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APPLIED FORENSIC RESEARCH SCIENCES

CSIR UGC - NET

UGC NET\JRF 2022

FORENSIC SCIENCE

UNIT – 02

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Unit – II • Microscopy: Polarizing, Comparison, Stereoscopic, Fluorescent and Electron

Microscopes • Spectrophotometry: UV, Visible, IR, Raman, Atomic absorption, Emission •
Neutron Activation Analysis • X – rays and x-ray based techniques such as XRD, XRF • Mass
Spectroscopy • Chromatographic Techniques: TLC, GLC, HPLC,HPTLC • Hyphenated
Techniques: GC-MS, LC-MS, IR-MS and ICP-MS • Electrophoresis: High and Low voltage
electrophoresis, Immunoelectrophoresis • Immunoassays: Principle, Types ,Techniques and
applications

MICROSCOPY
A microscope is an optical instrument that is used for magnifying objects
too small to be seen by naked eye.

Investigations or studies of cell architecture by means of the microscope

are called microscopy, and the person who pursuing the study is called
microscopist.

• Microscopes work on the physical principle of magnification

where the image of an object is magnified so that it can be visible.
• The substances that can only be seen with a microscope are called
microscopic substances.
• Microscopes are imperative in areas like microbiology that deals
with the structure and function of microscopic living beings.
• Microscopy is further divided into three branches; optical
microscopy, electron microscopy, and X-ray microscopy.
• X-ray microscopy is a fairly new technology that is responsible
for detailed imaging of subcellular organelles like the nucleus
and chromosomes.
• Microscopy, importantly optical microscopy, began with the
discovery of the first microscope by Anton Von Leeuwenhoek.
• The complexity of microscopy since then has increased rapidly
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with new and advanced microscopes with higher magnification

and resolution.
• In an optical microscope, the rays of light are passed through a
series of glass lenses to produce a magnified image on the
observer's eyes.
• Compound microscopes are the most common type of
microscope, mostly used for research and teaching purposes.
• In the case of an electron and X-ray microscope, an electron beam
is created which produced a digital magnified image of an object.
• Electron microscopes have very high magnification and
resolution which produces clear enlarged images of objects as
small as an atom,

• Depending on the nature of the sample, different types of

microscopes, including bright field microscope, fluorescence
microscope, phase contrast, and darkfield microscopes, are also
available.
• The magnification of these microscopes depends on the type of
lens used in the system which produces images of different
magnitude and resolution so that they can be viewed.
• Microscopy is important in different areas of science like
histology, cytology, and bacteriology. Microscopic examination
of the morphology and structure of cells has been used as an
essential technique for the identification of microorganisms.

History of Microscopy
1590: The two Janssen brothers of Holland, Francis Janssen and
Zacharias Janssen, who were spectacle makers built the first operational
light microscope.

1611: Kepler built the first compound microscope.

1665: Robert Hooke developed the first laboratory microscope which
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has a magnification of 14-42 X. He observed small pores in sections of

cork that he called cells.
1674: Leeuwenhoek discovered protozoa by his self-built microscope
with magnification of 270 X. He discovered bacteria for the first time 9
years later.
1905: Zsigmondy invented dark-field microscopy.

Properties of Microscope
A microscope has dual property i.e., magnification and resolution. The
usefulness of a microscope depends not so much on the degree of
magnification but rather on the resolution. Resolution has nothing to do
with the magnification.
(a) Magnification: Magnification or magnifying power of a microscope
is the degree of increase in size of optical image over the actual size of
object being viewed. Magnification = Size of retinal image seen with
microscope/Size of retinal image with naked eye Magnification of
microscope is calculated by multiplying the magnification of the
objective lens with that of the eye piece (ocular lens). For example, the
magnification of eye piece is 10x and the magnification of objective lens
is 40x, then the microscope magnifies the object by 10x40= 400 times
i.e. magnification is 400X.

The human eye has no power of magnification, so magnifying glasses

maybe used to magnify images up to about 10 times. A light microscope
in which combination of lens used has a magnification of 100-2000 X.
For higher magnification over 400x, oil immersion lens can be used in
which cedar wood oil placed between objective and the coverslip
increase the light gathering properties of the lens.

Units of Measurement used in Microscopy

1 meter (m) = 102 cm = 103 mm = 106 mm = 109 nm = 1010 A
1 centimeter (cm) = 1/100 meter (m) = 0.4 inch 1 millimeter (mm)
=1/1000 meter = 0.001 m - 10-3 m = 10-3 mm = 106 nm = 107 A 1
micrometer (mm)** = 1/1000 mm = 0.001mm = 10-mm = 10-6 m = 103
nm = 104 A 1 nanometer (nm) = 1/1000 mm = 0.001mm = 10-3 mm =
10-6 mm = 10-'m = 10 A

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1 angstrom (A)* = 1/10 nm = 0.1 nm = 10-2 nm = 10-7mm = 10-10m

Micrometers were formerly known as microns (u), and nanometers as
millimicrons (mu). The Angstrom is not an accepted measurement in
the International system of Units. It is included here, however, because
it was widely used in microscopy in the past.
(b)Resolution or Resolving power: Resolution (= resolving power or
resolving limit) of an optical device (eye or microscope) is its ability to
distinguish between two very closely placed objects as separate objects.
The resolving power of a microscope depends on (i) Wavelength of light
(A) and (it) numerical-aperture (NA) of the lens system used. Resolution
of a microscope can be calculated by Abbe Equation, after the name of
German Physicist Ernst Abbe in 1876.

Resolution = 0.611/NA= 0.611/ N Sino

0.61 = a trigonometric constant
1 = Wavelength of light used; 450-750 mm for visible light used in
compound microscope, Blue light has shortest wavelength (1 =450nm)
gives maximum resolution. Therefore, blue filter blue light commonly
used in microscopy.
NA-N Sin 0; where N is the refractive index of the medium (usually air
or oil) between the specimen and objective lens. For air N = 1.0 and for
immersion oil N = 1.5.

O or a- half angle of the cone of light entering the objective lens from
the specimen. The maximum value of 6 for the best objective lens is 70°
(Sin 70° = 0.94). The resolution of light microscope, using air and blue
light, will be
Lm=0.61x 450nm/ 1.0x 0.94= 292nm or -0.3um If oil and blue light
used, then Lm = 0.61x 450nm/ 1.0x 0.945 194nmor -0.2 um

Thus, light microscope can never resolve two closer particles less than
about 0.2 nm apart, no matter how many times the image is magnified.
The resolution of electron microscope is about 0.0005 um whereas the
human eye is about 100 um. It should be noted that lower the value of
Lm Higher will be resolution, which can be done by changing A, N or
6. Resolution will increase with a decrease in A and with an increase in

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NA; i.e. lm is inversely proportional to A and Lm is proportional to NA.

The numerical aperture (NA) is the light collecting ability of lens.

PRINCIPLES

Microscopy is necessary to evaluate the integrity of samples and to

correlate structure with function. Microscopy serves two independent
functions of enlargement (magnification) and improved resolution
(rendering of two objects as separate entities).

Light microscopes employ optical lenses to sequentially focus the image

of objects, whereas electron microscope uses electromagnetic lenses.

Light and electron microscopes work either in transmission or scanning

mode depending on whether the light or electron beam either passes
through the specimen and is diffracted or deflected by specimen surface.
Polarized light microscopes detect optically active substances in cells;
fo particles of silica or asbestos in lung tissue.

Phase contrast microscopes are often used to improve image contrast of

unstained material which is caused either by diffraction by the specimen
or even by differences in thickness of the specimen. At their point of
focus, the converging light rays shows interference, resulting in either
increase or decrease in the amplitude of the resultant wave (constructive
or destructive interference, respectively), which the eye detects as
differences in brightness.

Confocal microscopy is an imaging technique used to increase

micrograph contrast and/or to reconstruct three-dimensional images by
using a spatial pinhole to eliminate out-of-focus light or flare in
specimens that are thicker than the focal plane. This technique has been
gaining popularity in the scientific and industrial communities.

Typical applications include life sciences and semiconductor inspection.

In a conventional (i.e., widefield) fluorescence microscope, the entire
specimen is flooded in light from a light source. Due to the conservation
of light intensity transportation, all parts of specimen throughout the
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optical path will be excited and the fluorescence will be detected by a

photo detector or a camera.

In contrast, a confocal microscope uses point illumination and a pinhole

in an optically conjugate plane in front of the detector to eliminate out-
of-focus information. Only the light within the focal plane can be
detected, so the image quality is much better than that of wide-field
images. As only one point is illuminated at a time in confocal
microscopy, 2D or 3D imaging requires scanning over a regular raster
(i.e., a rectangular pattern of parallel scanning lines) in the specimen.
The thickness of the focal plane is defined mostly by the square of the
numerical aperture of the objective lens, and also by the optical
properties of the specimen and the ambient index of refraction.

Scanning electron microscopes (SEM) use a fine beam of electrons to

scan back and forth across the metal coated specimen surface. The
secondary electrons are generated from this surface collected by a
scintillation crystal, which converts each electron impact into a flash of
light. Each light flash inside the crystal is amplified by a photomultiplier
and used to build up an image on a fluorescent screen. The principle
application of SEM is the study of surfaces such as those of cells. The
resolution limit of scanning electron microscope is about 6 nm.

Specimen Preparation

This includes three steps:

1. Fixation

2. Sectioning, and

3. Staining.
TEM requires thin specimens, i.e., squashes, smear, hanging drops or
very thin sections. For preservation of cellular integrity first sample is
fixed either by rapid freezing or by chemical treatment. Generally used
fixatives are formaldehyde (primarily used in light microscopy) and
glutaraldehyde (for electron microscopy) is the result of formation of
methylene bridges with sidechain amino groups of proteins, whilst
osmium tetroxide, a common fixative in electron microscopy, cross

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links mainly with unsaturated fatty acid side chains. Fixed tissue is then
subjected to sequential process of sectioning and staining.
Fixed tissues, when not frozen, need support before proceeding for
sectioning. Embedding media such as waxes and epoxy resins (Araldite
or Epon) are immiscible with both water and alcohol, which necessitates
initial dehydration and equilibrium of fixed tissues by passing them
through increasing concentration of ethanol followed by transfer to
xylene or propylene oxide, before infiltration with an appropriate
embedding medium in its liquid phase.
Sections of 5-10 um thickness are obtained by cryostat from 20
refrigerated samples. Ultra-thin sections for TEM (less than 100 nm)
are cut with ultra microtomes using diamond or glass as cutting In
negative staining, a heavy metal stain, often phosphotungstic acid, is
allowed to dry in a puddle around the surfaces of isolated cell particles
supported on a thick carbon or plastic film. The stain molecules are
deposited into surface crevices in the specimen during drying and
produce ghost image in which the specimen appears light against dark
background, often outlining details very clearly. The contrast can
further improve by technique of shadowing.
Antibodies prepared against specific cellular proteins, which have
absorbed colloidal gold are frequently used for staining in TEM as gold
is electron dense. Freeze fracture techniques in which frozen samples are
cleaved with a knife along facture planes in a membrane, utilize this
shadowing techniques to produce replicas of broken cellular materials
that show the membrane surface structure on and within the cell
organelles.
Samples for ion probe analysis are prepared either by chemical fixation
or, more successfully, by ultrarapid freezing methods, such as
immersion in liquid propane or nitrogen slush, which serves to
immobilize ions. Where frozen tissue is used, sections must be ultra-
thin and are obtained by ultracryomicrotome.
Histological stains are used to produce contrast, which aids resolution.
Many stains used in light microscopy depend upon the anionic or
cationic behaviour of intracellular ampholytes and their efficiency to
influence pH. Cytoplasmic contents are generally cationic in slightly
acid pH range, implying preferential binding to anionic (acid) stains
such as eosin.

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Chromatin and DNA at pH 6.0 are anionic and thus binds to cationic
stains such as methylene blue. Contrast in material for TEM is improved
by incorporating heavy metal salts into the specimens to induce a greater
extent of electron absorption. This can be exploiting the binding of
uranium to nucleic acids and proteins and of lead to lipids.

POLARIZED LIGHT MICROSCOPE

Polarization microscope is quite similar to the interference microscope.

In polarizing microscope, Nicol prisms of calcite were formerly added
below the condenser or in the ocular. A simpler and cheaper system
utilizes sheets of Polaroid film, or a sheet of polyvinyl alcohol stained
with iodine and then stretched over gentle heat.
In this technique, polarized light is used to increase the contrast with the
light microscope. The structures differing in refractive index in different
planes can be visualized by this microscope. Molecular substructure of
cell organelles can be viewed through this microscope.
A polarizing microscope can be obtain from a compound or a normal
one by adding some pieces. The mainly differences between a
polarizing microscope and other microscopes:

▪ A polarizer and analyzer

▪ A circular rotating stage
▪ Special plates placed between the object and light path.
▪ Bertrand lens (if necessary)
A polarizer is a filter that only allows specific light waves or vibrations
to pass through it and focus them in a single plane. Polarizer is placed
below the condenser and sends only plane-polarized light into the
specimen. An analyzer, mainly used as a second polarizer located above
the sample, determines the quantity and the direction of the light that
illuminates a sample. Due to the use of these filters, the polarized light
waves vibrate in one single direction, instead of the normal ones that
vibrate in random directions. In this way the polarized light is more
concentrated and then more efficient to the study of minerals, for
example. By changing the relationship of the polarizer and the analyzer,
it's possible to determine the amount of absorbance, reflection and
refraction of the light through the microscope.

Analyzer is located above the objective. When it is rotated 360°, the

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visual field alternates between bright and dark at every 180° turn. When
the axes of polarizer and analyzer are parallel to one another, the two
positions of maximum light transmission are obtained. Rotation of
analyzer by 90° prevents the passage of polarized light and produces a
dark field.
The usual test is to rotate the specimen to find the maximum and
minimum brightness. The maximum brightness is obtained when the
axis of the specimen makes a + 45° angle with those of the polarizer and
analyzer. Polarized light may pass through material with the same
velocity in all directions, or the velocity may vary with direction. The
property of the material (cells and tissues) is the determining factor.
Isotropic material is that through which polarized light passes with the
same velocity, independent of the impinging direction. Such material
has the same refractive index in all directions. Anisotropic material is
that through which polarized light does not pass with the same velocity,
i.e., velocity of passage of polarized light varies with direction. Such
material is also called birefringent because it presents two different
indexes of refraction corresponding to the respective different velocities
of transmission.
Birefringence (B) may be expressed quantitatively as the difference
between the two indexes of refraction associated with the fast (Ne) and
slow (N.) rays. In practice, the retardation (r) of the light polarized in
one plane is measured relative to that light polarized in another
perpendicular plane with the polarizing microscope. The retardation
depends on the thickness of the specimen (t).

Thus, birefringence (B) = Ne - No = r/t

Thus, polarizing microscope is used to measure retardation of light
polarized in one plane relative to that in another perpendicular plane.
Measurement is in millimicrons and is facilitated by the use of a
compensator in the optical system. Retardation, which depends on the
thickness of the specimen, can be measured now to about 0.1 p (1Ao).
In biological fibers, the birefringence is positive if the index of is pxeater
along the length of the fiber than in the perpendicular plane, and it is
negative in the opposite case, i.e., when the index of refraction is greater
in the perpendicular plane.

Important Applications
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It is mostly used in the field of geology to study rocks and minerals.

Besides that can also be used in medicine, chemistry, biology and some
times in metallurgy. it is the best choice to study materials like minerals,
polymers, ceramics, wood, urea, substances of natural and synthetic
fibers with those birefringent properties, cellophane, and also botanical
and insect specimens and fish scales. With polarizing microscopy it is
possible to determine the color absorption, structure, composition and
refraction of light in isotropic (gases and liquids -one refractive index)
and anisotropic substances.

