely useful in the separation of charged compounds (even uncharged
molecules can be "charged" by variance of pH as we will see later
Gel permeation chromatography is a separation method dependent upon molecular size. The
method is also known as molecular sieve, gel filtration, or molecular exclusion chromatography
The advantages are: (i) gentleness of the technique permitting separation of a labile molecular
species, (iO almost 100% solute recovery, (iii) excellent reproducibility, and (iv) comparatively
short time and relatively inexpensive equipment needed for its operation
Gel permeation chromatography is based on a very simple principle. A column of gel beads or
porous glass granules is allowed to attain equilibrium with a solvent suitabfe forthe molecules to
be separated. If the mixture of molecules of different size is placed on the top of such an
equilibrated column, the larger molecules pass through the interstitial spaces between the beads
A gel filtration medium should possess the following characteristics:
(i) The gel material should be chemically inert.
(ii) It should preferably contain vanishingly small number of ionic groups.
(iii) Gel material should provide a wide choice of pore and particle sizes.
(iv) A given gel should have uniform particle and pore sizes.
(v) The gel matrix should have high mechanical rigidity.
Types of Gels
Sephadex.
Polyacrylamide
Agarose
Styragel
Molecular Weight Determination by Gel Filtration
Analytical Uses of Gel Permeation
ION-EXCHANGE CHROMATOGRAPHY
Ion exchange may be defined as the reversible exchange of ions in solution with ions
electrostatically bound to inert support medium. The governing factor in ion exchange reactions
is the electrostatic force of attraction, which in turn depends mainly on the relative charge. the
radius of the hydrated ions. and the degree of non-bonding interactions
Ion exchange separations are carried out usually in columns packed with an ion exchanger. Ion
exchangers can be divided into two groups: anion exchangers and cation exchangers.
This technique is extremely useful in the separation of charged compounds (even uncharged
molecules can be "charged" by variance of pH as we will see later
Two main groups of materials are used to prepare ion exchange resins: polystyrene, and
cellulose. Resins made from both of these materials differ in their flow properties, ion
accessibility, and chemical and mechanical stability
The choice of buffers. which maintain the pH of the column. is dictated by the compounds to be
separated and the type of ion exchange being carried out (anionic or cationic).
Anionic exchange chromatography should be carried out with cationic buffers, Reverse is true
for cation exchange chromatograph
Buffer -Ammonium acetate, Ammonium formate, Ammonium carbonate
Application
Use of ion exchange chromatography is in amino acid analysis. In fact the amino acid
"autoanalyser" is based on ion exchange principle
Ion exchange has been extensively used to determine the base composition of nucleic acids
For many biological applications, ultrapure, metal ion free reagents. are needed. This is
commercially performed by ion exchange chromatography
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
Ordinarily, a conventional chromatographic experiment takes an inordinately long time. To
reduce the time of experiment, one can increase the flow rate thereby reducing the retention
time of the solute components
cellulose. Resins made from both of these materials differ in their flow properties, ion
accessibility, and chemical and mechanical stability
The choice of buffers. which maintain the pH of the column. is dictated by the compounds to be
separated and the type of ion exchange being carried out (anionic or cationic).
Anionic exchange chromatography should be carried out with cationic buffers, Reverse is true
for cation exchange chromatograph
Buffer -Ammonium acetate, Ammonium formate, Ammonium carbonate
Application
Use of ion exchange chromatography is in amino acid analysis. In fact the amino acid
"autoanalyser" is based on ion exchange principle
Ion exchange has been extensively used to determine the base composition of nucleic acids
For many biological applications, ultrapure, metal ion free reagents. are needed. This is
commercially performed by ion exchange chromatography
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
Ordinarily, a conventional chromatographic experiment takes an inordinately long time. To
reduce the time of experiment, one can increase the flow rate thereby reducing the retention
time of the solute components
Six major components needed to perform HPLC are
(i) A solvent reservoir to store the mobile phase.
(ii) High pressure pump to push the mobile phase through the column.
(iii) A device to inject the sample into the mobile phase.
(iv) A column in which the separation will take place.
(v) A detector used in detecting the concentration of the sample components as they come out of
the column.
(vi) A potentiometric recorder to produce a chromatogram
Solvent Reservoir and the Solvents The solvent reservoir should meet the follOwing criteria:
(i) it must contain volume enough for repetitive analysis;
(ii) it must have a provision for degassing the solvents;
(iii) it must be inert to the solvent.
Pumping Systems Pumping system can be said to be the heart of HPLC. By producing
reproducible high pressures, the pump is a m~or factor in obtaining high resolution, high speed
analyses, and reproducible quantitative analyses.
(i) A pulseless stable flow. Absence of pulsations minimizes detector noise. (ii) A
suitable pump should provide solvent flow-rates of 0 .5-1 d mlIrnin, which is
compatible with most HPLC modes. (iii.) A constant volume delivery. (iv)
Amenability to high pressures of up to 6000 psi. (v) The pump should be adaptable
to gradient operation. Pumping systems available for HPLC are: (i) Liquid
(i) A solvent reservoir to store the mobile phase.
(ii) High pressure pump to push the mobile phase through the column.
(iii) A device to inject the sample into the mobile phase.
(iv) A column in which the separation will take place.
(v) A detector used in detecting the concentration of the sample components as they come out of
the column.
(vi) A potentiometric recorder to produce a chromatogram
Solvent Reservoir and the Solvents The solvent reservoir should meet the follOwing criteria:
(i) it must contain volume enough for repetitive analysis;
(ii) it must have a provision for degassing the solvents;
(iii) it must be inert to the solvent.
Pumping Systems Pumping system can be said to be the heart of HPLC. By producing
reproducible high pressures, the pump is a m~or factor in obtaining high resolution, high speed
analyses, and reproducible quantitative analyses.
(i) A pulseless stable flow. Absence of pulsations minimizes detector noise. (ii) A
suitable pump should provide solvent flow-rates of 0 .5-1 d mlIrnin, which is
compatible with most HPLC modes. (iii.) A constant volume delivery. (iv)
Amenability to high pressures of up to 6000 psi. (v) The pump should be adaptable
to gradient operation. Pumping systems available for HPLC are: (i) Liquid
displacement by compressed gases (holding coil). (ii) Pneumatic amplifier (iii)
Piston/diaphragm driven by a moving fluid. (iv) Reciprocating piston. (v) Syringe
pumps.