Pathway of Light
. The light passes through a polarizing filter called the polarizer (the
polarizer is fixed in an east to west vibrational way, but it can be rotated
if necessary. There is one more polarizing filter called the analyzer. It
is usually situated above the objectives and can be moved in and out of
the optical path).
. Passes through the birefringent specimen. The polarizer is usually fixed
in an east to west vibrational direction, but it can be rotated as required.
There is one more polarizing filter called the analyzer. It is usually
situated above the objectives and can be moved in and out of the optical
path.
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.
Image contrast arises from the interaction of plane-polarized light with
a birefringent (or doubly refracting) specimen to produce two individual
wave components that are each polarized in mutually perpendicular
planes. The velocities of these components are different and vary with
the propagation direction through the specimen. After exiting the
specimen, the light components become out of phase with each other,
but are recombined with constructive and destructive interference when
they pass through the analyzer.

When an anisotropic specimen is brought into focus and rotated through

360 degrees on a circular polarized light microscope stage, it will
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sequentially appear bright and dark (extinct), depending upon the

rotation position. When the specimen long axis is oriented at a 45-degree
angle to the polarizer axis, the maximum degree of brightness will be
achieved, and the greatest degree of extinction will be observed when
the two axes coincide. During rotation over a range of 360 degrees,
specimen visibility will oscillate between bright and dark four times, in
90-degree increments. This is due to the fact that when polarized light
impacts the birefringent specimen with a vibration direction parallel to
the optical axis, the illumination vibrations will coincide with the
principal axis of the specimen and it will appear isotropic (dark or
extinct). If the specimen orientation is altered by 45 degrees, incident
light rays will be resolved by the specimen into ordinary and
extraordinary components, which are then united in the analyzer to yield
interference patterns. Because interference only occurs when polarized
light rays have an identical vibration direction, the maximum
birefringence is observed when the angle between the specimen
principal plane and the illumination permitted vibrational direction
overlap. Interference between the recombining white light rays in the
analyzer vibration plane often produces a spectrum of color, which is
due to residual complementary colors arising from destructive
interference of white light. The colors observed under illumination with
white light in the microscope eyepiece can be utilized to quantitatively
draw conclusions about path differences and specimen thickness values
when the refractive indices of the specimen are known.
Polarized light microscopy is utilized to distinguish between singly
refracting (optically isotropic) and doubly refracting (optically
anisotropic) media. Anisotropic substances, such as uniaxial or biaxial
crystals, oriented polymers, or liquid crystals, generate interference
effects in the polarized light microscope, which result in differences of
color and intensity in the image as seen through the eyepieces and
captured on film, or as a digital image. This technique is useful for
orientation studies of doubly refracting media that are aligned in a
crystalline lattice or oriented through long-chain molecular interactions
in natural and synthetic polymers and related materials. Also
investigated in polarized light are stresses in transparent singly
refracting media (for example, glass) and the identification and
characterization of a wide spectrum of anisotropic substances through
their refractive index and birefringence.
The microscope is equipped with all of the standard accessories for
examination of birefringent specimens under polarized light. Although
similar to the common brightfield microscope, the polarized light
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microscope contains additional components that are unique to

instruments of this class. These include the polarizer and analyzer,
strain-free objectives and condenser, a circular graduated stage capable
of 360-degree rotation, and an opening in the microscope body or
intermediate tube for a full-wave retardation plate, quartz wedge, Berek
compensator, or quarter-wavelength plate. The monocular microscope
is designed with a straight observation tube and also contains a 360-
degree rotatable analyzer with a swing-out Bertrand lens, allowing both
conoscopic and orthoscopic examination of birefringent specimens. The
objectives (4x, 10, and 40x) are housed in mounts equipped with an
individual centering device, and the circular stage has a diameter of 140
millimeters with a clamping screw and an attachable mechanical stage.
Removal of the polarizer and analyzer (while other components remain
in place) from the light path renders the instrument equal to a typical
brightfield microscope with respect to the optical characteristics.
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.
Typical modern polarized (and brightfield) microscopes have a
lamphouse, which contains a 50 to 100-watt high-energy tungsten-
halogen lamp, attached to the base of the microscope. A transformer
providing direct current (DC) voltage to the lamp is usually built directly
into the microscope base and is controlled by a potentiometer positioned
near the lamp switch in bottom of the base (the lamp voltage control).
Between the lamphouse and the microscope base is a filter cassette that
positions removable color correction, heat, and neutral density filters in
the optical pathway. Also built into the microscope base is a collector
lens, the field iris aperture diaphragm, and a first surface reflecting
mirror that directs light through a port placed directly beneath the
condenser in the central optical pathway of the microscope. These
components control the size, intensity, and distribution of light in the
illumination field. The entire base system is designed to be vibration free
and to provide the optimum light source for Köhler illumination. In
general, the modern microscope illumination system is capable of
providing controlled light to produce an even, intensely illuminated
field of view, even though the lamp emits only an inhomogeneous
spectrum of visible, infrared, and near-ultraviolet radiation.
In some polarized light microscopes, the illuminator is replaced by a
plano-concave substage mirror. Almost any external light source can
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directed at the mirror, which is angled towards the polarizer positioned

beneath the condenser aperture. This configuration is useful when an
external source of monochromatic light, such as a sodium vapor lamp,
is required. Because the illumination intensity is not limited by a
permanent tungsten-halogen lamp, the microscope can be readily

adapted to high intensity light sources in order to observe weakly

birefringent specimens.

Polarizers
Polarized light microscopy was first introduced during the nineteenth
century, but instead of employing transmission-polarizing materials,
light was polarized by reflection from a stack of glass set at a 57-degree
angle to the plane of incidence. Later, more advanced instruments relied
on a crystal of doubly refracting material (such as calcite) specially cut
and cemented together to form a prism. A beam of white unpolarized
light entering a crystal of this type is separated into two components that
are polarized in mutually perpendicular directions. One of these light
rays is termed the ordinary ray, while the other is called the extraordinary
ray. The ordinary ray is refracted to a greater degree in the birefringent
crystal and impacts the cemented surface at the angle of total internal
reflection. As a result, this ray is reflected out of the prism and
eliminated by absorption in the optical mount. The extraordinary ray
traverses the prism and emerges as a beam of linearly polarized light
that is passed directly through the condenser and to the specimen
(positioned on the microscope stage). Several versions of this polarizing
device (which was also employed as the analyzer) were available, and
these were usually named after their designers. The most common
polarizing prism was named after William Nicol, who first cleaved and
cemented together two crystals of Iceland spar with Canada balsam in
1829. Nicol prisms were first used to measure the polarization angle of
birefringent compounds, leading to new developments in the
understanding of interactions between polarized light and crystalline
substances.
A crystal of doubly refracting (birefringent) material, usually calcite, is
cut along the plane labeled a-b-c-d and the two halves are then cemented
together to reproduce the original crystal shape. A beam of unpolarized
white light enters the crystal from the left and is split into two
components that are polarized in mutually perpendicular directions. One
of these beams (labeled the ordinary ray) is refracted to a greater degree

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and impacts the cemented boundary at an angle that results in its total
reflection out of the prism through the uppermost crystal face. The other
beam (extraordinary ray) is refracted to a lesser degree and passes
through the prism to exit as a plane-polarized beam of light. Other prism
configurations were suggested and constructed during the nineteenth
and early twentieth centuries, but are currently no longer utilized for
producing polarized light in most applications. Nicol prisms are very
expensive and bulky, and have a very limited aperture, which restricts
their

use at high magnifications. Instead, polarized light is now most

commonly produced by absorption of light having a set of specific
vibration directions in a dichroic medium. Certain natural minerals, such
as tourmaline, possess this property, but synthetic films invented by Dr.
Edwin H. Land light. Tiny crystallites of iodoquinine sulphate, oriented
in the same direction, are embedded in a transparent polymeric film to
prevent migration and reorientation of the crystals.
Land developed sheets containing polarizing films that were marketed
under the trade name of Polaroid', which has become the accepted
generic term for these sheets. Any device capable of selecting plane-
polarized light from natural (unpolarized) white light is now referred to
as a polar or polarizer, a name first introduced in 1948 by A. F.
Hallimond. Today, polarizers are widely used in liquid crystal displays
(LCDs), sunglasses, photography, microscopy, and for a myriad of
scientific and medical purposes. Light exiting the port in the microscope
base is first passed through a neutral linear Polaroid HN-type polarizer
to create plane-polarized light having a vibration vector that is confined
to a single plane. Hfilms are produced by stretching a sheet of polyvinyl
alcohol to align the long-chain polymeric molecules, which are
subsequently impregnated with iodine. These films are less effective
polarizing devices than a calcite prism, but do not restrict numerical
aperture. Typically, a pair of crossed polarizing H-films transmits
between 0.01 percent and 40 percent of the incident light, depending
upon the film thickness.
On most microscopes, the polarizer is located either on the light port or
in a filter holder directly beneath the condenser. The microscope has a
rotating polarizer assembly that fits snugly onto the light port in the base.
The polarizer can be rotated through a 360-degree angle and locked into
a single position by means of a small knurled locking screw, but is
generally oriented in an East-West direction by convention. Other
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microscopes typically have the polarizer attached to the substage

condenser assembly housing through a mount that may or may not allow
rotation of the polarizer. Some polarizers are held into place with a detent
that allows rotation in fixed increments of 45 degrees. Polarizers should
be removable from the light path, with a pivot or similar device, to allow
maximum brightfield intensity when the microscope is used in this
mode. Light diffracted, refracted, and transmitted by the specimen
converges at the back focal plane of the objective and is then directed
to an intermediate tube, which houses another polarizer, often termed
the "analyzer". The analyzer is another HN-type neutral linear Polaroid
polarizing filter positioned with the direction of light vibration oriented
at a 90-degree angle with respect to the polarizer beneath the condenser.
By convention, the vibration direction of the polarizer is set to the East
West (abbreviated E-W position), as illustrated in the birefringence
interactive Java tutorial. The same convention dictates that the analyzer
is oriented with the vibration direction in the North-South (abbreviated
N-S) orientation, at a 90-degree angle to the vibration direction of the
polarizer.
The analyzer is positioned after the specimen, either in a slot above the
objective or in an intermediate tube between the nosepiece and the
observation tubes. Older polarized light microscopes may have an
analyzer that is fitted into the eyepiece, either near the eye lens or
somewhere before the intermediate image plane. It is not wise to place
polarizers in a conjugate image plane, because scratches, imperfections,
dirt, and debris on the surface can be imaged along with the specimen.
Simple polarized light microscopes generally have a fixed analyzer, but
more elaborate instruments may have the capability to rotate the
analyzer in a 360-degree rotation about the optical axis and to remove
it from the light path with a slider mechanism. Analyzers of this type
are usually fitted with a scale of degrees and some form of locking
clamp.
Before using a polarized light microscope, the operator should remove
any birefringent specimens from the stage and check to ensure the
polarizer is secured in the standard position (often indicated by a click
stop), and that the light intensity is minimal when the analyzer is set to
the zero mark on the graduated scale. When properly configured, the
vibration direction of the analyzer is North-South when the polarizer
vibration plane is oriented in an East-West direction (this orientation is
now standardized). If the polarizer and analyzer are both capable of
rotation, it is possible that they may be crossed (with light intensity at a

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minimum when minus a specimen) even through their permitted

vibration directions are not East-West and North-South, respectively.
This situation may be rectified by moving the polarizer to its zero degree
click stop (or rotation angle), followed by re-setting the analyzer to this
reference point. It is essential that the polarizer and analyzer have
vibration planes oriented in the proper directions when retardation
and/or compensation plates are inserted into the optical path for
measurement purposes.
In older microscopes that are not equipped with graduated markings for
the polarizer and analyzer positions, it is possible to use the properties
of a known birefringent specimen to adjust the orientation of the
polarizer and analyzer. Recrystallized urea is excellent for this purpose,
because the chemical forms long dendritic crystallites that have
permitted vibration directions that are both parallel and perpendicular to
the long crystal axis. A small quantity (about 5 milligrams) of the
purified chemical can be sandwiched between a microscope slide and
cover glass, then carefully heated with a Bunsen burner or hot plate until
the crystals melt. Once liquefied, the cover glass can be pressed onto
the slide to minimize the thickness of the urea sandwich,

which is then allowed to cool. After recrystallization, the slide is placed

on a polarized light microscope stage and the long axes of the crystals
oriented East-West using the crosshairs in the eyepiece reticle as a
reference. The polarizer and analyzer are then rotated as a pair until both
the crystal and background are equally dark.

Condensers for Polarized Light Microscopy

Basic substage condenser construction in a polarized light microscope is
no different from an ordinary condenser used in brightfield microscopy.
In all forms of microscopy, the degree of condenser optical correction
should be consistent with that of the objectives. Typical laboratory
polarizing microscopes have an achromat, strain-free condenser with a
numerical aperture range between 0.90 and 1.35, and a swing-out lens
element that will provide even illumination at very low (2x to 4x)
magnifications. Removal of the swing lens alters the focal length of the
condenser to enable illumination of a much larger specimen area and to
allow the larger field of view provided by low magnification objectives
to be evenly illuminated. This is ideal for polarized light microscopy
where low magnifications are used to view crystals and other
birefringent materials in the orthoscopic mode.

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When interference patterns are to be studied, the swing lens can quickly
be brought into the optical path and a high numerical aperture objective
selected for use in conoscopic observation. It is important that the
numerical aperture of the condenser is high enough to provide adequate
illumination for viewing conoscopic images. Failure to insert the top
condenser lens when utilizing high magnification objectives will result
in poor illumination conditions and may lead to photomicrographs or
digital images that have an uneven background. Also, because the cone
of illumination and condenser numerical aperture are reduced without
the top lens, resolution of the microscope will be compromised,
resulting in a loss of fine specimen detail.

The condenser aperture diaphragm controls the angle of the illumination

cone that passes through the microscope optical train. Reducing the
opening size of this iris diaphragm decreases the cone angle and
increases the contrast of images observed through the eyepieces. It
should be noted, however, that the condenser aperture diaphragm is not
intended as a mechanism to adjust the intensity of illumination, which
should be controlled by the voltage supplied to the lamp. Some polarized
light microscopes are equipped with a fixed condenser (no swing-lens)
that is designed to provide a compromise between the requirements for
conoscopic and orthoscopic

illumination. Variation in the degree of illumination convergence can be

accomplished by adjusting the condenser aperture diaphragm or by
raising or lowering the condenser (although the latter technique is not
recommended for critical examinations).

Rotating Circular Stages

Early polarized light microscopes utilized fixed stages, with the polarizer
and analyzer mechanically linked to rotate in synchrony around the
optical axis. Although this configuration was cumbersome by today's
standards, it had the advantage of not requiring coincidence between the
stage axis and the optical axis of the microscope. Modern polarized light
microscopes are often equipped with specially designed 360-degree
rotatable circular stages, which ease the task of performing orientation
studies in polarized light. The circular stage features a goniometer
divided into 1-degree increments, and has two verniers (not shown)
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placed 90 degrees apart, with click (detent or pawl) stops positioned at

45-degree steps. Use of a precision ball bearing movement ensures
extremely fine control over the verniers, which allow the microscopist
to read angles of rotation with an accuracy near 0.1 degree. A clamp is
used to secure the stage so specimens can be positioned at a fixed angle
with respect to the polarizer and analyzer.

The most critical aspect of the circular stage alignment on a polarizing

microscope is to ensure that the stage is centered within the view field
and the optical axis of the microscope. This is accomplished with the
two centering knobs located on the front of the stage. The first step in
the alignment process is to center the microscope objectives with respect
to the condenser, the field of view, and the optical axis of the
microscope. A pair of small setscrews in the nose piece of most
research-grade polarizing microscopes allows centering of individual
objectives by means of an Allen wrench. Each objective should be
independently centered to the optical axis, according to the
manufacturer's suggestions, while observing a specimen on the circular
stage. Some microscopes provide for individual objective centration,
while other centration systems operate on the nose piece as a unit. After
the objectives are centered, the stage should be centered in the view field,
which will coincide with the optical axis of the microscope. When the
stage is properly centered, a specific specimen detail placed in the center
of a cross hair reticle should not be displaced more than 0.01 millimeter
from the microscope optical axis after a full 360-degree rotation of the
stage. In general, microscopes are designed to allow adjustment of either
the stage or the objectives to coincide with the optical axis, but not both.
Some designs have objectives that are in fixed position in the nose piece
with an adjustable circular stage, while others lock the stage into position
and allow centration of the objectives. Errors in centration of the
rotating circular stage can lead to aggravation when examining
birefringent specimens with a polarized light microscope. If the center
of stage rotation does not coincide with the center of the field view, a
feature being examined may disappear when the stage is rotated. As
objective magnification increases (leading to a much smaller field of
view), the discrepancy between the field of view center and the axis of
rotation becomes greater. At the highest magnifications (60x and 100x),
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even minute errors in centration can lead to huge differences in

specimen placement as the stage is rotated.