Holding coil. This unit is usually available with less expensive HPLC systems. A large holding
coil made up of stainless steel tubing is filled up with the solvent. Compressed gas from a
cylinder forces the liquid at constant pressure from the holding coil into the chromatographic
column.
Sample Injection
The first method employs a micro syringe designed to withstand high pressures. With the help
of this micro syringe. the sample is introduced either directly onto the column or onto an inert
material directly above the column
The Guard Column The resolution power of HPLC is so high that an elaborate sample
preparation before chromatography is not necessary. Thus. sera. or other biological materials
can be applied tothe column without any pretreatment. This. however. clogs the column after a
few applications as the column during separation retains many undesirable components of the
biological samples. To circumvent this problem. a short column (2-10 cm) precedes the main
column. This short column is known as guard colwnn and its function is to retain those
biological components which would otherwise clog the main column.
Detectors
UVVIS photometers can be used for HPLC. These detectors are inexpensive. sensitive.
insensitive to normal flow and temperature fluctuations. and well suited for gradient elution.
UVNVIS spectrophotometers with wavelength selection range of 200-800 nm are very popular
HPLC detectors
Application
HPLC has been successfully applied to the separation of proteins, nucleic acids,
polysaccharides, plant pigments, amino acids, pesticides, steroids, drugs and their metabolites,
animal and plant hormones and complex lipids.
Supercritical Fluid Chromatography
A supercritical fluid chromatography is a material that can be either liquid or gas used in state
above critical temperature or critical pressure where gases or liquid can co exist
Principles are similar to those of High Performance Liquid Chromatography (HPLC), however
SFC typically utilizes carbon dioxide as the mobile phase; therefore the entire chromatographic
flow path must be pressurized
Because the supercritical phase represents a state in which liquid and gas properties converge,
supercritical fluid chromatography is sometimes called "convergence chromatography.
INSTRUMENTATION 1.Stationaryphase 2.Mobile phase 3.Pumps 4.Injectors 5.Ovens
6.Columns 7.Detector
Piston/diaphragm driven by a moving fluid. (iv) Reciprocating piston. (v) Syringe
pumps.
Holding coil. This unit is usually available with less expensive HPLC systems. A large holding
coil made up of stainless steel tubing is filled up with the solvent. Compressed gas from a
cylinder forces the liquid at constant pressure from the holding coil into the chromatographic
column.
Sample Injection
The first method employs a micro syringe designed to withstand high pressures. With the help
of this micro syringe. the sample is introduced either directly onto the column or onto an inert
material directly above the column
The Guard Column The resolution power of HPLC is so high that an elaborate sample
preparation before chromatography is not necessary. Thus. sera. or other biological materials
can be applied tothe column without any pretreatment. This. however. clogs the column after a
few applications as the column during separation retains many undesirable components of the
biological samples. To circumvent this problem. a short column (2-10 cm) precedes the main
column. This short column is known as guard colwnn and its function is to retain those
biological components which would otherwise clog the main column.
Detectors
UVVIS photometers can be used for HPLC. These detectors are inexpensive. sensitive.
insensitive to normal flow and temperature fluctuations. and well suited for gradient elution.
UVNVIS spectrophotometers with wavelength selection range of 200-800 nm are very popular
HPLC detectors
Application
HPLC has been successfully applied to the separation of proteins, nucleic acids,
polysaccharides, plant pigments, amino acids, pesticides, steroids, drugs and their metabolites,
animal and plant hormones and complex lipids.
Supercritical Fluid Chromatography
A supercritical fluid chromatography is a material that can be either liquid or gas used in state
above critical temperature or critical pressure where gases or liquid can co exist
Principles are similar to those of High Performance Liquid Chromatography (HPLC), however
SFC typically utilizes carbon dioxide as the mobile phase; therefore the entire chromatographic
flow path must be pressurized
Because the supercritical phase represents a state in which liquid and gas properties converge,
supercritical fluid chromatography is sometimes called "convergence chromatography.
INSTRUMENTATION 1.Stationaryphase 2.Mobile phase 3.Pumps 4.Injectors 5.Ovens
6.Columns 7.Detector
STATIONARY PHASE
• Both packed and open tubular columns are used.
• Packed columns can provide more theoretical plates and
handle large volume than open tubular columns.
• Because of low viscosity of super critical media.
MOBILE PHASE
own is too non-polar to effectively elute many analytes, co-solvents are added to modify the
mobile phase polarity.
PUMPS Here mainly flow control is necessary so syringe pumps are used for capillary SFC for
consistent pressure and for packed columns for easier blending of the mobile phase or
introduction of modifier fluids reciprocating pumps are used.
INJECTORS
In capillary SFC small sample should be quickly injected into the column and so
pneumatically driven valves are used.
OVENS
Conventional GAS chromatography & liquid chromatography ovens are used
DETECTORS
Flame ionization detectors and flame photometry detector, liquid-phase detectors like refractive
index detector, ultraviolet-visible spectrophotometric detectors and light scattering detectors
have been employed for SFC.
ADVANTAGES
romatography and
liquid chromatography for analysis of thermal liable or non volatile compounds
Centrifugation
The particles in the solution will then sediment faster because a centrifugal force is acting upon
them in addition to the gravitational force.
It was in 1923 that Svedberg and Nicols employed a centrifuge for the first time to increase the
gravitational force so as to speed up the rate of sedimentation for the purpose of measuring
particle sizes.
The applications of this technique range from collection and separation of cells. organelles. and
molecules to the study of molecular weights of macromolecules.
• Both packed and open tubular columns are used.
• Packed columns can provide more theoretical plates and
handle large volume than open tubular columns.
• Because of low viscosity of super critical media.
MOBILE PHASE
own is too non-polar to effectively elute many analytes, co-solvents are added to modify the
mobile phase polarity.
PUMPS Here mainly flow control is necessary so syringe pumps are used for capillary SFC for
consistent pressure and for packed columns for easier blending of the mobile phase or
introduction of modifier fluids reciprocating pumps are used.
INJECTORS
In capillary SFC small sample should be quickly injected into the column and so
pneumatically driven valves are used.
OVENS
Conventional GAS chromatography & liquid chromatography ovens are used
DETECTORS
Flame ionization detectors and flame photometry detector, liquid-phase detectors like refractive
index detector, ultraviolet-visible spectrophotometric detectors and light scattering detectors
have been employed for SFC.