The circular microscope stage contains a pair of spring clips intended to

secure the specimen during observation with the microscope. An
optional mechanical stage intended for use on the circular stage. This
stage is a low-profile model that has a cross-travel motion of about 25
x 25 millimeters, with a graduated vernier to log specific locations on
the specimen. The mechanical stage is fastened to pre-drilled holes on
the circular stage and the specimen is translated with two rack-and-
pinion gear sets controlled by the x- and y translational knobs. Use of a
mechanical stage allows precise positioning of the specimen, but the
protruding translation knobs often interfere with free rotation of
objectives and can even collide with them.

In the past, several manufacturers offered a universal attachment for

circular polarized microscope stages. This accessory allows a mineral
thin section to be secured between two glass hemispheres and rotated
about several axes in order to precisely orient selected grains in the
optical path. The universal stage is employed to observe selected
optical, crystallographic, and textural features that yield clues to the
structure of semi-crystalline specimens. Another stage that is sometimes
of utility in measuring birefringence and refractive index is the spindle
stage adapter, which is also mounted directly onto the circular stage.
Specimen grains are secured to the spindle tip, which is positioned on a
base plate that allows the spindle to pivot around

a horizontal axis while holding the grain immersed in oil between a glass
window and a coverslip. Although these stages are presently difficult to
obtain, they can prove invaluable to quantitative polarized light
microscopy investigations.

Objectives for Polarized Light Microscopy

Optical correction of polarized light objectives can be achromatic, plan

achromatic, or plan fluorite. Apochromatic objectives from older fixed
tube length microscopes should be avoided because it is difficult to
remove all residual stress and strain from the numerous lens elements
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and tight mounts. Recently however, advances in objective design for

infinity-corrected microscopes have yielded high quality strain free
apochromatic objectives that are useful for differential interference
contrast or examination of birefringent specimens with crossed
polarized illumination. The average numerical aperture of 20x and 40x
polarized light objectives is usually 10 to 25 percent higher than those
for ordinary microscopes because observations of conoscopic
interference patterns require high numerical apertures. Objectives
designed for polarized light microscopy must be stress and strain free.
Most manufacturers thoroughly test objectives designed for use on
polarized microscopes, selecting only those that pass the rigorous
tests.

Unwanted birefringence in microscope objectives can arise primarily by

two mechanisms. The first is "natural" birefringence, which is an artifact
of the inherent anisotropic character of glasses, crystals and other
materials used to make the lenses. To circumvent this problem,
manufacturers choose strain-free optical glass or isotropic crystals to
construct lens elements. The second type is "strain" birefringence,
which occurs when multiple lenses are cemented together and mounted
in close proximity with tightly fitting frames. Strain birefringence can
also occur as a result of damage to the objective due to dropping or
rough handling.

Those objectives that pass the stress test are marked P or POL, and are
usually labeled with red engraved letters. Several manufacturers also
use a flat black or dark gray barrel (with or without red letters) for quick
identification of strain-free polarized light objectives. When both the
objectives and the condenser are stress and strain-free, the microscope
view field background appears a deep solid black when observed
through the eyepieces without a specimen between crossed polarizers.
Any stress in these optical components can give rise to an appreciable
degree of anisotropic character, termed internal birefringence. This
results in a contribution to specimen interference effects by the
microscope optical system itself, and can often make interpretation of
images very difficult. Evidence for stress and/or strain in the optical
system can be obtained by the presence a blue, gray, or brownish
background when observing specimens that ordinarily would have a
black background.

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A pair of typical objectives designed exclusively for polarized light

microscopy. The objective barrels are painted flat black and are
decorated with red lettering to indicate specific capabilities of the
objectives and to designate their strain-free condition for polarized light.
Cut-away diagrams of the objectives reveal internal lens elements,
which are corrected for chromatic and spherical aberration. The
objective on the left is a low-power 4x objective designed to view
birefringent specimens at lower magnifications. The front lens element
is larger than the 40x objective on the right because illumination
requirements for the increased field of view enjoyed by lower power
objectives. Polarized light objectives range in magnification from about
2x to 100x, with the most common being 4x, 10x, 20, and 40x, a
selection that serves a majority of purposes for specimen examination in
both orthoscopic and conoscopic modes.

Retardation and Accessory Plates

Almost all polarized light microscopes are equipped with a slot in the
body tube above the nosepiece and between the polarizer and analyzer.
The purpose of this slot is to house an accessory or retardation plate in
a specific orientation with respect to the polarizer and analyzer vibration
directions. Originally, the slot was oriented with its long axis directed
Northeast-southwest as observed from the eyepieces, but more recent
microscopes have the direction changed to Southeast-Northwest. In
older microscopes, the slot dimensions were 10 x 3 millimeters, but the
size has now been standardized (DIN specification) to 20 ~ 6
millimeters. When the accessory/retardation plates are not inserted into
the body tube, a cover is often fitted to prevent dust from entering the
microscope through the slots.
Retardation plates are composed of optically anisotropic quartz, mica, or
gypsum minerals ground to a precise thickness and mounted between
two windows having flat (plane) faces. These plates produce a specific
optical path length difference (OPD) of mutually perpendicular plane-
polarized light waves when inserted diagonally in the microscope
between crossed polarizers. The three most common retardation plates
produce optical path length differences of an entire wavelength (ranging
between 530 and 570 nanometers), a quarter wavelength (137-150
nanometers), or a variable path length obtained by utilizing a wedge-
shaped design that covers a wide spectrum of wavelengths (up to six
orders or about 3000 nanometers).

The quartz wedge is the simplest example of a compensator, which is

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utilized to vary the optical path length difference to match that of the
specimen, either by the degree of insertion into the optical axis or in
some other manner. A whole-wave plate is often referred to as a
sensitive tint or first-order red plate, because it produces the interference
color having a tint similar to the first-order red seen in the Michel-Levy
chart. Older compensators were made by cleaving gypsum to the
appropriate thickness to achieve the first-order red color, and may be
marked gypsum plate, Gips, Gyps, one , or A = 530 nm on the frame
housing. If the plate originated in Germany, it will probably be labeled
Rot I. Quarter wave plates (sometimes referred to as a mica plate) are
usually fashioned from quartz or muscovite crystals sandwiched
between two glass windows, just as the first-order plates. Depending
upon the manufacturer, quarter wave plates may be marked Mica,
Glimmer, 1/4 A, or A = 147 nm. First-order red and quarter wavelength
plates are usually mounted in long rectangular frames that slide the plate
through the compensator slot and into the optical pathway. Late model
microscopes combine these plates into a single framework that has three
openings: one for the firstorder red plate, one for the quarter wave plate,
and a central opening without a plate for use with plane-polarized light
without compensators. In addition, these plate frames have knobs at each
end that are larger than the slot dimensions to ensure the plates cannot
be dropped, borrowed, or stolen.
A primary consideration when using compensation plates is to establish
the direction of the slow permitted vibration vector. By convention, this
direction will be Northeast-southwest, in the image, that is tilted about
the horizontal axis by means of a calibrated micrometer drum to enable
precise measurements of retardation. Twin quartz plates are substituted
for calcite in the Ehringhaus compensator, which operates in a manner
similar to the Berek compensator. The Berek, and Ehringhaus
compensators are standard tools for fiber analysis with polarized light
microscopy.

The Bertrand Lens

Advanced polarized light microscopes are often equipped with a
Bertrand lens (sometimes referred to as an Amici-Bertrand lens)
positioned on a movable sliding or tilting mount that is located between
the analyzer and the eyepieces. In some cases, there is also a provision
for focusing the Bertrand lens. When coupled to the eyepiece, the
Bertrand lens provides a system that focuses on the objective rear focal
plane, allowing the microscopist to observe illumination alignment,
condenser aperture size, and conoscopic polarized light images. In
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Köhler illumination, an image of the lamp filament is formed in the

objective rear focal plane, together with the image of the condenser
aperture, so the Bertrand lens is often utilized to adjusting the
illuminating (condenser) aperture diaphragm for optimum specimen
contrast. The primary function in polarized light microscopy, however,
is to view interference figures (conoscopic images). These images
appear in the objective rear focal plane when an optically anisotropic
specimen is viewed between crossed polarizers using a high numerical
aperture objective/condenser combination.
Microscope Older polarized light microscopes may have a provision for
centration of the Bertrand lens to allow the center of the objective rear
aperture to coincide with the intersection of the eyepiece crosshairs.
Some of the older microscopes also have an iris diaphragm positioned
near the intermediate image plane or Bertrand lens, which can be
adjusted (reduced in size) to improve the clarity of interference figures
obtained from small crystals when the microscope is operated in
conoscopic mode. If the diaphragm is not opened again after conoscopic
observations, the field of view is restricted when the microscope is
returned to orthoscopic viewing mode. This diaphragm, if present, is
operated by a lever or knurled ring mounted either in the microscope
body tube or the viewing head (near or within the intermediate image
plane). Later model microscopes often mount the Bertrand lens in a
turret along with lenses that change the image magnification factor.
Adjustment is made with a small knob that is labeled B or Ph for the
Bertrand lens position, and 0 or some other number for the
magnification lens. A Bertrand lens can also serve as a telescope for
configuring phase contrast objectives by providing a magnified image
of the objective rear focal plane with the phase rings superimposed over
the condenser phase plate annulus.

Eyepieces (Oculars)
Early polarized light microscopes, like their brightfield counterparts,
were often equipped with monocular observation tubes and a single
eyepiece. Coupled to a reflecting substage mirror for illumination, these
microscopes did not provide adequate illumination to visualize and
photograph very weakly birefringent specimens. Although low-cost
student microscopes are still equipped with monocular viewing heads,
a majority of modern research-grade polarized light microscopes have
binocular or trinocular observation tube systems. The eye tubes are
usually adjustable for a range of interocular distances to accommodate
the interpupillary separation of the microscopist (usually between 55
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and 75 millimeters).

Many polarized light microscopes are equipped with an eyepiece diopter

adjustment, which should be made to each of the eyepieces individually.
Some microscopes have a graded scale on each eyepiece that indicates
the position of the eye lens with respect to main body of the eyepiece.
Other models hold the body of the eyepiece in a fixed position securely
in the eye tube with a pin and slot. The first step in diopter adjustment
is to either line up the graded markings on eyepieces equipped with such
markings or turn the eye lenses clockwise to the shortest focal length
position. Next, focus the specimen with the 10x objective and then rotate
the nosepiece until a lower magnification objective (usually the 5x) is
above the specimen. At this point, refocus each eye lens individually
(do not use the microscope coarse or fine focus mechanisms) until the
specimen is in sharp focus. Rotate the 20x objective into the optical path
and refocus the microscope with the fine focus knob. Repeat the diopter
eye lens adjustments with the 5x objective (again not disturbing the
microscope fine focus mechanism), and the microscope should be
adjusted to the correct diopter settings. These settings will vary from
user to user, so record the position of the eye lenses if the eyepiece has
a graded scale for quick return to the proper adjustment.
Best results in polarized light microscopy require that objectives be used
in combination with eyepieces that are appropriate to the optical
correction and type of objective. Microscopes with a fixed tube length
often have eyepieces (termed compensating eyepieces) that help to
correct for chromatic difference of magnification when coupled to
objectives designed specifically for that purpose. Newer microscopes
with infinity-corrected optical systems often correct aberrations in the
objectives themselves or in the tube lens. Inscriptions on the side of the
eyepiece describe its particular characteristics and function, including
the magnification, field number, and whether the eyepiece is designed
for viewing at a high eye point.
Modern microscopes feature vastly improved plan-corrected objectives
in which the primary image has much less curvature of field than older
objectives. In addition, most polarized light microscopes now feature
much wider body tubes that have greatly increased the size of
intermediate images. To address these new features, manufacturers now
produce wide-eyefield eyepieces that increase the viewable area of the
specimen by as much as 40 percent. Because the strategies of eyepiece
objective correction techniques vary from manufacturer to
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manufacturer, it is very important to use only eyepieces recommended

by a specific manufacturer for use with their objectives.

Care should be taken in choosing eyepiece/objective combinations to

ensure the optimal magnification of specimen detail without adding
unnecessary artifacts. For instance, to achieve a magnification of 200x,
the microscopist could choose a 20x eyepiece coupled to a 10x
objective. An alternative choice for the same magnification would be a
10x eyepiece with a 20x objective. Because the 20x objective has a
higher numerical aperture (approximately 0.45 to 0.55) than does the
10x objective (approximately 0.25), and considering that numerical
aperture values define an objective's resolution, it is clear that the latter
choice would be the best. If photomicrographs or digital images of the
same viewfield were made with each objective/eyepiece combination
described above, it would be obvious that the 10x eyepiece/20x
objective duo would produce images that excelled in specimen detail
and clarity when compared to the alternative combination. Housing
Eyepieces designed for polarized light microscopy are usually equipped
with a crosshair reticle (or graticule) that locates the center of the field
of view. A pin or slot system, described above, is often utilized to couple
the eyepiece to a specific orientation in the observation tube so that the
crosshairs may be quickly located and brought into a North-South and
East-West direction with respect to the microscopist's view. These
eyepieces can be adapted for measurement purposes by exchanging the
small circular disk-shaped glass reticle with crosshairs for a reticle
having a measuring rule or grid etched into the surface. Because the
reticle lies in the same plane as specimen and the field diaphragm, it
appears in sharp focus superimposed over the image of the specimen.
Eyepieces using reticles must contain a focusing mechanism (usually a
helical screw or slider) that allows the image of the reticle to be brought
into focus. Each objective must be individually calibrated to the ruled
reticle by comparison with a stage micrometer, which is a microscope
slide containing an etched millimeter scale. The calibration is conducted
by focusing the microscope on the stage micrometer and determining
how many millimeters is represented by each division on the ocular
reticle rule.
Many modern microscopes are designed with inclined observation tubes
in an effort to position the eyepieces at an ergonomically reasonable
height above the laboratory bench. The result is a convenient viewing
angle that allows the stage to remain horizontal, but these designs
require several prisms to be interpolated into the optical path.
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Depending upon the glass utilized in manufacture, the prisms may

produce considerable depolarization effects, which are offset by
inclusion of high-order retardation plates in the observation tube optical
system.

Adjusting the Polarized Light Microscope

Crossing the polarizers in a microscope should be accomplished when

the objectives, condenser, and eyepieces have been removed from the
optical path. If the analyzer is restricted to a fixed position, then it is a
simple matter to rotate the polarizer while peering through the eye tubes
until maximum extinction is achieved. For microscopes equipped with
a rotating analyzer, fixing the polarizer into position, either through a
graduated goniometer or click-stop, allows the operator to rotate the
analyzer until minimum intensity is obtained. If markings are not
provided on either the analyzer or polarizer, the microscopist should
remember that simply crossing the polarizers in order to obtain
minimum intensity in not sufficient. It is necessary to restrict the
permitted vibration directions of the polarizer in the North-South
orientation, and the analyzer in the East-West direction.
As described above, a thin preparation of well-shaped prismatic urea
crystallites can be oriented either North-South or East-West by
reference to the crosshairs in the eyepiece. Then, the polarizers can be
rotated as a pair in order to obtain the minimum intensity of background
and crystal in combination. If both polarizers can be rotated, this
procedure may yield either a North-South or an East-West setting for the
polarizer. The former orientation is preferred because it can be set by
comparison with a polarizer whose vibration direction is known.
The condenser can focused and centered by reducing the size of the
illuminated field diaphragm (located in front of the collector lens), then
translating the condenser so that the image of the diaphragm edge is
sharp when observed through the eyepieces. Next, the field diaphragm
should be centered in the viewfield by using the condenser adjusting
thumbscrews mounted on the substage housing that secures the
condenser. After the diaphragm (and condenser) is centered, the leaves
may be opened until the entire field of view is illuminated.
The lamp filament should be focused into the front focal plane of the
condenser (a requirement of Köhler illumination) by altering the focus
of the collector lens so that the tungsten helices are visible. The
condenser front focal plane lies in or near the plane of the illuminating
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aperture (condenser) diaphragm. Because the rear focal plane of the

objective is in a plane conjugate to the condenser, it is possible to
observe the filament image by removing the eyepiece or inserting the
Bertrand lens.
When viewing interference fringes in conoscopic mode, it is often
convenient to employ a section of opal glass or a frosted filter near the
lamp collector lens in order to diffuse the filament image in the objective
rear focal plane.

In order to match the objective numerical aperture, the condenser

aperture diaphragm must be adjusted while observing the objective rear
focal plane. Again, the Bertrand lens provides a convenient mechanism
of observing the relationship between the condenser illuminating
aperture and the objective aperture. For most studies in polarized light,
the diameter of the condenser aperture should be set to about 90 percent
of the objective numerical aperture.
Although it is not essential, centering the rotating stage is very
convenient if measurements are to be conducted or specimens rotated
through large angles. The simplest method is to locate a small specimen
feature (as a marker) and move the feature into the center of the rotation
axis of the stage. This location may not coincide with the viewfield
center, as defined by the eyepiece crosshairs. Using the centration knobs
or keys near the stage, the marker feature can be translated (through trial
and error) until its center of rotation coincides with the viewfield center.
Some polarized light microscopes allow independent centering of the
objectives in the nosepiece. If so, this task should be accomplished prior
to attempting stage centration.

COMPARISON MICROSCOPE
The comparison microscope is the mainstay of forensic science
allowing two objects or samples to be compared side by side.
While the primary use of this type of instrument is criminology as in
ballistics, other scientific fields including paleontology and archaeology
utilize these special compound microscopes.
Forensic experts used to have this problem as well, prior to the invention
of the comparison microscope, a microscope that allows for the direct
assessment and comparison of two substances in one field of view at the
same time. What is a comparison Microscope? to an optical bridge,
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which results in a split view window. The comparison microscope is

used in forensic sciences to compare microscopic patterns and identify
or deny their common origin. Without this device, the identification of
toolmarks and firearms would be such a cumbersome process that it
would be carried out on a very limited basis. The idea behind the
comparison microscope is simple. Two microscopes are placed next to
each other and the optical paths of each microscope are connected
together by the optical bridge.
The optical bridge consists of a series of lenses and a mirror that brings
the two images back together at the single eyepiece. The user looks
through the eyepiece as with a regular microscope, except that a line in
the middle separates the circular view field into two parts. The left side
of the view field is the image produced by the left microscope, and the
right side of the view field is the image produced by the right
microscope. In some more modern or sophisticated comparison
microscopes, it is also possible to super-impose the view fields
generated by the two microscopes. This is particularly convenient when
the forensic scientist compares impressed patterns rather than striated
patterns.
It is important that the two microscopes are identical. In order for a
comparison to be valid, the two images produced in the circular view
field needs to be at the same magnification and present the same lens
distortion (if any). Comparison microscopes are mostly used in a
reflected light setting, but a transmitted light setting is also available in
some instances, and fluorescent light settings are found on higher-end
models. This allows for comparison of more than just bullets and
toolmarks. Use of a comparison microscope is straightforward. The
incriminated impression, typically a bullet or casing found at a crime
scene or a toolmark's cast from a crime scene, is placed under the left
microscope and thus, appears in the left part of the circular view field.
A comparison impression, such as a bullet fired from a revolver found
on a suspect, is placed under the right microscope and thus, appears in
the right part of the view field. When comparing striations, the forensic
scientist moves the comparison object until the striations match the ones
present on the incriminated object. If the striations do not present
similarities, then the two objects cannot be associated with a common
origin. If the striations match, then a common source between the two
objects is established. When comparing impression marks, the forensic
scientist can use the superimposition option and, again, by moving the
comparison object on the right, try to find common characteristics
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between the two objects. The comparison microscope is used to

compare impression evidence that requires a magnification ranging
from 5x to approximately 100x. Items that are commonly observed
under the comparison microscope are fired bullets, fired casings, and
toolmarks. These items are observed under a reflected light setting.
Other evidence, including impressions of serial numbers or characters
from a typewriter, can also be compared using the comparison
microscope.
These are compared using a reflected light setting. This comparison
might allow for the link between a stamped serial number and a die or
between a sheet of paper bearing characters and the typewriter that was
used to write it. The comparison microscope is also used to compare
layers of a paint chip. This might allow for the identification of the
vehicle from which the paint originated. Finally, when used in a
transmitted light setting, hair, fibers, or the extruding striations of plastic
bags can be compared. This allows the comparison of fibers found on a
seat with the clothing of a suspect, for example.
Plastic bag striations might establish links between different plastic bags
and to demonstrate that they originate from the same batch. This is
particularly useful with the small bags used to sell drugs. When dealing
with fibers and plastic bags, the comparison microscope can also be
used in an ultraviolet light setting or a polarized light setting. The
comparison microscope was invented in the 1920s by American Army
Colonel Calvin Goddard (1891–1955) who was working for the Bureau
of Forensic Ballistics of the City of New York. Goddard also benefited
from the help of Colonel Charles Waite, Philip Gravelle, and John
Fisher. At that time, the comparison microscope was used to compare
fired bullets and casings. In the late 1920s, Swedish criminalist Harry
Söderman (1902–1956) drastically improved the comparison
microscope by inventing a system for rotating the bullets under the
objectives.
This allowed for a much faster comparison of lands of grooves of bullets
by simultaneous rotation of both the suspect and comparison bullets.
Söderman gave the name Hastoscope to his invention.

Comparison Microscope Structure

Before the days of comparison microscopes, forensic experts had a much
tougher time comparing two samples. For example, they would need to
sequentially, as opposed to simultaneously, examine the different
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samples under the same microscope. This would be like looking at one
shirt at the store, putting it away, and then looking at another shirt and
trying to compare the two things.
Forensic examiners were later able to take photographs of one sample
prior to looking at another sample. This would be like comparing a
photograph of a shirt at home to a real 'live' shirt at the store. Also, not
the best thing.
Of course, you could also take a photo of one specimen and then another
and compare the photos side by side at the same time. But who likes to
compare photos side by side? It's not the same 'feel' as comparing the
actual things to one another, right? Also, the photographs are two-
dimensional, the real things are 3-dimensional, and this adds an
important layer of complexity as a result. So that's where comparison
microscopes come in.
A comparison microscope is basically two microscopes connected by
something known as an optical bridge. The optical bridge has mirrors
and prisms that direct the light from each microscope to a common
viewing area and a common set of oculars.
RCM: Radical Forensic Comparison Microscope were developed about
four decades ago in consultation with the leading Scientists and
Criminologists of world repute, thereafter the instruments are being
continually updated. Comparison Microscope are the most advanced &
versatile instruments invented for comparative Micro study in Forensic
Science, geology, Metallurgy, Mineralogy, Crystallography and
Chemical Microscopy. By means of this sophisticated instrument, two
micro samples can be studied under one Eye. It combines the latest opto-
mechanical developments in light microscopy. This instrument consist
of two microscopes & their split images can be viewed simultaneously,
with the image from one being on the left side & the other image on the
right side.

The primary use of these types of dual microscopes is in forensics, a

discipline that uses science and technology to help solve crimes by
providing factual evidence and linking crime scene evidence to a
suspect or location.

Tools create specific marks, bullets have signature marks, hair can be
human or animal and using two compound microscopes to study a
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known sample against a collected piece of evidence can confirm an

objects origin.

Having multiple eyepieces, image magnifications ranging from 6x to

400x depending on the microscope model and image viewing options
including left or right views only, side by side views of both samples
and superimposed viewing places one image on top of the other for
comparison.

Availability

A number of forensic microscope companies use components from the

primary microscope providers, including Leica, Zeiss and Olympus.
Leeds Forensic Systems offers a number of systems that use Olympus
and Zeiss optical components.

• Leica - "motorized comparison macroscope is used for a wide range

of forensic investigations including ballistics, toolmark, and document
comparison. Intelligent Automation combines with outstanding
ergonomic design. The five Apochromatically-corrected macro
objectives feature a high numerical aperture and built-in, adjustable iris
diaphragms for top optical performance. Different imaging modes (split
image, superimposed image and combined image) are set at the press of
a button. This convenient platform provides increased reproducibility,
accuracy, and efficiency."
• FX C is a motorized macroscope that provides detailed study of a
small section of a larger object. Intelligent Automation software
provides a wide range of computerized options including virtual
microscopy. Leeds Forensic Systems - Provides a wide range of
forensic microscopes many utilize Olympus BX microscopic
components that provide ergonomic eyepieces, uniform illumination
and a variety of observation tubes.
• Leeds Firearms and Toolmarks - Microscope systems provide
built in aperture diaphragms, large viewing area and ergonomic
design with single knob focus.

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• With image magnification of 5.5x to 55x as well as 6x to 102x and

16:1 zoom ratio and built-in aperture diaphragms, the Trace
Evidence Comparison Microscope is a modular unit that allows it to
be utilized by all forensic aspects of comparison microscopy.
• Some units provide a 22mm view and are capable of many
contrasting options including bright and dark fields, phase contrast,
polarized light and fluorescence. Dual view components allow
systems to be used as a training microscope or for multiple-person
viewing.
• Ken-a-Vision - (see below) Provides a cordless teaching comparison
instrument that can be used for macro and micro samples. View
options include independent and dual side viewing with illumination
from the top or bottom. A full charge allows this teaching forensic
instrument to be in use without power for forty hours.
• Forensic Comparison Microscopes - Offers three comparison
instruments specifically designed for use in the field of criminal
investigation. Their 10x by 144x magnification unit is portable and
includes a USB digital camera system with ports for still photo or
videotaping.

The 40x by 600x forensic unit offers multiple illumination options

including light direction allowing this unit to be used for
metallurgical and epi-illumination applications. The 40x by 1000x
compound unit includes both CCD video and USB still photo
camera, unique screen options permit image reversal plus left and
right side image views at 100%. This unit has a clean thin-line screen
division allowing better comparison of samples without
compromising clarity.
• GX Microscopes: Units feature integrated micro and macro systems
that includes two tilt-able stages, vernier controls and comes with
multiple eyepieces and objective lenses. Accessories for units may
include digital camera systems, and holding brackets for all types of
items including bullets and cartridges that can be mounted to the
microscope.
DISSECTING MICROSCOPE (STEREO OR STEREOSCOPIC
MICROSCOPE)

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These are also known as stereoscopic microscopes. This is a type of

digital optical microscope designed with a low magnification power
(5x-250x), by use of light reflected from the surface of the specimen,
and not the light reflected the specimen. Its primary role is for dissection
of specimens and viewing and qualitatively analyzing the dissected
samples.
It was first designed by Cherudin d'Orleans in 1677 by making a small
microscope with two separate eyepieces and objective lenses. Later in
1852, an inventor named Charles Wheatstone describe the principle of
the stereoscopic visualization and published it with the title 'On Some
Remarkable, and Hitherto Unobserved, Phenomena of Binocular
Vision'. It was later advanced by John Leornld Riddell who equally
published it on the Journal of Microscopical Science as 'On Binocular
Microscope'. Horatio S. Greenough, an American Biologist developed
the stereo Microscope, with two separate but identical optical paths and
manufactured it with the Carl Zeiss Company, to which the scope of the
Dissecting Microscope is built to date.

Principle of Dissecting microscope (Stereo microscope)

▪ The working principle of the dissecting Microscope depends on
the two types of light paths used by the microscope's objectives
and eyepiece. Each light path provides a different angle of
viewing. They have the top light which is used while dissecting
and the bottom light that is used to view the images.
▪ This lighting is enabled by the construction of two eyepieces
(binocular stereoscope) each showing a different type of light
pathway, each providing a viewing comfort area.
▪ Being a digital microscope, the images are viewed live on a
computer monitor screen in 3dimensional visuals. They also offer
a very close observation of small specimens such as insects where
the image produced is normally larger than the sample size, an
effect known as macrophotography. The image is recorded and in
complex samples, the topography (surface) is analyzed in 3D.
▪ The dissecting microscope works with two magnification
systems: Fixed (primary) magnification where two objective
lenses provide a degree of magnification and the Zoom
(pancratic) magnification which offers a continuous
magnification at variant ranges, using the auxiliary objectives
whose function is to increase total magnification depending on
some factors. The variance between the zoom and the fixed
magnification can be achieved by changing the eyepiece lenses.
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Between the fixed and the zoom magnification is an optical

system known as the Galilean optical system which has fixed-
focus lenses that confer fixed magnification for different sets of
1. Foot or Base: It is the basal, horse-shoe shaped or circular part of
dissecting microscope. It is made of heavy material. It provides support
to other parts of microscope.
2. Stand: It is short but strong, hollow cylindrical rod. Its one end is
fixed provides support to the mirror, adjustment screw and other parts.
3. Folder Arm: It is a horizontal arm. Its one end is attached with the
vertical limb and on it's another end is attached lens. Folded arm is
movable. It can be moved up and down as well as left and right.
4. LED illuminators- For some of the dissecting Microscopes, they
have an inbuilt LED illuminator as a source of light.

5. Eyepieces- They have two eyepieces each focusing different

pathways of the light into and out of the specimen, each with its own
magnification power. To increase the magnification, the use of auxiliary
eyepieces can be used.
6. Objective lenses- They also have different magnifications, focusing
the image on the digital camera, and to improve magnification, the
addition of auxiliary objectives is used. It is a simple convex lens of
either 2x, 3X, 5X, 10X or 20x magnification.
7. Stage- This is the apparatus for placing the specimen, they are large
hence they can hold large specimen apparatus. It is rectangular glass
plate attached to the upper end of the stand or limb. Slide or the object,
to be observed, is kept on the stage.
8. Optical system- This is the system that is found between the fixed
and the zoom magnification which offers fixed-focus lensing.
9. Digital camera- It is temporarily fixed for most dissecting
microscopes for capturing images and recording the outcome of the
images. It captures 2D and 3D images depending on the type of digital
camera that is installed.
WORKING:
Place the slide or the object, to be observed, on the stage. Bring the lens
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over the slide with the help of folded arm. Put the clips on two ends of
the slide to keep it in position. Move the vertical limb up and down by
adjustment screw to bring the slide into desired focus. Adjust the light
over the slide with the help of the mirror and observe. In case some
material is to be dissected, place the material over the slide, repeat the
entire process mentioned above, dissect the material with the help of
needles or other instruments, and observe. .
UTILITY:
Dissecting microscope is used to dissect small organisms or organs, e.g.,
embryo dissection. Its special utility is to observe such materials where
high magnification is not needed.

TYPES OF DISSECTING MICROSCOPES (STEREO MICROSCOPE)

1. Stereo Zoom Dissecting Microscope- They are trinocular or
binocular dissecting microscopes with a zooming range of 6.7x-45x.
They can be attached to the digital camera which takes photos of the
viewing images. They have a dual-LED illuminator and they can rotate
at 360o. Magnification can be changed by adding auxiliary objectives
or different eyepieces.
2. Digital Tablet Dissection Microscope- These are high-end dissecting
microscopes. They have a touch screen LCD tablet camera with a
continuous magnification of 6.7x-45x. They have auxiliary eyepieces
whose magnification ranges can be increased or decreased. Auxiliary
lenses can also be added to the objectives for modifying the
magnification. They have a 5.0-megapixel digital camera for capturing
images and recording videos directly into the tablet or a USB cable.
They have an in-built LED illuminators both at the top and bottom of the
microscope, each operating separately.
3. Stereo zoom boom stand microscopes- The have a large base and
the largest stage for viewing large samples. They have LED lighting or
optional dual-pipe lighting. They have a zooming range of 6x-45x which
can be altered upward by adding auxiliary lenses or eyepieces.
4. Stereo Zoom Dissecting Microscope- this is a compact stereo zoom
dissecting Microscope with a zooming range of 10x-30x producing
sharp parfocalled images. They have a rotating head hence the eyepiece
can be positioned away and toward the specimen, they have a halogen
lamp of 10 watts and fluorescent lighting of 5 watts.