ADVANTAGES
romatography and
liquid chromatography for analysis of thermal liable or non volatile compounds
Centrifugation
The particles in the solution will then sediment faster because a centrifugal force is acting upon
them in addition to the gravitational force.
It was in 1923 that Svedberg and Nicols employed a centrifuge for the first time to increase the
gravitational force so as to speed up the rate of sedimentation for the purpose of measuring
particle sizes.
The applications of this technique range from collection and separation of cells. organelles. and
molecules to the study of molecular weights of macromolecules.
The basic components of a centrifuge are (i) a metal rotor with holes in it to accommodate a
vessel of liquid and a motor or alternative means of spinning the rotor at a selected speed
BASIC PRINCIPLES OF SEDIMENTATION
An object moving in a Circle at a steady angular velocity will experience a force. F. directed
outwards. This is the basis of centrifugation
Angular velocity in radians. Ѡ. and the radius of rotation. r. in centimeters. collectively
determine the magnitude of the force F
F= Ѡ
2
r
F might be expressed in terms of earth's gravitational force if it is divided by 980. The resultant
is referred to as the relative centrifugal force. RCF. RCF is more frequently referred to as the
'number times g'.
RCF = Ѡ
2
r/980
For everyday use. the relationship will be better if expressed in terms of 'revolutions per
minute'. rpm. the common way in which the operating speed of a centrifuge rotor is expressed.
Radius. w. and rpm share the following relationship
Ѡ=
π(rpm)/30
Substituting for Ѡ= (1. 119 x 10
-5
) (rpm)
2
r
If one considers the above relationship. it becomes clear that since all other values are constants.
the RCF depends upon the rpm and the radius of rotation. r.
Apart from RCF, the rate of sedimentation of a given particle would also depend upon its own
characteristics such as its density, and its radius
INSTRUMENTATION
centrifuges can be roughly categorized into three different types on the basis of their operating
speed.
Desk Top Centrifuges
They are normally used to collect rapidly sedimenting substances such as red blood cells, yeast
cells or bulky precipitates of chemical reactions. These are also known as clinical centrifuges
since most of the clinical work is done by these models. Their maximum speed is usually 3000
rpm and they do not have any temperature regulatory system
It is therefore very important that the contents of the centrifuge tubes are balanced accurately
and that they are never loaded with an odd number of tubes.
High Speed Centrifuges
vessel of liquid and a motor or alternative means of spinning the rotor at a selected speed
BASIC PRINCIPLES OF SEDIMENTATION
An object moving in a Circle at a steady angular velocity will experience a force. F. directed
outwards. This is the basis of centrifugation
Angular velocity in radians. Ѡ. and the radius of rotation. r. in centimeters. collectively
determine the magnitude of the force F
F= Ѡ
2
r
F might be expressed in terms of earth's gravitational force if it is divided by 980. The resultant
is referred to as the relative centrifugal force. RCF. RCF is more frequently referred to as the
'number times g'.
RCF = Ѡ
2
r/980
For everyday use. the relationship will be better if expressed in terms of 'revolutions per
minute'. rpm. the common way in which the operating speed of a centrifuge rotor is expressed.
Radius. w. and rpm share the following relationship
Ѡ=
π(rpm)/30
Substituting for Ѡ= (1. 119 x 10
-5
) (rpm)
2
r
If one considers the above relationship. it becomes clear that since all other values are constants.
the RCF depends upon the rpm and the radius of rotation. r.
Apart from RCF, the rate of sedimentation of a given particle would also depend upon its own
characteristics such as its density, and its radius
INSTRUMENTATION
centrifuges can be roughly categorized into three different types on the basis of their operating
speed.
Desk Top Centrifuges
They are normally used to collect rapidly sedimenting substances such as red blood cells, yeast
cells or bulky precipitates of chemical reactions. These are also known as clinical centrifuges
since most of the clinical work is done by these models. Their maximum speed is usually 3000
rpm and they do not have any temperature regulatory system
It is therefore very important that the contents of the centrifuge tubes are balanced accurately
and that they are never loaded with an odd number of tubes.
High Speed Centrifuges
High speed centrifuges can operate with maximum speed of up to 25,000 rpm providing about
90,000 g centrifugal force in the process. They are usually equipped with refrigeration
equipment to remove heat generated due to friction between the air and the spinning rotor
The temperature can easily be maintained in the range a 4°C by means of a thermocouple. The
highest carrying capacity may be 1.5 dm3 • These instruments are routinely used to collect
microorganisms, cell debris, cells, large cellular organelles, precipitates of chemical reactions
and immunoprecipitates.
The Ultracentrifuge
The ultracentrifuge can operate at speeds up to 75,000 rpm providing centrifugal force in excess
of 500,000 g. At such speed the friction between air and the spinning rotor generates significant
amount of heat.
To eliminate this source of heating, the rotor chamber is sealed and evacuated by two pumps
working in tandem making it possible to attain and hold vacuums of 1 to 2 ~. Apart from this
the ultracentrifuge has a refrigeration system which can maintain the temperature of the rotor
between 0° and 4°C.
To prevent the rotor from operating at speeds which exceed its maximum rated speed, all
centrifuges possess an overspeed device. Operation of rotor at excessive speeds can result in an
explosion with the rotor being torn apart. To contain such explosions the rotor chamber is
always enclosed in a heavy armor plate
The ultracentrifuges also are of two types - the preparatory ultracentrifuge, and the analytical
ultracentrifuge.
Rotors
BaSically, rotors come in four varieties: fixed-angle rotors, vertical tube rotors, swingingbucket
rotors, and zonal rotors.
Fixed-angle rotors: These rotors have holes within their body and one can slide the centrifuge
tubes within these holes. Since the holes are at an angle (between 14° and 40°) to the vertical, the
tubes and the solution within also take the same angle
Vertical-tube rotors: These rotors too have holes within their body in which one can slide the
centrifuge tubes. However, these holes lie parallel to the rotor shaft and not at an angle. As the
rotor accelerates and centrifugal field is applied, the solution within the tube reorients through
90°
90,000 g centrifugal force in the process. They are usually equipped with refrigeration
equipment to remove heat generated due to friction between the air and the spinning rotor
The temperature can easily be maintained in the range a 4°C by means of a thermocouple. The
highest carrying capacity may be 1.5 dm3 • These instruments are routinely used to collect
microorganisms, cell debris, cells, large cellular organelles, precipitates of chemical reactions
and immunoprecipitates.
The Ultracentrifuge
The ultracentrifuge can operate at speeds up to 75,000 rpm providing centrifugal force in excess
of 500,000 g. At such speed the friction between air and the spinning rotor generates significant
amount of heat.
To eliminate this source of heating, the rotor chamber is sealed and evacuated by two pumps
working in tandem making it possible to attain and hold vacuums of 1 to 2 ~. Apart from this
the ultracentrifuge has a refrigeration system which can maintain the temperature of the rotor
between 0° and 4°C.
To prevent the rotor from operating at speeds which exceed its maximum rated speed, all
centrifuges possess an overspeed device. Operation of rotor at excessive speeds can result in an
explosion with the rotor being torn apart. To contain such explosions the rotor chamber is
always enclosed in a heavy armor plate
The ultracentrifuges also are of two types - the preparatory ultracentrifuge, and the analytical
ultracentrifuge.
Rotors
BaSically, rotors come in four varieties: fixed-angle rotors, vertical tube rotors, swingingbucket
rotors, and zonal rotors.
Fixed-angle rotors: These rotors have holes within their body and one can slide the centrifuge
tubes within these holes. Since the holes are at an angle (between 14° and 40°) to the vertical, the
tubes and the solution within also take the same angle
Vertical-tube rotors: These rotors too have holes within their body in which one can slide the
centrifuge tubes. However, these holes lie parallel to the rotor shaft and not at an angle. As the
rotor accelerates and centrifugal field is applied, the solution within the tube reorients through
90°
Swinging-bucket rotors- As against fixed hole type rotors we have seen above, these rotors have
buckets that swing out to a horizontal position when the rotor accelerates. The solution in the
tube reorients to lie perpendicular to the axis of rotation and parallel to the applied centrifugal
field. When the rotor decelerates, the tubes fall back to their original position and the solution
too regains its original orientation
Preparative centrifugation is concerned with the actual isolation of biological material for
subsequent biochemical investigations.
Separations carried out in a suspending medium which is homogenous are known as differential
centrifugation while those carried out in a suspending medium having density gradients are
known as density gradient centrifugations
A mixture of homogeneous particles can be separated by c:entrifugation on the basis of their
densities and/or their size. this can be achieved either by the time required for their complete
sedimentation in a fixed centrifugal field or on the extent of fueir sedimentation after a given
time in a fixed centrifugal field.
Differential centrifugation
We can choose the centrifugal field in such a manner that a particular organelle sediments
during the already known time of centrifugation to give a pellet. The pellet and supernatant are
separated at the end of each step and the supernatant recentrifuged to sediment another lighter
intracellular organelle. This is the essence of differential centrifugation.
buckets that swing out to a horizontal position when the rotor accelerates. The solution in the
tube reorients to lie perpendicular to the axis of rotation and parallel to the applied centrifugal
field. When the rotor decelerates, the tubes fall back to their original position and the solution
too regains its original orientation
Preparative centrifugation is concerned with the actual isolation of biological material for
subsequent biochemical investigations.
Separations carried out in a suspending medium which is homogenous are known as differential
centrifugation while those carried out in a suspending medium having density gradients are
known as density gradient centrifugations
A mixture of homogeneous particles can be separated by c:entrifugation on the basis of their
densities and/or their size. this can be achieved either by the time required for their complete
sedimentation in a fixed centrifugal field or on the extent of fueir sedimentation after a given
time in a fixed centrifugal field.
Differential centrifugation
We can choose the centrifugal field in such a manner that a particular organelle sediments
during the already known time of centrifugation to give a pellet. The pellet and supernatant are
separated at the end of each step and the supernatant recentrifuged to sediment another lighter
intracellular organelle. This is the essence of differential centrifugation.
DENSITY GRADIENT CENTRIFUGATION
As opposed to differential centrifugation. where a homogenous medium is used for separation.
density gradient centrifugation employs medium which has gradients. The separation under the
centrifugal field is therefore dependent upon the buoyant densities of the particles.
Density gradient centrifugation has two variations rate-zonal centrifugation. and isopycnic
centrifugation.
Rate-Zonal Centrifugation
The gradient used here has maximum density below that ofleast dense sedimenting particle. The
density gradient is reasonably shallow. The technique involves careful layering of a sample
solution on top of a preformed liquid density gradient whose denSity continuously increases
towards the bottom of the sample tube. Centrifugation is then performed at a comparatively low
speed for a short time.
Isopycnic centrifugation
If a density gradient is now prepared in a tube in such a manner that the density goes on
increasing toward the bottom of the tube and a solution of different particles is centrifuged in
this medium, different particles differing in their buoyant densities will travel different lengths
and become stationary at a region where the density of the layer below them is greater than
their own buoyant density. This is the essence of isopycnic centrifugation also known as the
sedimentation equilibrium centrifugation
As opposed to differential centrifugation. where a homogenous medium is used for separation.
density gradient centrifugation employs medium which has gradients. The separation under the
centrifugal field is therefore dependent upon the buoyant densities of the particles.
Density gradient centrifugation has two variations rate-zonal centrifugation. and isopycnic
centrifugation.
Rate-Zonal Centrifugation
The gradient used here has maximum density below that ofleast dense sedimenting particle. The
density gradient is reasonably shallow. The technique involves careful layering of a sample
solution on top of a preformed liquid density gradient whose denSity continuously increases
towards the bottom of the sample tube. Centrifugation is then performed at a comparatively low
speed for a short time.
Isopycnic centrifugation
If a density gradient is now prepared in a tube in such a manner that the density goes on
increasing toward the bottom of the tube and a solution of different particles is centrifuged in
this medium, different particles differing in their buoyant densities will travel different lengths
and become stationary at a region where the density of the layer below them is greater than
their own buoyant density. This is the essence of isopycnic centrifugation also known as the
sedimentation equilibrium centrifugation
Pickels (1943). Brakke (1951) and Kahler and Lloyd (1951) were the first few scientists to use
stabilizing gradients. They used sucrose for these gradients. Sucrose still remains the material in
most general use
Radiation Biology
Atomic nuclei are composed of two major components; protons which are positively charged,
and neutrons which are neutral. The number of electrons (negatively charged) in an atom is
always equal to the number of protons in the nucleus thereby making the atom electrically
neutral. This number is known as the atomic number (Z).