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5. Dual Power Dissecting Microscope- It has a dual-powered

dissecting microscope of 10x and 30x, with a 360° rotation ability, for
focussing and viewing. They have a dual objective pair, parfocalled,
parcentered, and achromatic. Rotating the lenses allows a change of
image magnification. It also uses a high LED intensity light ring which
fills the surface with light. With a flexible stand, it can be lifted head
high for viewing larger specimens.
6. Single Power Stereo Dissection Microscope- they have very low
magnification power ranging from 10X-40x with inclined eyepieces at
45°C. they also have diopter adjustments of 50mm to 70mm.
7. Single Magnification Handheld Pocket Microscope - This is a
single powered handheld dissecting microscope with two magnification
powers and it does not require light. Manufactured in Japan, it has high
optical quality glass with a lot of ease in useability and its compact size
makes it portable.

APPLICATIONS OF DISSECTING MICROSCOPE (STEREO

MICROSCOPE)
Like most microscopes, it is used in a wide range of fields including
manufacturing, medical, quality control, inspection and biomedical
studies like the entomological study of insects and some of its functions
include:

1. Studying the topography of solid samples

2. For dissection .
3. For microsurgical procedures
4. For the manufacturing of watches, circuit boards, and their
inspections

5. Used for inspection of fractures (fractography)

6. Used in forensic engineering

ADVANTAGES
1. It can be used in a wide range of fields making it one of the most
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important microscopic techniques.

2. The use of two light pathways offers great magnification differences
for visualizing the image.
3. The attachment of a digital camera allows recording and imaging the
image produced.

4. It is easily portable and easy to use.

5. They are used to view whole specimens and not in pieces.
DISADVANTAGES
1. The Galilean Optical Systems which are an essential part of the
microscope are quite expensive to purchase.

2. Generally, the microscope is costly to purchase.

3. They have a low magnification power hence they are not able to view
images of high magnification, above 100x hence they can't be used to
view tissue structures and other structures.

FLUORESCENCE MICROSCOPY
Fluorescence microscopy is a very powerful analytical tool that
combines the magnifying properties of light microscopy with
visualization of fluorescence. Fluorescence is a phenomenon that
involves absorbance and emission of a small range of light wavelengths
by a fluorescent molecule known as a fluorophore. Fluorescence
microscopy is accomplished in conjunction with the basic light
microscope by the addition of a powerful light source, specialized filters,
and a means of fluorescently labeling a sample. This video describes the
basic principles behind fluorescence microscopy including the
mechanism of fluorescence, the Stoke's shift, and photobleaching. It
also gives examples of the numerous ways to fluorescently label a
sample including the use of fluorescently tagged antibodies and
proteins, nucleic acid fluorescent dyes with, and the addition of naturally
fluorescent proteins to a specimen. The major components of the
fluorescence microscope including a xenon or mercury light source,
light filters, the dichroic mirror, and use of the shutter to illuminate the
sample are all described.
A fluorescence microscope is an optical microscope that uses
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fluorescence and phosphorescence instead of, or in addition to, reflection

and absorption to study properties of organic or inorganic substances.
Fluorescence is the emission of light by a substance that has absorbed
light or other electromagnetic radiation while phosphorescence is a
specific type of photoluminescence related to fluorescence. Unlike
fluorescence, a phosphorescent material does not immediately re-emit
the radiation it absorbs. The fluorescence microscope was devised in the
early part of the twentieth century by August Köhler, Carl Reichert, and
Heinrich Lehmann, among others.

PRINCIPLE OF FLUORESCENCE MICROSCOPY

1. Most cellular components are colorless and cannot be clearly
distinguished under a microscope. The basic premise of fluorescence
microscopy is to stain the components with dyes.
2. Fluorescent dyes, also known as fluorophores or fluorochromes, are
molecules that absorb excitation light at a given wavelength (generally
UV), and after a short delay emit light at a longer wavelength. The delay
between absorption and emission is negligible, generally on the order
of nanoseconds.
3. The emission light can then be filtered from the excitation light to
reveal the location of the fluorophores.
Fluorescence microscopy uses a much higher intensity light to
illuminate the sample.
This light excites fluorescence species in the sample, which then emit
light of a longer wavelength. The image produced is based on the second
light source or the emission wavelength of the fluorescent species rather
than from the light originally used to illuminate, and excite, the sample.
Working Light of the excitation

wavelength is focused on the specimen through the objective lens. The

fluorescence emitted by the specimen is focused on the detector by the
objective. Since most of the excitation light is transmitted through the
specimen, only reflected excitatory light reaches the objective together
with the emitted light.
Forms The "fluorescence microscope" refers to any microscope that uses
fluorescence to generate an image, whether it is a more simple set up
like an epifluorescence microscope, or a more complicated design such
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as a confocal microscope, which uses optical sectioning to get better

resolution of the fluorescent image. Most fluorescence microscopes in
use are epifluorescence microscopes, where excitation of the
fluorophore and detection of the fluorescence are done through the same
light path (i.e. through the objective).

Typical components of a fluorescence microscope are:

• Fluorescent dyes (Fluorophore)

• A fluorophore is a fluorescent chemical compound that can re-emit light upon
light.
• Fluorophores typically contain several combined aromatic groups, or
plane or cyclic molecules with several n bonds. Many fluorescent stains
have been designed for a range of biological molecules. Some of these
are small molecules that are intrinsically fluorescent and bind a
biological molecule of interest.
Major examples of these are nucleic acid stains like DAPI and Hoechst,
phalloidin which is used to stain actin fibers in mammalian cells.
• A light source Four main types of light sources are used, including
xenon arc lamps or mercury-vapor lamps with an excitation filter, lasers,
and high-power LEDs.
• Lasers are mostly used for complex fluorescence microscopy
techniques, while xenon lamps, and mercury lamps, and LEDs with a
dichroic excitation filter are commonly used for wide-field
epifluorescence microscopes.

• The excitation filter

• The exciter is typically a bandpass filter that passes only the
wavelengths absorbed by the fluorophore, thus minimizing the
excitation of other sources of fluorescence and blocking excitation light
in the fluorescence emission band.

• The dichroic mirror

• A dichroic filter or thin-film filter is a very accurate color filter used to

selectively pass light of a small range of colors while reflecting other
colors.

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• The emission filter. The emitter is typically a bandpass filter that passes
only the wavelengths emitted by the fluorophore and blocks all
undesired light outside this band - especially the excitation light. By
blocking unwanted excitation energy (including UV and IR) or sample
and system autofluorescence, optical filters ensure the darkest
background. Applications of Fluorescence Microscope

To identify structures in fixed and live biological samples.

• Fluorescence microscopy is a common tool for today's life science
research because it allows the use of multicolor staining, labeling of
structures within cells, and the measurement of the physiological
state of a cell.
• Working Principle of Fluorescence Microscope Many substances
absorb light. However some of them, after absorbing light of a
particular wavelength and energy, emit light of a longer wavelength
and lesser energy. Such substances are called 'fluorescent
substances'.
Application of this phenomenon is the basis of fluorescence microscope.
In practice, microbes are stained with a fluorescent dye and then
illuminated with blue light. The dye absorbs blue light (shorter
wavelength) and emits green light (longer wavelength). In a
fluorescence microscope, a high intensity mercury arc lamp is used as
the light source.
It emits white light, which is passed through an 'exciter filter'. It allows
only the blue component of the white light (the white light consists of
seven colors, which in the decreasing order of wavelength are violet,
indigo, blue, green, yellow, orange and red) to pass through it and blocks
out all other color components.
A dichroic mirror, which reflects blue light, but allows green light is
used on the path of the blue light. The mirror is fixed at such an angle
that the blue light is reflected downward to the specimen. The specimen
is previously stained with a fluorescent dye, such as acridine orange
NO, acridine yellow, acriflavine, thioflavine S, thioflavine T or titan
yellow G. Certain portions of the specimen retain the dye, while others
do not. The portions, which retain the fluorescent dye, absorb blue light
and emit green light. The emitted green light goes upward and passes
through the dichroic mirror.

It reflects back blue light, if any, and allows only green light to pass
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through. Then, the light reaches a 'barrier filter'. It allows green light to
pass to eye and blocks out any residual blue light from the specimen,
which might not have been completely reflected by the dichroic mirror.
Thus, the eye perceives the stained portions of the specimen as glowing
green object against a jetblack background, whereas the unstained
portions of the specimen remain invisible.

ADVANTAGES
1. Fluorescence microscopy is the most popular method for studying the
dynamic behavior exhibited in live-cell imaging.
2. This stems from its ability to isolate individual proteins with a high
degree of specificity amidst non-fluorescing material.
3. The sensitivity is high enough to detect as few as 50 molecules per
cubic micrometer.
4. Different molecules can now be stained with different colors, allowing
multiple types of the molecule to be tracked simultaneously.
5. These factors combine to give fluorescence microscopy a clear
advantage over other optical imaging techniques, for both in vitro and
in vivo imaging.
LIMITATIONS

Fluorophores lose their ability to fluoresce as they are illuminated in a

process called photobleaching. Photobleaching occurs as the fluorescent
molecules accumulate chemical damage from the electrons excited
during fluorescence. Cells are susceptible to phototoxicity, particularly
with short wavelength light. Furthermore, fluorescent molecules have a
tendency to generate reactive chemical species when under illumination
which enhances the phototoxic effect. Unlike transmitted and reflected
light microscopy techniques fluorescence microscopy only allows
observation of the specific structures which have been labeled for
fluorescence.

ELECTRON MICROSCOPE
1. The electron microscope is an instrument which utilizes the short
wavelength of an electron beam, rather than light waves, to obtain very
high magnification and resolution of minute structures for which a light
microscope is inadequate. It contains an electric gun whose beam is
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refracted and focused onto a specimen by an electron lens system. The

image of the specimen is magnified and projected onto a stage or
fluorescent screen.
2. On the basis of several earlier researches in Physics, Knoll and Ruska
(1931) of Berlin were the first to develop an electron microscope. With
the help of their electron microscope, they could magnify objects up to
12,000 times. By an Improved type of electron microscope developed
by Borries and Ruska (1938), they could obtain pictures of 20,000 times
magnification. In this die magnification of objective coil was 100 and
the projector coil was 200. The resolution of this microscope was 100Ă.
The electron microscopes with 4-1 OÅ or even better resolution are now
available.
3. A magnification as high as 1, 60,000 times can be achieved by vising
intermediate coils between the objective and projector coils of the
electron microscope.
An electron microscope is a microscope that uses a beam of accelerated
electrons as a source of illumination.
It is a special type of microscope having a high resolution of images,
able to magnify objects in nanometers, which are formed by controlled
use of electrons in vacuum captured on a phosphorescent screen. Ernst
Ruska (1906-1988), a German engineer and academic professor, built
the first Electron Microscope in 1931, and the same principles behind
his prototype still govern modern EMs.
WORKING PRINCIPLE
Electron microscopes use signals arising from the interaction of an
electron beam with the sample to obtain information about structure,
morphology, and composition.

1. The electron gun generates electrons.

2. Two sets of condenser lenses focus the electron beam on the specimen
and then into a thin tight beam.
3. To move electrons down the column, an accelerating voltage (mostly
between 100 kV-1000 kV) is applied between tungsten filament and
anode.
4. The specimen to be examined is made extremely thin, at least 200
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times thinner than those used in the optical microscope. Ultra-thin

sections of 20-100 nm are cut which is already placed on the specimen
holder.

5. The electronic beam passes through the specimen and electrons are
scattered depending upon the thickness or refractive index of different
parts of the specimen.

6. The denser regions in the specimen scatter more electrons and

therefore appear darker in the image since fewer electrons strike that
area of the screen. In contrast, transparent regions are brighter.
7. The electron beam coming out of the specimen passes to the objective
lens, which has high power and forms the intermediate magnified
image.
8. The ocular lenses then produce the final further magnified image.
Construction An electron microscope consists of an electric gun,
microscope column, electromagnetic coils, a fluorescent screen and
some other accessories described below:
(a) The electron gun is located at the top of the body of microscope. It is
the source of electrons. It is made up of a tungsten filament surrounded
by a negatively biased shield with an aperture. The electron beam is
drawn off through this aperture.
(b) The microscope column or central column is made up of an
evacuated metal tube. It protects the person operating the microscope
from X-rays that are generated when the electrons strike the surface of
the metal tube.
(c) The electromagnetic coils or lenses include projector coils, objective
and condenser. In each coil, the coils of electric wire are wound on a
hollow metallic cylinder. The magnetic field, produced by passing the
electric current through the magnetic coil, functions as a magnifying
lens.
(d) The fluorescent screen is used for observing the magnified image of
the object. It remains coated with a chemical which, on being excited,
forms the image as on the screen of television.
(e) Some other essential accessories of the electron microscope Include
high voltage transformers (for developing high voltage current for the
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electron gun and electromagnetic coils), vacuum pumps (for

maintaining high vacuum inside the microscope column), a water
cooling system (for prevention from overheating of various parts), a
circulating pump, a refrigeration plant and also' a filter system.
microscope All these parts require elaborate arrangements and
contribute to the massive size of the electron microscope.
(f) The image formation in this microscope occurs by the scattering of
electrons. The electrons strike the atomic nuclei and get dispersed.
These dispersed electrons form the electron image. By projecting on a
fluorescent screen or photographic film, this electron image is converted
into a visible image of the object.

(g) The electron beam in this microscope is made by accelerating

electrons through a potential difference of from 1-1500kV in an electron
gun.
(h) Only dried specimens are studied by electron microscope. Living
cells cannot be studied with this microscope because they possess water
which causes large scale scattering of electrons.
(i) Ultra thin sections (10-50 nm thickness), which are more than 200
times thinner than those routinely used for light microscopy, are cut for
electron microscopy. These are cut with the help of diamond or glass
knives of an ultra-microtome.
PARTS OF ELECTRON MICROSCOPE

EM is in the form of a tall vacuum column which is vertically mounted.

It has the following components:

1. Electron gun
• The electron gun is a heated tungsten filament, which
generates electrons.

2. Electromagnetic lenses

• Condenser lens focuses the electron beam on the specimen. A second

condenser lens forms the electrons into a thin tight beam. The electron
beam coming out of the specimen passes down the second of magnetic
coils called the objective lens, which has high power and forms the

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intermediate magnified image. The third set of magnetic lenses called

projector (ocular) lenses produce the final further magnified image.
Each of these lenses acts as an image magnifier all the while maintaining
an incredible level of detail and resolution.

3. Specimen Holder
• The specimen holder is an extremely thin film of carbon or collodion
held by a metal grid.

4. Image viewing and Recording System.

• The final image is projected on a fluorescent screen.
• Below the fluorescent screen is a camera for recording the image.
Applications Electron microscopes are used to investigate the
ultrastructure of a wide range of biological and inorganic specimens
including microorganisms, cells, large molecules, biopsy samples,
metals, and crystals. Industrially, electron microscopes are often used
for quality control and failure analysis.
• Modern electron microscopes produce electron micrographs using
specialized digital cameras and frame grabbers to capture the images.
Science of microbiology owes its development to the electron
microscope. Study of microorganisms like bacteria, virus and other
pathogens have made the treatment of diseases very effective.

ADVANTAGES
• Very high magnification
• Incredibly high resolution
• Material rarely distorted by preparation
• It is possible to investigate a greater depth of field

• Diverse applications Limitations

• The live specimen cannot be observed.
• As the penetration power of the electron beam is very low, the object
should be ultra-thin. For this, the specimen is dried and cut into ultra-
thin sections before observation.

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• As the EM works in a vacuum, the specimen should be

completely dry.

• Expensive to build and maintain

• Requiring researcher training Image artifacts resulting from specimen

preparation.
• This type of microscope is a large, cumbersome extremely sensitive to
vibration and external magnetic fields. Some differences between light
microscope and electron microscope:

LIGHT MICROSCOPE
1. Visible light is used in this microscope.

2. Source of illumination is situated at the bottom.

3. For magnification in this microscope the lens system consists of

glass lenses.

4. The lenses arc ocular, objective and condenser.

5. The image is either seen with the eye or recorded on a photographic

film with a camera in this microscope.

ELECTRON MICROSCOPE
1. Electrons are used in electron microscope.
2. Source of illumination is situated at the top in this
microscope.

3. The lens system consists of electromagnetic coils in this

microscope.

4. This microscope has projector coils, an objective and a

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condenser.

5. The image in an electron microscope is either recorded on a

fluorescent screen or recorded on a photographic film.

TYPES OF ELECTRON MICROSCOPE

There are two types of electron microscopes, with different operating styles:
1. THE TRANSMISSION ELECTRON MICROSCOPE (TEM)
Parts of Transmission Electron Microscope (TEM) Their working
mechanism is enabled by the high-resolution power they produce which
allows it to be used in a wide variety of fields. It has three working parts
which include:

1. Electron gun

2. Image producing system

3. Image recording system

1. Electron gun
• This is the part of the Transmission Electron Microscope responsible
for producing electron beams. Electrons are produced by a cathode that
is a tungsten filament that is V-shaped and it is normally heated. The
tungsten filament is covered by a control grid known as a Wehnelt
cylinder made up of a central hole which lies columnar to the tube. The
cathode lies on top of or below the cylindrical column hole. The cathode
and the control grid are negatively charged with an end of the anode
which is disk-shaped that also has an axial hole. When electrons are
transmitted from the cathode, they pass through the columnar aperture
(hole) to the anode at high voltage with constant energy, which is
efficient for focusing the specimen to produce an accurately defined
image.
• It also has the condenser lens system which works to focus the electron
beam on the specimen by controlling the energy intensity and the
column hole of the electron gun. The TEM uses two condenser lenses
to converge the beam of electrons to the specimen. The two condenser
lens each function to produce an image i.e the first lens which has strong
magnification, produces a smaller image of the specimen, to the second
condenser lens, directing the image to the objectives.

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2. Image- Producing system

• Its made up of the objective lens, a movable stage or holding the
specimen, intermediate and projector lenses. They function by focusing
the passing electrons through the specimen forming a highly magnified
image.
• The objective has a short focal length of about 1-5mm and it produces
an intermediate image from the condenser which are transmitted to the
projector lenses for magnification.
• The projector lenses are of two types, i.e., the intermediate lens which
allows great magnification of the image and the projector lens which
gives a generally greater magnification over the intermediate lens.
• To produce efficient high standard images, the objectives and the
projector lenses need high power supplies with high stability for the
highest standard of resolution. 3. Image-Recording System Its made up
of the fluorescent screen used to view and to focus on the image.
• They also have a digital camera that permanently records the images
captured after viewing. They have a vacuum system that prevents the
bombardment or collision of electrons with air molecules disrupting
their movement and ability to focus.
• A vacuumed system facilitates the straight movement of electrons to
the image. The vacuumed system is made up of a pump, gauge, valves
and a power supply. The image that is formed is called a monochromatic
image, which is greyish or black and white.

• The image must be visible to the human eye, and therefore, the
electrons are allowed to pass through a fluorescent screen fixed at the
base of the microscope. The image can also be captured digitally and
displayed on a computer and stored in a JPEG or TIFF format.
• During the storage, the image can be manipulated from its
monochromatic state to a colored image depending on the recording
apparatus e.g., use of pixel cameras can store the image in color. The
presence of colored images allows easy visualization, identification, and
characterization of the images.
How does a Transmission Electron Microscope (TEM) work?
From the instrumentation described, the working mechanism is a
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sequential process of the parts of the TEM mentioned above. To mean:

• A heated tungsten filament in the electron gun produces electrons that
get focus on the specimen by the condenser lenses.
• Magnetic lenses are used to focus the beam of electrons of the
specimen. By the assistance offered by the column tube of the condenser
lens into the vacuum creating a clear image, the vacuum allows
electrons to produce a clear image without collision with any air
molecules which may deflect them.
• On reaching the specimen, the specimen scatters the electrons focusing
them on the magnetic lenses forming a large clear image, and if it passes
through a fluorescent screen it forms a polychromatic image.
• The denser the specimen, the more the electrons are scattered forming
a darker image because fewer electron reaches the screen for
visualization while thinner, more transparent specimens appear brighter.

NOTE:
If the screen is moved aside, a photographic image can be captured in
pixels forming a permanent image. Preparation of specimen for
visualization by TEM The specimen to be viewed under the TEM must
undergo a special preparation technique to enable visualization and
creation of a clear image.
Electrons are easily absorbed and easily scattered on solid elements,
showing poor visualization for thick specimens. And therefore, very
thin specimens are used for accurate and clear visualization forming a
clear image as well. The specimen should be about 20-100nm thin and
0.025-0.1nm diameter, as small as that of a bacterial cell. Thin
specimens allow interaction with electrons in a vacuumed space, are able
to maintain their innate structure.
To get thin slice specimens, the specimen is first fixed on a plastic
material with glutaraldehyde or osmium tetraoxide. These chemical
agents stabilize the structure of the cell and maintain its originality. The
addition of an organic solvent like alcohol such as ethanol will
dehydrate the cell completely for embedding the specimen to the
plastics.
• The specimen is then permeated by adding an unpolymerized liquid
epoxy plastic making it hardened like a solid block. This is where thin
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sections are cut from using a glass knife with a piece of special
equipment known as an ultramicrotome. The specimen is then stained
appropriately (with the appropriate stain) for the uniform scattering of
electrons. The thin sections are then soaked in heavy metallic elements
such as lead citrate and uranyl acetate allowing the lean and aluminum
ions to bind to the cell structures. This forms an opaque layer against
the electrons on the cell structures to increase contrast. The stained thin
sections are then mounted on copper grids for viewing. The primary
staining techniques that are applied for viewing under the TEM is
Negative staining coupled with heavy metallic elements coating. The
metallic coating scatters electrons which appears on the photographic
film while uncoated sections and used to study bacterial, viral cell
morphologies and structures.
Freeze-itching treatment
To reduce the possible dangers of artifacts, freeze-itching is used
especially for the treatment of microbial cells, unlike chemical fixation,
dehydration, and embedding, where most specimens get contaminated.
Microbial cell organelles undergo special treatment known as Freeze-
itching whereby the specimens are prepared with liquid nitrogen and
then warmed at -100°C in a vacuum chamber.
• The sections are then cut with a precooled knife in liquid nitrogen at -
196°C. After warming up the sectioned specimen in a high vacuum for
about 2 minutes, it can then coated ith platinum and carbon layer
forming replicas. These are then be viewed under the TEM displaying
more detailed internal structures of the cell in 3D.
This step of treatment with Liquid nitrogen is known as freeze-itching.
Applications of Transmission Electron Microscope (TEM) TEM is used
in a wide variety of fields From Biology, Microbiology,
Nanotechnology, forensic studies, etc. Some of these applications
include:

1. To visualize and study cell structures of bacteria, viruses, and fungi

2. To view bacteria flagella and plasmids

3. To view the shapes and sizes of microbial cell organelles

4. To study and differentiate between plant and animal cells.

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5. Its also used in nanotechnology to study nanoparticles such as ZnO

nanoparticles
6. It is used to detect and identify fractures, damaged microparticles
which further enable repair mechanisms of the particles. Advantages of
Transmission Electron

ADVANTAGES
1. It has a very powerful magnification of about 2 million times that of
the Light microscope.
2. It can be used for a variety of applications ranging from basic Biology
to Nanotechnology, to education and industrial uses.

3. It can be used to acquire vast information on compounds and their

structures.

4. It produces very efficient, high-quality images with high clarity.

5. It can produce permanent images.

6. It is easy to train and use the Transmission Electron Microscope

LIMITATIONS

1. Generally, the TEMs are very expensive to purchase

2. They are very big to handle.

3. The preparation of specimens to be viewed under the TEM is very tedious.
4. The use of chemical fixations, dehydrators, and embedments can
cause the dangers of artifacts.

5. They are laborious to maintain.

6. It requires a constant inflow of voltage to operate.

7. They are extremely sensitive to vibrations and electro-magnetic
movements hence they are used in isolated areas, where they are not
exposed.

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8. It produces monochromatic images, unless they use a fluorescent

screen at the end of visualization.

2. THE SCANNING ELECTRON MICROSCOPE (SEM)

The first Scanning Electron Microscope was initially made by Mafred
von Ardenne in 1937 with an aim to surpass the transmission electron
Microscope. He used high resolution power to scan a small raster using
a beam of electrons that were focused on the raster. He also aimed at
reducing the problems of chromatic aberrations images produced by the
Transmission electron Microscopes. More studies followed by scientists
and research institutions such as Cambridge Scientific Instrument
Company who eventually developed a fully constructed Scanning
electron Microscope, in 1965 and named it a Stereoscan. The price of
the Scanning Electron Microscope (SEM) is approximately $1 million.
Scanning Electron Microscope (SEM) is a type of electron microscope
that scans surfaces of microorganisms that uses a beam of electrons
moving at low energy to focus and scan specimens. The development
of electron microscopes was due to the inefficiency of the wavelength
of the light microscopes, electron microscopes have vert short
wavelengths in comparison to the light microscope which enables better
resolution power.
• Conventional scanning electron microscopy depends on the emission
of secondary electrons from the surface of a specimen. Because of its
great depth of focus, a scanning electron microscope is the EM analog
of a stereo light microscope. It provides detailed images of the surfaces
of cells and whole organisms that are not possible by TEM. It can also
be used for particle counting and size determination, and for process
control. It is termed a scanning electron microscope because the image
is formed by scanning a focused electron beam onto the surface of the
specimen in a raster pattern.

The Principle of the Scanning Electron Microscope

Unlike the Transmission Electron Microscope which uses transmitted
electrons, the scanning electron Microscope used emitted electrons. The
Scanning electron microscope works on the principle of applying
kinetic energy to produce signals on the interaction of the electrons.
These electrons are secondary electrons, backscattered electrons and
diffracted backscattered electrons which are used to view crystallized
elements and photons. Secondary and backscattered electrons are used
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to produce an image. The secondary electrons are emitted from the

specimen play the primary role of detecting the morphology and
topography of the specimen while the backscattered electrons show
contrast in the composition of the elements of the specimen.
How does the Scanning Electron Microscope (SEM) work?

These electrons are secondary electrons, backscattered electrons and

diffracted backscattered electrons which are used to view crystallized
elements and photons. Secondary and backscattered electrons are used
to produce an image. The secondary electrons are emitted from the
specimen play the primary role of detecting the morphology and
topography of the specimen while the backscattered electrons show
contrast in the composition of the elements of the specimen.
Vacuum pump electron detector
The source of the electrons and the electromagnetic lenses are from
tungsten filament lamps that are placed at the top of the column and it
is similar to those of the transmission electron Microscope. The electrons
are emitted after thermal energy is applied to the electron source and
allowed to move in a fast motion to the anode, which has a positive
charge. The beam of electrons activates the emission of primary
scattered (Primary) electrons at high energy levels and secondary
electrons at low-energy levels from the specimen surface.
The beam of electrons interacts with the specimen to produce signals
that give information about the surface topography and composition of
the specimen. The specimen does not need special treatment for
visualization under the SEM, even air dried samples can be examined
directly. However, microbial specimens need fixation, dehydration, and
drying in order to maintain the structural features of the cells and to
prevent collapsing of the cells when exposed to the high vacuum of the
microscope. The samples are mounted and coated with thin layer heavy
metal elements to allow spatial scattering of electric charges on the
surface of the specimen allowing better image production, with high
clarity. Scanning by this microscope is attained by tapering a beam of
electrons back and forth over a thin section of the microscope. When
the electrons reach the specimen, the surface releases a tiny staw of
electrons known as secondary electrons which are then trapped by a
special detector apparatus.
When the secondary electrons reach and enter the detector, they strike a
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scintillator (a luminescence material that fluoresces when struck by a

charged particle or high energy photon). This emits flashes of light
which get converted into an electric current by a photomultiplier,
sending a signal to the cathode ray tube. This produces an image that
looks like a television picture that can be viewed and photographed. The
quantity of secondary electrons that enter the detector is highly defined
by the nature of the specimen i.e., raised surfaces receive high quantities
of electrons, entering the detector
• Therefore raised surfaces will appear brighter on the screen while
depressed surfaces appear darker. Parts of a Scanning Electron
Microscope (SEM) The major components of the Scanning Electron
Microscope include;
• Electron Source - This is where electrons are produced under thermal
heat at a voltage of 1 40kV. the electrons the condense into a beam that
is used for the creation of ana image and analysis.
• There are three types of electron sources that can be used i. e Tungsten
filament, Lanthanum hexaboride, and Field emission gun (FEG) Lenses
- it has several condenser lenses that focus the beam of electrons from
the source through the column forming a narrow beam of electrons that
form a snot called a not size while depressed surfaces have fewer
electrons reaching the surface and hence fewer electrons enter the
detector.
• Its made up of several detectors that are able to differentiate the
secondary electrons, backscattered electrons, and diffracted
backscattered electrons.
• The functioning of the detectors highly depends on the voltage speed,
the density of the specimen.

• The display device (data output devices)

• Power supply
• Vacuum system Like the transmission electron Microscope, the
Scanning electron microscope should be free from vibrations and any
electromagnetic elements.

Scanning Electron Microscope (SEM)

• Some modern SEMs are able to generate digital data that can be portable.
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. It is easy to acquire data from the SEM, within a short period of time
of about 5 minutes. Limitations

• They are very expensive to purchase

. They are bulky to carry
• They must be used in rooms that are free of vibrations and free of
electromagnetic elements

• They must be maintained with a consistent voltage

• They should be maintained with access to cooling systems The
combination of the working principles of the Scanning Electron
Microscope (SEM) and the Transmission Electron Microscope (TEM)
formed the Scanning-Transmission Electron Microscope (STEM). The
Scanning- Transmission Electron Microscope (STEM), uses a
convergent beam of electrons to focus on a probe on the specimen, and
the probe is then scanned on its surface collecting signals which are then
collected as point-to-point to form an image.

TRANSMISSION ELECTRON MICROSCOPE OR STEM

1. In scanning transmission electron microscope or STEM, the specimen
is scanned in the same way as in scanning electron microscope or SEM
but transmitted electrons are collected and utilized for the formation of
picture on the screen.
2. The main advantage of this electron microscope is that it is fitted with
detector systems. Due to this facility, it can separately detect the
scattered electrons, un scattered electrons, and also those electrons
which are there as a result of the combination of scattered and un-
scattered electrons.