Neutrons are uncharged particles with a mass approximately equal to that of a proton.
The sum of protons and neutrons in a given nucleus is known as the mass number (A). Thus, A
= Z + N. Where N denotes the number of neutrons.
Isotopes - Isotopes are atoms of a given element, with identical number protons in the
nucleus, the same pattern of electrons in the clouds around the nucleus and therefore the same
chemical characteristics, but with different number of neutrons and consequently different
atomic mass
The number of stable isotopes that elements possess varies widely, for example, calcium
possesses six, carbon has three, while sodium has only one.
Radioactivity
Stability of an isotope of a given element is dictated by the ratio of neutrons to protons in the
nucleus, The relationship between the number of protons and neutrons in a nucleus can be seen
by plotting the values of N against Z for known isotopes.
It can thus be surmised that N : Z ratio of a stable isotope lies within narrow limits so that an
isotope outside these limits will be unstable. Such a nucleus will try to adjust its N : Z ratio
towards stability, giving out radiation in the process. Thus the phenomenon of radioactivity can
be seen as an attempt on the part of the isotope to achieve stability
stabilizing gradients. They used sucrose for these gradients. Sucrose still remains the material in
most general use
Radiation Biology
Atomic nuclei are composed of two major components; protons which are positively charged,
and neutrons which are neutral. The number of electrons (negatively charged) in an atom is
always equal to the number of protons in the nucleus thereby making the atom electrically
neutral. This number is known as the atomic number (Z).
Neutrons are uncharged particles with a mass approximately equal to that of a proton.
The sum of protons and neutrons in a given nucleus is known as the mass number (A). Thus, A
= Z + N. Where N denotes the number of neutrons.
Isotopes - Isotopes are atoms of a given element, with identical number protons in the
nucleus, the same pattern of electrons in the clouds around the nucleus and therefore the same
chemical characteristics, but with different number of neutrons and consequently different
atomic mass
The number of stable isotopes that elements possess varies widely, for example, calcium
possesses six, carbon has three, while sodium has only one.
Radioactivity
Stability of an isotope of a given element is dictated by the ratio of neutrons to protons in the
nucleus, The relationship between the number of protons and neutrons in a nucleus can be seen
by plotting the values of N against Z for known isotopes.
It can thus be surmised that N : Z ratio of a stable isotope lies within narrow limits so that an
isotope outside these limits will be unstable. Such a nucleus will try to adjust its N : Z ratio
towards stability, giving out radiation in the process. Thus the phenomenon of radioactivity can
be seen as an attempt on the part of the isotope to achieve stability
RADIOACTIVE DECAY
An unstable radioactive nucleus may reach or approach stability through emission of radiation.
This emission of radiation is known as radioactive decay.
A radioactive compound may decay by anyone or more of the several ways described below
Negatron emission. In this case a neutron is converted to a proton resulting in the ejection of a
negatively charged beta particle known as a negatron β-ve
NEUTRON -----PROTON + NEGATRON
The negatron emission results in loss of a neutron and gain of a proton. Consequently the N : Z
ratio decreases while the atomic number (Z) increases by one. The mass number (A) remains
unchanged.
15P
32
------------16S
32
+ β-ve
Positron emission. The positron is understood to be a positively charged β particle. Isotopes
like 22Na and IIC decay by emitting positrons. During positron emission a proton is converted
to a neutron.
PROTON -------7 NEU1RON + POSITRON
Positrons have a transient life and are annihilated on coming in contact with electrons. The
mass and energy of the two particles is then converted to two gamma rays emitted at 1800 to
each other
Positron emission results in loss of a proton and gain of a neutron. Consequently, the N : Z ratio
increases, the atomic number decreases by one, while the mass number remains constant.
6C
11
-------------- 5B
11
+ β +ve
An unstable radioactive nucleus may reach or approach stability through emission of radiation.
This emission of radiation is known as radioactive decay.
A radioactive compound may decay by anyone or more of the several ways described below
Negatron emission. In this case a neutron is converted to a proton resulting in the ejection of a
negatively charged beta particle known as a negatron β-ve
NEUTRON -----PROTON + NEGATRON
The negatron emission results in loss of a neutron and gain of a proton. Consequently the N : Z
ratio decreases while the atomic number (Z) increases by one. The mass number (A) remains
unchanged.
15P
32
------------16S
32
+ β-ve
Positron emission. The positron is understood to be a positively charged β particle. Isotopes
like 22Na and IIC decay by emitting positrons. During positron emission a proton is converted
to a neutron.
PROTON -------7 NEU1RON + POSITRON
Positrons have a transient life and are annihilated on coming in contact with electrons. The
mass and energy of the two particles is then converted to two gamma rays emitted at 1800 to
each other
Positron emission results in loss of a proton and gain of a neutron. Consequently, the N : Z ratio
increases, the atomic number decreases by one, while the mass number remains constant.
6C
11
-------------- 5B
11
+ β +ve
Alpha emission. The alpha particles are heavier than other emitted particles because they
consist of two neutrons and two protons. Alpha emission results in a decrease in atomic number
of two and a decrease in mass number of four. Decay of 238U is cited as an example
Emission of gamma rays. y-rays are usually emitted when the nucleus of an atom is
transformed. This frequently accompanies alpha or beta .particle emission. Gamma emission
does not lead to a change in atomic or mass numbers
Isotopes emitting alpha-particles are most energetic, their energies falling in the range of 4.0-8.0
MeV, while - β and y-emitters generally possess decay energies of less than 3.0 MeV
Rate of Radioactive Decay
For a particular isotope, the proportion of nuclei that decay in a given time is a constant known
as its disintegration constant. If the decline in activity of a radioactive isotope is plotted against
time (Figure 13.2) a typical exponential form is obtained.
This may be mathematically be expressed as a simple first order process
-dN/ dt =λN
dN/ dt = the number of atoms decaying per small increment of time
N = the total number of radioactive atoms present at any given time
λ = a decay constant, characteristic for a given isotope,
t = time.