SPECTROPHOTOMETER

The spectrophotometer is an instrument which measures an amount of

light that a sample absorbs. The spectrophotometer works by passing a
light beam through a sample to measure the light intensity of a sample.
These instruments are used in the process of measuring colour and used
for monitoring colour accuracy throughout production. They are
primarily used by researchers and manufacturers everywhere. The major
Spectrophotometer Applications are limitless as they are used in
practically every industrial and commercial field. However, it finds its
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major applications in liquids, plastics, paper, metals and fabrics. This

helps in ensuring that the colour chosen remains consistent from its
original conception to the final, finished product.
• A spectrophotometer is an instrument that measures the amount of light
absorbed by a sample. Spectrophotometer techniques are mostly used to
measure the concentration of solutes in solution by measuring the
amount of the light that is absorbed by the solution in a cuvette placed
in the spectrophotometer. Scientist Arnold J. Beckman and his
colleagues at the National Technologies Laboratory (NTL) invented the
Beckman DU spectrophotometer in 1940.
A spectrophotometer is made up of two instruments: a spectrometer and
a photometer. The spectrometer is to produce light of any wavelength,
while the photometer is to measure the intensity of light. The
spectrophotometer is designed in a way that the liquid or a sample is
placed between spectrometer and photometer. The photometer measures
the amount of light that passes through the sample and delivers a voltage
signal to the display. If the absorbing of light change, the voltage signal
also changes. Spectrophotometers come in a variety of shapes and sizes
and have multipurpose uses to them. The different types of
spectrophotometers available are all different from one another, based
on their application and desired functionality. The most popular
spectrophotometers are 45 degrees, sphere and multi-angle
spectrophotometers. Another closely related concept is Spectroscopy,
that simply measures the absorption of light from its source and the
intensity of light as well. The basic spectrophotometer instrument
consists of a light source, a digital display, a monochromator, a
wavelength sector to transmit a selected wavelength, a collimator for
straight light beam transmission, photoelectric detector and a cuvette to
place a sample. The intensity of light is symbolized as lo measure the
number of photons per second. When the light is passed through the
blank solution, it does not absorb light and is symbolized as (1). Other
important factors are Absorbance (A) and Transmittance (T).

A = -log10 T
Here, we need to measure the intensity of light that passes a blank
solution, and later measures the intensity of light passing a sample.
Calculate the transmittance and the absorbance. For the measurement of
absorbance, we can use an isosbestic point where the absorbance and
wavelength of two or more species are the same. A number of protons

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transmit and absorb totally depended on the length of the cuvette and
the concentration of the sample. The transmittance and absorption
relation is:
Absorbance (A) = -log(T)
Light intensity before passing via cuvette Further, there are several
varieties of spectrophotometer devices such as UV Spectrometry,
atomic emission spectrophotometry and atomic absorption
spectrophotometry and much more. It can also be classified into two
types based on the range of light source wavelengths like IR
spectrophotometer and UV-visible spectrophotometer. Some major real-
life applications of spectrophotometry in various fields are laundry soap,
carpeting and production of small parts such as toys or intricate
machinery. The major types of spectrophotometers are categorized into
2, these are mainly portable spectrophotometers and bench
spectrophotometers, they both are unique and have their own uses.
Principle of Spectrophotometer The spectrophotometer technique is to
measure light intensity as a function of wavelength. It does this by
diffracting the light beam into a spectrum of wavelengths, detecting the
intensities with a charge coupled device, and displaying the results as a
graph on the detector and then on the display device.
1. In the spectrophotometer, a prism (or) grating is used to split the
incident beam into different wavelengths.
2. By suitable mechanisms, waves of specific wavelengths can be
manipulated to fall on the test solution. The range of the wavelengths of
the incident light can be as low as 1 to 2nm.
3. The spectrophotometer is useful for measuring the absorption
spectrum of a compound, that is, the absorption of light by a solution at
each wavelength.
Instrumentation of Spectrophotometer
The essential components of spectrophotometer instrumentation
include:

1. A table and cheap radiant energy source

• Materials that can be excited to high energy states by a high voltage

electric discharge (or) by electrical heating serve as excellent radiant

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energy sources.
2. A monochromator, to break the polychromatic radiation into
component wavelength (or) bands of wavelengths. A monochromator
resolves polychromatic radiation into its individual wavelengths and
isolates these wavelengths into very narrow bands. Prisms:
• A prism disperses polychromatic light from the source into its
constituent wavelengths by virtue of its ability to reflect different
wavelengths to a different extent
• Two types of Prisms are usually employed in commercial instruments.
Namely, 600 cornu quartz prism and 300 Littrow Prism. Grating:
• Gratings are often used in the monochromators of spectrophotometers
operating ultraviolet, visible and infrared regions.

3. Transport vessels (cuvettes), to hold the sample

• Samples to be studied in the ultraviolet (or) visible region are usually
glasses (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.

4. A Photosensitive detector and an associated readout system

Most detectors depend on the photoelectric effect. The current is then
proportional to the light intensity and therefore a measure of it.
Radiation detectors generate electronic signals which are proportional
to the transmitter light.

• These signals need to be translated into a form that is easy to interpret.

• This is accomplished by using amplifiers, Ammeters, Potentiometers
and Potentiometric recorders. Applications Some of the major
applications of spectrophotometers include the following:
Detection of concentration of substances Detection of impurities
Structure elucidation of organic compounds Monitoring dissolved
oxygen content in freshwater and marine ecosystems Characterization
of proteins Detection of functional groups Respiratory gas analysis in
hospitals Molecular weight determination of compounds The visible
and UV spectrophotometer may be used to identify classes of
compounds in both the pure state and in biological preparations.
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ULTRAVIOLET-VISIBLE SPECTROSCOPY
Ultraviolet-visible spectroscopy or ultraviolet-visible
spectrophotometry (UV/VIS) involves the spectroscopy of photons and
spectrophotometry. It uses light in the visible and adjacent to ultra violet
(UV) and near infrared (NIR) ranges. In this region of energy space,
molecules undergo electronic transitions. All atoms absorb in the UV
region because photons are energetic enough to excite outer electrons. If
the frequency is high enough, photo-ionisation takes place. UV
spectroscopy is also used in quantifying protein and DNA concentration
as well as the ratio of protein to DNA concentration in a solution.
Several amino acids usually found in protein, such as tryptophan, absorb
light in the 280 nm range and DNA absorbs light in the 260 nm range.
For this reason, the ratio of 260/280 nm absorbance is a good general
indicator of the relative purity of a solution in terms of these two
macromolecules.
Reasonable estimates of protein or DNA concentration can also be made
this wav using Beer's law. UV/Vis spectroscopy is routinely used in the
quantitative determination of solutions of transition metal ions and
highly conjugated organic compounds. Solutions of transition metal
ions can be coloured (i.e., absorb visible light) because electrons within
the metal atoms can be excited from one electronic state to another.
The colour of metal ion solutions is strongly affected by the presence of
other species, such as certain anions or ligands. For instance, the colour
of a dilute solution of copper sulphate is very light blue; adding
ammonia intensifies the colour and changes the wavelength of
maximum absorption (a max). Organic compounds, especially those
with a high degree of conjugation, also absorb light in the UV or visible
regions of the electromagnetic spectrum. The solvents for these
determinations are often water for water soluble compounds, or ethanol
for organic soluble compounds. (Organic solvents may have significant
UV absorption; not all sol vents are suitable for use in UV spectroscopy.
(a) Small molecules can be recognized by antibody but cannot stimulate
the immune system to produce antibody. They must combine with
adjuvants (usually proteins) to form an immunogen. Proteins can always
be an immunogen.
(b) Antibody can recognize structural specific areas on antigen, which
is called epitope. The recognition areas on the antibody are called
paratopes.
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(c) An antibody is a Y-shaped molecular.

(d) An antibody consists of two heavy and two light chains linked by
disulfide bonds. The Fab fragments of H and L chains are highly
variable and the Fc fragments are conservative.

INFRARED SPECTROPHOTOMTER

An IR spectrum is essentially a graph plotted with the infrared light

absorbed on the Y-axis against. frequency or wavelength on the X-axis.
IR Spectroscopy detects frequencies of infrared light that are absorbed
by a molecule. Molecules tend to absorb these specific frequencies of
light since they correspond to the frequency of the vibration of bonds in
the molecule. The energy required to excite the bonds belonging to a
molecule, and to make them vibrate with more amplitude, occurs in the
Infrared region. A bond will only interact with the electromagnetic
infrared radiation, however, if it is polar.

The presence of separate areas of partial positive and negative charge in

a molecule allows the electric field component of the electromagnetic
wave to excite the vibrational energy of the molecule. The change in the
vibrational energy leads to another corresponding change in the dipole
moment of the given molecule. The intensity of the absorption depends
on the polarity of the bond. Symmetrical non-polar bonds in N≡N and
O=O do not absorb radiation, as they cannot interact with an electric
field.

Most of the bands that indicate what functional group is present are
found in the region from 4000 cm-1 to 1300 cm-1. Their bands can be
identified and used to determine the functional group of an unknown
compound. Bands that are unique to each molecule, similar to a
fingerprint, are found in the fingerprint region, from 1300 cm-1 to 400
cm-1. These bands are only used to compare the spectra of one
compound to another.

SAMPLE

The samples used in IR spectroscopy can be either in the solid, liquid,

or gaseous state.

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• Solid samples can be prepared by crushing the sample with a

mulling agent which has an oily texture. A thin layer of this mull
can now be applied on a salt plate to be measured.
• Liquid samples are generally kept between two salt plates and
measured since the plates are transparent to IR light. Salt plates
can be made up of sodium chloride, calcium fluoride, or even
potassium bromide.
• Since the concentration of gaseous samples can be in parts per
million, the sample cell must have a relatively long pathlength,
i.e. light must travel for a relatively long distance in the sample
cell.

Thus, samples of multiple physical states can be used in Infrared

Spectroscopy.

PRINCIPLE

The IR spectroscopy theory utilizes the concept that molecules tend to

absorb specific frequencies of light that are characteristic of the
corresponding structure of the molecules. The energies are reliant on the
shape of the molecular surfaces, the associated vibronic coupling, and
the mass corresponding to the atoms.

For instance, the molecule can absorb the energy contained in the
incident light and the result is a faster rotation or a more pronounced
vibration.

APPLICATIONS

Because much of the evidence that is left at a crime scene consists of

organic compounds, infrared spectroscopy is useful in forensic
investigations. Paint, ink, sweat, fuels, and hair are all examples of
substances that can be identified this way. Over time, the technology has
also been refined so that sample sizes can be very small, and portable IR
equipment can be taken directly to a scene for on-site analysis. IR doesn't
alter the evidence since very small amounts of energy are applied.

Infrared spectroscopy can be used to identify forged or altered

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documents by shining a beam of infrared light on the document's ink. In

a study published in the June 2002 issue of the Journal of Applied
Spectroscopy, researchers at Berkeley lab collaborated with the U.S.
Secret Service to show that changes in ink could be identified without
having to extract the ink from the paper, which older methods required.

Other studies done at the Berkeley Lab showed that sweat prints, which
are composed of salts, oils, and proteins, could also be identified for
specific individuals by using infrared light.

Fibers of various kinds are often found at crime scenes, but they may be
from fabric, animals, human hair, or from other sources. IR makes it
possible to identify what kind of fiber has been found. If it is hair, it can
then be compared to a known sample, perhaps taken from a suspect. It's
even possible to determine if the hair has traces of hairspray or other
hair-styling products. With IR, sample preparation is simple, and as
noted earlier, the sample size can be very small.

RAMAN SCPECTROPHOTOMETER

Raman spectroscopy is a molecular spectroscopic technique that utilizes

the interaction of light with matter to gain insight into a material's make
up or characteristics, like FTIR. The information provided by Raman
spectroscopy results from a light scattering process, whereas IR
spectroscopy relies on absorption of light. Raman spectroscopy yields
information about intra- and inter-molecular vibrations and can provide
additional understanding about a reaction. Both Raman and FTIR
spectroscopy provide a spectrum characteristic of the specific vibrations
of a molecule ("molecular fingerprint') and are valuable for identifying
a substance. However, Raman spectroscopy can give additional
information about lower frequency modes, and vibrations that give
insight into crystal lattice and molecular backbone structure.

Inline Raman spectroscopy is used to monitor crystallization processes

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and reveal reaction mechanisms and kinetics. Combined with analysis

tools, this data enables informed reaction understanding and
optimization.

PRINCIPLE

When light interacts with molecules in a gas, liquid, or solid, the vast
majority of the photons are dispersed or scattered at the same energy as
the incident photons. This is described as elastic scattering, or Rayleigh
scattering. A small number of these photons, approximately 1 photon in
10 million will scatter at a different frequency than the incident photon.
This process is called inelastic scattering, or the Raman effect, named
after Sir C.V. Raman who discovered this and was awarded the 1930
Nobel Prize in Physics for his work. Since that time, Raman has been
utilized for a vast array of applications from medical diagnostics to
material science and reaction analysis. Raman allows the user to collect
the vibrational signature of a molecule, giving insight into how it is put
together, as well as how it interacts with other molecules around it.

HOW DOES IT WORK?

Unlike FTIR Spectroscopy that looks at changes in dipole moments,

Raman looks at changes in a molecular bonds polarizability. Interaction
of light with a molecule can induce a deformation of its electron cloud.
This deformation is known as a change in polarizability. Molecular
bonds have specific energy transitions in which a change of
polarizability occurs, giving rise to Raman active modes. As an
example, molecules that contain bonds between homonuclear atoms
such as carbon-carbon, sulfur-sulfur, and nitrogen-nitrogen bonds
undergo a change in polarizability when photons interact with them.
These are examples of bonds that give rise to Raman active spectral
bands, but would not be seen or difficult to see in FTIR.

Because Raman is an inherently weak effect, the optical components of

a Raman Spectrometer must be well matched and optimized. Also, since
organic molecules may have a greater tendency to fluoresce when
shorter wavelength radiation is used, longer wavelength monochromatic
excitation sources, such as solid state laser diodes that produces light at
785 nm, are typically used.

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APPLICATION

Renishaw's inVia Raman spectrometer has numerous applications

within forensic science, including the identification of illicit drugs,
gunshot residue, accelerants in arson cases, inks used in counterfeiting,
or explosives.

(1) Explosive and Gunshot Residue

Raman data can be obtained from almost any surface, allowing minute
traces of explosives or a firearm’s discharge to be detected without
attempting to lift samples from evidence. Very small discharge residues
(1 micrometer in diameter) may be assessed in the laboratory. One of
the greatest benefits of using a Renishaw Raman system is gaining
access to an extensive forensic database. This makes identifying
compounds easier, faster, and more accurate than ever.

(2) Fraudulent Documents

The sophistication of document fraud has risen dramatically in recent

years. Documents may contain added terms and conditions after the date
of signing, overlapping text, or multiple signatures that must be
analyzed.

Rapid Raman imaging, such as Renishaw's StreamLine approach, is the

best available method to investigate questionable documents and
identify fraud. The use of a Raman microscope is non-destructive,
preserving the evidence in its original form.

Additionally, Raman spectroscopy is highly sensitive to minute

chemical differences between inks. Images are generated in minutes,
making this technique one of the fastest to characterize ink chemical
structure. Furthermore, Raman imaging is significantly better at
determining the order of ink deposition than competing methods.

(3) Illicit Drugs

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Traditional methods used to identify narcotics and other illicit

substances use IR spectroscopy or a gas chromatography-mass
spectrometer. These approaches are destructive, requiring samples to be
extensively pre-processed, and take a long time. In comparison, Raman
spectroscopy represents a rapid and nondestructive method of
identifying organic chemical compounds.

The Raman method is effective in analysis of tablets, powders, and

liquids. Importantly, Raman data are not strongly affected by the
proximity of plastics or glass, meaning that substances can be analyzed
in their original packaging. This maintains evidence integrity and
prevents sample contamination.

One of the challenges in identifying illicit drugs is that they often

represent a mixture of compounds. For example, narcotics are typically
cut with lactose or mannitol, complicating their analysis. Raman
spectroscopy is capable of separating these components, therefore
increasing accuracy of drug identification.

ATOMIC ABSORPTION SPECTROMETER

Atomic absorption spectrometry (AAS) detects elements in either liquid

or solid samples through the application of characteristic wavelengths of
electromagnetic radiation from a light source. Individual elements will
absorb wavelengths differently, and these absorbances are measured
against standards. In effect, AAS takes advantage of the different
radiation wavelengths that are absorbed by different atoms.

In AAS, analytes are first atomized so that their characteristic

wavelengths are emitted and recorded. Then, during excitation, electrons
move up one energy level in their respective atoms when those atoms
absorb a specific energy. This energy corresponds to a specific
wavelength that is characteristic of the element. Depending on the light
wavelength and its intensity, specific elements can be detected and their
concentrations measured.
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AAS has an unlimited number of applications and is still a popular

choice for uncomplicated trace elemental analysis. Flame atomic
absorption spectrometry (FAAS) is widely accepted in many industries,
which continue to utilize the unique and specific benefits of this
technology. Graphite furnace atomic absorption spectrometry (GFAAS)
is an established technology for measuring elements at parts per billion
(ppb or µg/l) concentrations with incredibly low sample volumes.