The negative sign is essential because the activity is decreasing
consist of two neutrons and two protons. Alpha emission results in a decrease in atomic number
of two and a decrease in mass number of four. Decay of 238U is cited as an example
Emission of gamma rays. y-rays are usually emitted when the nucleus of an atom is
transformed. This frequently accompanies alpha or beta .particle emission. Gamma emission
does not lead to a change in atomic or mass numbers
Isotopes emitting alpha-particles are most energetic, their energies falling in the range of 4.0-8.0
MeV, while - β and y-emitters generally possess decay energies of less than 3.0 MeV
Rate of Radioactive Decay
For a particular isotope, the proportion of nuclei that decay in a given time is a constant known
as its disintegration constant. If the decline in activity of a radioactive isotope is plotted against
time (Figure 13.2) a typical exponential form is obtained.
This may be mathematically be expressed as a simple first order process
-dN/ dt =λN
dN/ dt = the number of atoms decaying per small increment of time
N = the total number of radioactive atoms present at any given time
λ = a decay constant, characteristic for a given isotope,
t = time.
The negative sign is essential because the activity is decreasing
The above equation means that A is that fraction of radioactive atoms which decays per unit
time.
Another constant for any given radioisotope is the time that is required for the original activity
to fall by a half. This is referred to as the half-life and is used more commonly than the
disintegration constant because it is of much more practical utility
The above relationships allow us to calculate half-life of a given isotope if its decay constant is
known and vice versa.
The unit of radioactivity is the curie (Ci). It is defined as the quantity of radioactive material in
which the number of nuclear disintegrations per second
The curie is a large unit and only millicuries (mCi) are needed for tracer applications. The curie
refers to the number of disintegrations actually occurring in a sample rather than the number
of disintegrations counted in a radiation counter
The international system of units (SI) has specified the term bequerel (Bq) to replace the term
curie, which is currently in use. The bequerel is defined as one disintegration per second. A
curie is therefore equal to 3.7 X 1010 Bq.
PRODUCTION OF ISOTOPES
time.
Another constant for any given radioisotope is the time that is required for the original activity
to fall by a half. This is referred to as the half-life and is used more commonly than the
disintegration constant because it is of much more practical utility
The above relationships allow us to calculate half-life of a given isotope if its decay constant is
known and vice versa.
The unit of radioactivity is the curie (Ci). It is defined as the quantity of radioactive material in
which the number of nuclear disintegrations per second
The curie is a large unit and only millicuries (mCi) are needed for tracer applications. The curie
refers to the number of disintegrations actually occurring in a sample rather than the number
of disintegrations counted in a radiation counter
The international system of units (SI) has specified the term bequerel (Bq) to replace the term
curie, which is currently in use. The bequerel is defined as one disintegration per second. A
curie is therefore equal to 3.7 X 1010 Bq.
PRODUCTION OF ISOTOPES
Bombarding the target nuclei with alpha-particles from naturally radioactive sources was the
way in which artificial isotopes were produced initially.
f Cockroft. and Walton. In 1931, these workers bombarded lithium target nuclei with hydrogen
nuclei and found that many such collisions resulted in the emission of high energy alpha
particles:
The cyclotron. an instrument that accelerates protons and other charged particles to very high
energies. was developed in the late 1930s. Although this development made a large number of
nuclear transformations possible. perhaps the most important development was the advent of
nuclear reactors
SYNTHESIS OF LABELED COMPOUNDS
A prerequisite for these isotopes to be used in biological studies is their incorporation in the bio-
organic compound which is to be studied. For example. 14C has to be incorporated into glucose
if we want to study the fate of the glucose in the body
The glucose molecule which has one or more of its carbon atoms (l2C) replaced by (l4C) is
known as a labeled molecule (glucose). while 14C is the label. It is also necessary that the label
should be incorporated in the position. which is appropriate for the experiment being carried
out.
INTERACTION OF RADIOACTIVITY WITH MATTER
Interaction of radioactivity with matter can be divided into two broad categories
(i) Excitation-This interaction may be weak. capable only of lifting an electron to a higher
energy orbital from its ground state. This electron eventually descends to its ground
state and the energy difference between the ground state and the higher energy orbital
is emitted as electromagnetic radiation. This type of interaction is known as excitation
(ii) Ionization. A closer interaction of radiation with matter can impart so much energy to
the orbital electron that it leaves the atom completely. This results in the formation of
an ion pair (a positive atom. i.e .• ion and a negative electron). This process is termed
ionization.
Alpha-particles-have a considerable energy (3-8 MeV). On account of their great mass. such a
kinetic energy means a relatively low velocity Uust 1% of the velocity of light}. Since they
dissipate their energy quickly they are not known to be very penetrating. Ordinarily. interaction
of an alpha particle with matter will result predominantly in ionization.
way in which artificial isotopes were produced initially.
f Cockroft. and Walton. In 1931, these workers bombarded lithium target nuclei with hydrogen
nuclei and found that many such collisions resulted in the emission of high energy alpha
particles:
The cyclotron. an instrument that accelerates protons and other charged particles to very high
energies. was developed in the late 1930s. Although this development made a large number of
nuclear transformations possible. perhaps the most important development was the advent of
nuclear reactors
SYNTHESIS OF LABELED COMPOUNDS
A prerequisite for these isotopes to be used in biological studies is their incorporation in the bio-
organic compound which is to be studied. For example. 14C has to be incorporated into glucose
if we want to study the fate of the glucose in the body
The glucose molecule which has one or more of its carbon atoms (l2C) replaced by (l4C) is
known as a labeled molecule (glucose). while 14C is the label. It is also necessary that the label
should be incorporated in the position. which is appropriate for the experiment being carried
out.
INTERACTION OF RADIOACTIVITY WITH MATTER
Interaction of radioactivity with matter can be divided into two broad categories
(i) Excitation-This interaction may be weak. capable only of lifting an electron to a higher
energy orbital from its ground state. This electron eventually descends to its ground
state and the energy difference between the ground state and the higher energy orbital
is emitted as electromagnetic radiation. This type of interaction is known as excitation
(ii) Ionization. A closer interaction of radiation with matter can impart so much energy to
the orbital electron that it leaves the atom completely. This results in the formation of
an ion pair (a positive atom. i.e .• ion and a negative electron). This process is termed
ionization.
Alpha-particles-have a considerable energy (3-8 MeV). On account of their great mass. such a
kinetic energy means a relatively low velocity Uust 1% of the velocity of light}. Since they
dissipate their energy quickly they are not known to be very penetrating. Ordinarily. interaction
of an alpha particle with matter will result predominantly in ionization.
Negatrons possess a single negative charge. have a small mass and high velocities. They
interact with matter to cause ionization and excitation in a manner exactly similar to that of a
particles. due to their extreme speed and vanishingly small Size. their probability of interacting
with matter is smaller as compared to a-particles. They are therefore less ionizing but more
penetrating than the a-particles
Gamma rays are electromagnetic radiation and are therefore devoid of any charge or mass.
Due to the above properties and because of their high velocity. the probability of their
interaction with intervening atoms is rather less.
Travel great distances before their energy is dissipated. This makes them highly penetrating.
They interact with matter in three important ways which lead to production of secondary
electrons. These electrons can in tum cause ionization and excitation.
Photoelectric absorption consists of the transfer of the entire energy of the radiation to a single
electron. The electron is then ejected as a photoelectron which behaves as a negatron. Relatively
low energy y-radiation indulges in such interactions.
MEASUREMENT OF RADIOACTIVITY
The simplest arrangement to measure radioactivity involves measurement of the ionization
caused by radiation in a gas fllied chamber. The number of ionized particles generated in the
gas by direct interaction (primary interaction) with radiation can be increased several fold by
various methods discussed
SCINTILLATION COUNTERS - exploit the excitation caused by radioactive interaction in
order to measure radioactivity.
The electrons are not made to leave the atom completely, but are merely catapulted to higher
energy levels. When these electrons return to their ground state, they emit electromagnetic
radiation.
This light can be observed and the number of such flashes gives an index of the intensity of the
radiation.
Absolute activity refers to the number of disintegrations actually taking place in the sample,
whereas the relative activity refers to the number of disintegrations accounted for.
Ionization chambers are not used in biochemistry on a large scale (moreover, with the
development of scintillation counters these chambers have now become obsolete). Yet a short
description of their mode of action is required for a good understanding of the-proportional and
the Geiger-Muller counters.
Principle of the ionization chamber lies in the measurement of the number of ions and electrons
produced by the radiation in a gas filled chamber.
Scintillation can be counted by two different techniques
In solid or external scintillation counting,the sample is placed close to a fluor
crystal(crystallized zinc sulphide for α.-emitters; sodium for y-emitters; anthracene for β-
emitters), which in tum is placed adjacent to a photomultiplier. This photomultiplier is
interact with matter to cause ionization and excitation in a manner exactly similar to that of a
particles. due to their extreme speed and vanishingly small Size. their probability of interacting
with matter is smaller as compared to a-particles. They are therefore less ionizing but more
penetrating than the a-particles
Gamma rays are electromagnetic radiation and are therefore devoid of any charge or mass.
Due to the above properties and because of their high velocity. the probability of their
interaction with intervening atoms is rather less.
Travel great distances before their energy is dissipated. This makes them highly penetrating.
They interact with matter in three important ways which lead to production of secondary
electrons. These electrons can in tum cause ionization and excitation.
Photoelectric absorption consists of the transfer of the entire energy of the radiation to a single
electron. The electron is then ejected as a photoelectron which behaves as a negatron. Relatively
low energy y-radiation indulges in such interactions.
MEASUREMENT OF RADIOACTIVITY
The simplest arrangement to measure radioactivity involves measurement of the ionization
caused by radiation in a gas fllied chamber. The number of ionized particles generated in the
gas by direct interaction (primary interaction) with radiation can be increased several fold by
various methods discussed
SCINTILLATION COUNTERS - exploit the excitation caused by radioactive interaction in
order to measure radioactivity.
The electrons are not made to leave the atom completely, but are merely catapulted to higher
energy levels. When these electrons return to their ground state, they emit electromagnetic
radiation.
This light can be observed and the number of such flashes gives an index of the intensity of the
radiation.
Absolute activity refers to the number of disintegrations actually taking place in the sample,
whereas the relative activity refers to the number of disintegrations accounted for.
Ionization chambers are not used in biochemistry on a large scale (moreover, with the
development of scintillation counters these chambers have now become obsolete). Yet a short
description of their mode of action is required for a good understanding of the-proportional and
the Geiger-Muller counters.
Principle of the ionization chamber lies in the measurement of the number of ions and electrons
produced by the radiation in a gas filled chamber.
Scintillation can be counted by two different techniques
In solid or external scintillation counting,the sample is placed close to a fluor
crystal(crystallized zinc sulphide for α.-emitters; sodium for y-emitters; anthracene for β-
emitters), which in tum is placed adjacent to a photomultiplier. This photomultiplier is
connected to a high voltage supply and a scalar. Solid scintillation counting is particularly
useful for measurement of y-emitting isotopes.
In liquid or internal scintillation counting, the radioactive sample is suspended in a scintillation
system composed of the solvent and an appropriate Scintillator. Liquid scintillation counting is
extremely useful for quantitating soft β-emitters.
THE TRACER TECHNIQUE
Such labled compounds remain chemically identical to similar compounds within the system
with which they will mingle, they differ from unlabeled compounds in as much as that they emit
easily measurable radiation or have slightly different masses
The tracer studies in general consist of :
(i) Preparation of a labeled compound
(ii) Introduction of the labeled compound into a biological system,
(iii) Separation and determination of labeled species in various biochemical fractions at
a later time.
Clinical Application
Diagnosis. Radioisotopes are very widely used for diagnostic test
Therapy. Radioisotopes have been particularly useful in treating cancers. 6OCO radiations are
given to tumors and many tumors have been known to regress to quite some extent by this
treatment
Radioisotopes in Sterilization of Foods and Equipments
Radioimmunoassay
(RIA) is a highly sophisticated technique and can detect extremely small amounts of non-
radioactive material. It can achieve this even if the mixture contains huge amounts of other
materials in which the investigator is not interested.
• A fixed concentration of labeled tracer antigen is incubated with a constant amount of
antiserum
• If unlabeled antigen is added to the system, there is competition between labeled tracer
and unlabeled antigen for the limited number of binding sites on the antibody
• The amount of tracer bound to antibody will decrease as the conc. of unlabeled antigen
increases
• This can be measured after separating antibody bound from free tracer and counting
the bound fraction
The binding of radioactively labeled antigen (Ag·) to a fixed amount of antibody (Ab) can be
partially inhibited by addition of unlabeled antigen (Ag).
useful for measurement of y-emitting isotopes.
In liquid or internal scintillation counting, the radioactive sample is suspended in a scintillation
system composed of the solvent and an appropriate Scintillator. Liquid scintillation counting is
extremely useful for quantitating soft β-emitters.
THE TRACER TECHNIQUE
Such labled compounds remain chemically identical to similar compounds within the system
with which they will mingle, they differ from unlabeled compounds in as much as that they emit
easily measurable radiation or have slightly different masses
The tracer studies in general consist of :
(i) Preparation of a labeled compound
(ii) Introduction of the labeled compound into a biological system,
(iii) Separation and determination of labeled species in various biochemical fractions at
a later time.
Clinical Application
Diagnosis. Radioisotopes are very widely used for diagnostic test
Therapy. Radioisotopes have been particularly useful in treating cancers. 6OCO radiations are
given to tumors and many tumors have been known to regress to quite some extent by this
treatment
Radioisotopes in Sterilization of Foods and Equipments
Radioimmunoassay
(RIA) is a highly sophisticated technique and can detect extremely small amounts of non-
radioactive material. It can achieve this even if the mixture contains huge amounts of other
materials in which the investigator is not interested.
• A fixed concentration of labeled tracer antigen is incubated with a constant amount of
antiserum
• If unlabeled antigen is added to the system, there is competition between labeled tracer
and unlabeled antigen for the limited number of binding sites on the antibody
• The amount of tracer bound to antibody will decrease as the conc. of unlabeled antigen
increases
• This can be measured after separating antibody bound from free tracer and counting
the bound fraction
The binding of radioactively labeled antigen (Ag·) to a fixed amount of antibody (Ab) can be
partially inhibited by addition of unlabeled antigen (Ag).
The extent of this inhibition is a measure of the unlabeled material added.
Thus, if the medium consists of 100% antigen in the radioactive form, all the antibodies will be
found bound to the Ag*. If we now make th¢ mixture 50% with respect to Ag* by adding Ag,
only 50% of the antibody will be found binding ! to Ag*; the other 50% would have bound Ag.
If the medium is made more poor with respect to Ag* - 25% Ag* and 75% Ag - only 25% of the
antibody will associate with Ag*; the other 75% will bindAg.
Most of the time the antigen or the hapten used for radioimmunoassay is labeled with
125
I.
There is a very good reason for this
125
I. is a strong y-emitter.
Advantages:
- Extremely sensitive method
- Large number of samples can be processed
- Small changes in hormone concentrations can be reproducibly measured
AUTORADIOGRAPHY
Autoradiography is the bio-analytical technique used to visualize the distribution of radioactive
labelled substance with radioisotope in a biological sample
It is a method by which a radioactive material can be localized within a particular tissue, cell,
cell organelles or even biomolecules.
Autoradiography, although used to locate the radioactive substances, it can also be used for
quantitative estimation by using densitometer
Micro autoradiography, has been developed for studying subcellular structures, even those as
small as individual strands of deoxyribonucleic acid (DNA).
The cells being studied are given a nutrient solution containing molecules that have been
labeled, usually with radioactive tritium, carbon, or phosphorus
PRINCIPLES OF AUTO RADIOGRAPHY
• Resolution and radioisotope characteristics
• Film emulsion and sensitivity
• Determination of exposure time
• Tissue preparation and artifacts
Thus, if the medium consists of 100% antigen in the radioactive form, all the antibodies will be
found bound to the Ag*. If we now make th¢ mixture 50% with respect to Ag* by adding Ag,
only 50% of the antibody will be found binding ! to Ag*; the other 50% would have bound Ag.
If the medium is made more poor with respect to Ag* - 25% Ag* and 75% Ag - only 25% of the
antibody will associate with Ag*; the other 75% will bindAg.
Most of the time the antigen or the hapten used for radioimmunoassay is labeled with
125
I.
There is a very good reason for this
125
I. is a strong y-emitter.
Advantages:
- Extremely sensitive method
- Large number of samples can be processed
- Small changes in hormone concentrations can be reproducibly measured
AUTORADIOGRAPHY
Autoradiography is the bio-analytical technique used to visualize the distribution of radioactive
labelled substance with radioisotope in a biological sample
It is a method by which a radioactive material can be localized within a particular tissue, cell,
cell organelles or even biomolecules.
Autoradiography, although used to locate the radioactive substances, it can also be used for
quantitative estimation by using densitometer
Micro autoradiography, has been developed for studying subcellular structures, even those as
small as individual strands of deoxyribonucleic acid (DNA).
The cells being studied are given a nutrient solution containing molecules that have been
labeled, usually with radioactive tritium, carbon, or phosphorus
PRINCIPLES OF AUTO RADIOGRAPHY
• Resolution and radioisotope characteristics
• Film emulsion and sensitivity
• Determination of exposure time
• Tissue preparation and artifacts
BASIC COMPONENTS
• Specimen
• Tracer
• Detector
Autoradiography is based upon the ability of radioactive substance to expose the photographic
film by ionizing it.
In this technique a radioactive substance is put in direct contact with a thick layer of a
photographic emulsion (thickness of 5-50 mm) having gelatin substances and silver halide
crystals.
It is then left in dark for several days for proper exposure
The silver halide crystals are exposed to the radiation which chemically converts silver halide
into metallic silver (reduced) giving a dark colour band
The resulting radiography is viewed by electron microscope, pre flashed screen, intensifying
screen, electrophoresis, digital scanners etc.
Applications
To find and investigate the various properties of DNA
To find the location and amount of particular substance within a cell including cell
organelle, metabolites etc.
Tissue localization of radioactive substance.
To find the site and performance of targeted drug.
• Specimen
• Tracer
• Detector
Autoradiography is based upon the ability of radioactive substance to expose the photographic
film by ionizing it.
In this technique a radioactive substance is put in direct contact with a thick layer of a
photographic emulsion (thickness of 5-50 mm) having gelatin substances and silver halide
crystals.
It is then left in dark for several days for proper exposure
The silver halide crystals are exposed to the radiation which chemically converts silver halide
into metallic silver (reduced) giving a dark colour band
The resulting radiography is viewed by electron microscope, pre flashed screen, intensifying
screen, electrophoresis, digital scanners etc.
Applications
To find and investigate the various properties of DNA
To find the location and amount of particular substance within a cell including cell
organelle, metabolites etc.
Tissue localization of radioactive substance.
To find the site and performance of targeted drug.
To locate the metabolic activity site in the cell.
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