PRINCIPLE

The basic principles of AAS can be expressed as follows. Firstly, all

atoms or ions can absorb light at specific, unique wavelengths. When a
sample containing copper (Cu) and nickel (Ni), for example, is exposed
to light at the characteristic wavelength of Cu, then only the Cu atoms
or ions will absorb this light. The amount of light absorbed at this
wavelength is directly proportional to the concentration of the absorbing
ions or atoms.

The electrons within an atom exist at various energy levels. When the
atom is exposed to its own unique wavelength, it can absorb the energy
(photons) and electrons move from a ground state to excited states. The
radiant energy absorbed by the electrons is directly related to the
transition that occurs during this process. Furthermore, since the
electronic structure of every element is unique, the radiation absorbed
represents a unique property of each individual element and it can be
measured.

An atomic absorption spectrometer uses these basic principles and

applies them in practical quantitative analysis. A typical atomic
absorption spectrometer consists of four main components: the light
source, the atomization system, the monochromator and the detection
system.

In a typical experiment, the sample, either liquid or solid, is atomized in

either a flame or a graphite furnace. The free atoms are then exposed to
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light, typically produced by a hollow-cathode lamp, and undergo

electronic transitions from the ground state to excited electronic states.
The light produced by the lamp is emitted from excited atoms of the
same element that is to be determined, therefore the radiation energy
corresponds directly to the wavelength absorbed by the atomized
sample. A monochromator is placed between the sample and the detector
to reduce background interference. From here, the detector measures the
intensity of the beam of light and converts it to absorption data.

While solid samples can be used for AAS this analysis is usually
restricted to the more expensive graphite furnaces where the sample can
be heated by controlled electrical heating as opposed to a direct flame.
Also, AAS is normally only used to analyse metal atoms. The main
reason for this is that metals have narrow, bright and clear single
emission and absorption lines.

APPLICATIONS

Application of atomic absorption spectroscopy to detect multimetal

traces in injured skin is a promising tool for investigation of fatalities
caused by electrocution. The present paper is aimed at testing the
reliability of this method for metal traces detection in electric current
marks and is focused on study of peculiarities of metal penetration into
the skin exposed to a current impact. Bare aluminum wire, tin-lead
coated copper multistrand wire, and zinc-plated steel rope were used to
make electrical marks on pig skin. It is demonstrated that amount of
copper, zinc, lead, and iron may serve as statistically reliable indicators
for the type of wire, which caused the electrical mark, in spite of the
background content of these metals in the skin without injury. Different
penetration rates for different metals contained in the wire inflicting an
electrical mark were observed.

EMISSION SPECTROMETER

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Atomic (or optical) emission spectrometry (AES, OES) is an important

technique for the multielement analysis of a wide range of materials.
Many elements have been discovered using emission spectrometry and
it is the most commonly used procedure for the measurement of trace
elements in rocks, water, soil, manufactured goods, and biological
specimens. The technique is used to monitor the levels of different
chemicals and trace elements in the environment and to determine the
compositions of solids, liquids, and gases. In geo analysis, emission
spectrometry has been instrumental in the exploration of
economic mineral deposits. In metallurgy and in the semiconductor
industry, emission spectrometry is of prominent importance in the
production control of both raw materials and finished products. Finally,
emission spectrometry allows the elements present in the sun and stars
to be identified, helping us to understand better the nature of the
universe. These are only a few examples of scientific and technical
disciplines in which the technique of emission spectrometry has made a
significant contribution.

PRINCIPLE

The theory or working principle of Atomic Emission Spectroscopy

involves the examination of the wavelengths of photons discharged by
atoms and molecules as they transit from a high energy state to a low
energy state. A characteristic set of wavelengths is emitted by each
element or substance which depends on its electronic structure. A study
of these wavelengths can reveal the elemental structure of the sample.

Flame Atomic Emission Spectroscopy

In this method, a sample of the material to be analyzed is brought into

flame in the form of a sprayed solution or gas. Free atoms of the material
are produced when the flame heat evaporates the solvent and breaks
the chemical bonds of the analyte. The heat also changes the atoms into
electronically charged particles which emits light when they get back to
the ground electronic state. Light is emitted at a wavelength
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characteristic to each element which is then dispersed by a prism or

grating and detected in spectrometer.

Flame emission spectroscopy is frequently used while studying alkali

metals for pharmaceutical research and analysis.

Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-

AES)

Inductively coupled plasma atomic emission spectroscopy (ICP-AES)

employs the use of inductively coupled plasma for producing excited
ions and atoms that radiate electromagnetically charged particles at
wavelengths characteristic to a definite element.

APPLICATION

• It can be used to determine metals, metalloids, and some non-metals

simultaneously.

• It does not need a light source.

• It is helpful in analysis of ferrous and nonferrous alloys.

• It is also help in identification of metals in geological, environmental,

and biological materials.

• Water analysis ICP instruments have been coupled with mass

spectrometers to provide a powerful analytical technique.

NEUTRON ACTIVATION ANALYSIS

Neutron activation analysis (NAA) is a sensitive analytical technique for

determining the amount of different elements (major, minor, or trace)
present in a sample. The technique is most accurately viewed as a

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method of quantitative chemical analysis based on the nuclear properties

of constituent elements. Briefly, NAA involves placing a small amount
of sample material in a flood of neutrons (typically generated by a
nuclear reactor) in order to activate or create radioactive isotopes of the
elements present (Box 1). As these excited isotopes return to a stable
state, they emit charged particles and noncharged γ-rays, in a process
known as radioactive decay. The detection of those decays permits both
the identification of elements originally present in the sample and the
precise determination of their quantity.

PRINCIPLE

Neutron activation analysis is a physical technique that is based on

nuclear reactions. The sample becomes radioactive when neutrons react
with the nuclei of the elements’ atoms. Radionuclides are formed and
subsequently decay by emitting gamma rays that are unique in half-life
and energy. Gamma-ray intensity is proportional to the element content
in the sample. Instrumental neutron activation analysis (INAA) and k0-
INAA are the most sensitive analytical techniques used for the
quantitative multielement analysis of major, minor, and trace elements
in samples from almost every conceivable field of scientific or technical
interest.

WORKING

To conduct a Neutron Activation Analysis experiment, the sample is

exposed to neutrons in a nuclear reactor, causing a portion of the atoms
to undergo neutron capture: this produces high energy compound nuclei
which rapidly transform to radioactive forms of the original chemical
element(s). As the short-lived radioactive isotopes undergo decay to
reach stable ground state configurations, the sample is placed on a high
purity germanium detector which records the intensities and energies of
the gamma rays that are emitted. Because a given radioactive isotope
always emits gamma rays at certain specific energies and intensities, the
radioisotopes present, and hence the parent chemical element(s) present
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in the sample can be determined quantitatively. This “Short-Lived

Neutron Activation Analysis” (SLNAA) technique can be used to
quantify dozens of chemical elements including metals, non-metals and
metalloids at the parts per million level.

Variations on the SLNAA technique include Prompt Gamma Neutron

Activation Analysis, which uses the “prompt” gamma rays emitted
during neutron capture, rather than the gamma rays that are emitted as
the resulting radioisotope undergoes decay, in order to determine
elemental composition; PGNAA is particularly useful for quantifying
boron, cadmium, and certain lanthanoids (Eu, Gd, Sm).

A third permutation is Delayed Neutron Counting NAA, which is used

primarily for the quantification of uranium. Unlike of the lighter
elements in the Periodic Table, when uranium absorbs a neutron, the
neutron capture event is followed immediately by nuclear fission. At the
uranium atom splits, it ejects an average of 2.8 neutrons per fission; by
quantifying (counting) the ejected neutrons, the amount of uranium
initially present in the sample can be determined.

NAA is a highly sensitive analytical technique, particularly with the high

neutron fluxes available at the McMaster Nuclear Reactor, and can be
applied to any element that possesses a suitable activation product
(radioisotope). With appropriate experimental parameters, excellent
sensitivity is possible for some 70 elements; depending on sample
composition, multi-element analysis at the ppm level for up to 30
elements can be accomplished with a single 1 gram sample. Typical
analytical applications of SLNAA and PGNAA include determining
major and trace elements in a wide variety of materials, including rocks
and other geological samples, as well as ceramics, oils, plastics, metals,
water, biological and botanical materials. DNC NAA is most commonly
used for determining uranium content in soil samples for the uranium
mining industry. A significant advantage of NAA over other analytical
techniques such as ICP-MS is the simplicity of sample treatment before
analysis: in most cases, the only requirement is that the sample be
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reduced to a size suitable for encapsulation (< 2 mL). Activation analysis

is also non-destructive, and can therefore be used for expensive or
irreplaceable samples such as archaeological artifacts.

APPLICATIONS

Personnel of the NAA laboratory have considerable experience in the

forensic analysis of evidentiary materials. Bullet fragments, gunshot
residue, plastic, hair and fingernails, and geological materials are
included among recent examples. Comparing materials non-
destructively is a chief advantage of NAA for forensics.

High-Purity Materials

Materials such as high-purity silica, silicon, aluminium, other materials

and their compounds that do not form long-lived radionuclides,
cellulose air filters, as well as graphite are excellent matrices for high-
sensitivity NAA. Such materials can be irradiated in graphite rabbits for
many hours in PT-1 for determinations of many elements at the sub-ppb
level. Silicon wafers and SiO2 used in fibre optics are examples that
have been analysed.

Radiochemical separations:

Extremely low quantities of certain elements (such as Ir) can be

measured utilizing microwave digestion facilities available at ORNL
and straightforward chemical separation techniques.

X-RAY AND X-RAY BASED TECHNIQUES

An x-ray examination creates images of your internal organs or bones

to help diagnose conditions or injuries. A special machine emits (puts
out) a small amount of ionising radiation. This radiation passes through
your body and is captured on a special device to produce the image.

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The dose of radiation you will receive depends on the area of your body
being examined. Smaller areas such as the hand receive a lesser dose
compared to a larger area such as the spine. On average, the dose of
radiation is roughly the same as you would receive from the general
environment in about one week.

WORKING

A small amount of ionising radiation is passed through the body. In the

past, this went onto a sheet of special film. Nowadays x-ray
examinations are more likely to use a device that will capture
transmitted x-rays to create an electronic image.

The calcium in bones blocks the passage of radiation, so healthy bones

show up as white or grey. On the other hand, radiation passes easily
through air spaces, so healthy lungs appear black.

APPLICATIONS

X-ray is the most common, basic and essential imaging method used in
forensic medicine. It serves to display and localize the foreign objects
in the body and helps to detect various traumatic and pathological
changes. X-ray imaging is valuable in anthropological assessment of an
individual. X-ray allows non-invasive evaluation of important findings
before the autopsy and thus selection of the optimal strategy for
dissection. Basic indications for postmortem X-ray imaging in forensic
medicine include gunshot and explosive fatalities (identification and
localization of projectiles or other components of ammunition,
visualization of secondary missiles), sharp force injuries (air embolism,
identification of the weapon) and motor vehicle related deaths. The
method is also helpful for complex injury evaluation in abused victims
or in persons where abuse is suspected. Finally, X-ray imaging still
remains the gold standard method for identification of unknown
deceased. With time modern imaging methods, especially computed
tomography and magnetic resonance imaging, are more and more
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applied in forensic medicine. Their application extends possibilities of

the visualization the bony structures toward a more detailed imaging of
soft tissues and internal organs. The application of modern imaging
methods in postmortem body investigation is known as digital or virtual
autopsy. At present digital postmortem imaging is considered as a
bloodless alternative to the conventional autopsy.

XRD

X-ray diffraction (XRD) is a powerful tool widely used in research and

industry. While XRD is usually well known for qualitative and
quantitative analyses of crystalline phases in materials, far more
information can be obtained from a careful analysis of the diffraction
patterns or by using specific XRD settings: i.e., characterization of solid
solutions, crystallite size and shape, crystal orientation, internal elastic
strains/stresses at different levels, effect of temperature, close surface
characterization etc. The objectives of this paper are first to summarize
some basic principles of X-ray diffraction, and next to provide some
examples of applications of XRD in the field of ceramics materials.

PRINCIPLE

Max von Laue, in 1912, discovered that crystalline substances act as

three-dimensional diffraction gratings for X-ray wavelengths similar to
the spacing of planes in a crystal lattice. X-ray diffraction is now a
common technique for the study of crystal structures and atomic
spacing.

X-ray diffraction is based on constructive interference of

monochromatic X-rays and a crystalline sample. These X-rays are
generated by a cathode ray tube, filtered to produce monochromatic
radiation, collimated to concentrate, and directed toward the sample.
The interaction of the incident rays with the sample produces
constructive interference (and a diffracted ray) when conditions
satisfy Bragg's Law (nλ=2d sin θ). This law relates the wavelength of
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electromagnetic radiation to the diffraction angle and the lattice spacing

in a crystalline sample. These diffracted X-rays are then detected,
processed and counted. By scanning the sample through a range of
2θangles, all possible diffraction directions of the lattice should be
attained due to the random orientation of the powdered material.
Conversion of the diffraction peaks to d-spacings allows identification
of the mineral because each mineral has a set of unique d-spacings.
Typically, this is achieved by comparison of d-spacings with standard
reference patterns.

All diffraction methods are based on generation of X-rays in an X-ray

tube. These X-rays are directed at the sample, and the diffracted rays
are collected. A key component of all diffraction is the angle between
the incident and diffracted rays. Powder and single crystal diffraction
vary in instrumentation beyond this.

WORKING

X-ray diffractometers consist of three basic elements: an X-ray tube, a

sample holder, and an X-ray detector.

X-rays are generated in a cathode ray tube by heating a filament to

produce electrons, accelerating the electrons toward a target by
applying a voltage, and bombarding the target material with electrons.
When electrons have sufficient energy to dislodge inner shell electrons
of the target material, characteristic X-ray spectra are produced. These
spectra consist of several components, the most common being Kα and
Kβ. Kα consists, in part, of Kα1 and Kα2. Kα1 has a slightly shorter
wavelength and twice the intensity as Kα2. The specific wavelengths
are characteristic of the target material (Cu, Fe, Mo, Cr). Filtering, by
foils or crystal monochrometers, is required to produce monochromatic
X-rays needed for diffraction. Kα1and Kα2 are sufficiently close in
wavelength such that a weighted average of the two is used. Copper is
the most common target material for single-crystal diffraction, with
CuKα radiation = 1.5418Å. These X-rays are collimated and directed
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onto the sample. As the sample and detector are rotated, the intensity of
the reflected X-rays is recorded. When the geometry of the incident X-
rays impinging the sample satisfies the Bragg Equation, constructive
interference occurs and a peak in intensity occurs. A detector records
and processes this X-ray signal and converts the signal to a count rate
which is then output to a device such as a printer or computer monitor.

The geometry of an X-ray diffractometer is such that the sample rotates

in the path of the collimated X-ray beam at an angle θ while the X-ray
detector is mounted on an arm to collect the diffracted X-rays and
rotates at an angle of 2θ. The instrument used to maintain the angle and
rotate the sample is termed a goniometer. For typical powder patterns,
data is collected at 2θ from ~5° to 70°, angles that are preset in the X-
ray scan.

APPLICATIONS

As criminal investigators work to stem global drug trafficking, one of

the key tools for the forensic analysis of drugs is X-ray diffraction
(XRD). Using forensic XRD, forensic scientists can quickly obtain
detailed phase and structural information of crystalline materials by
bombarding drug samples with incident X-rays and then measuring the
intensities and scattering angles of the X-rays scattered by these
samples.

XRD is an important part of the analytical workflow for the

identification of drug samples. Using XRD, forensic scientists can
confirm findings from complementary analytical techniques such as
mass spectrometry, while providing additional information about the
crystalline structure of drug samples.

XRD can be used to confirm the specific drugs present in a seized

substance, while determining their chemical forms. It can also quantify
the concentration of the main drug component and additional chemicals
found in a drug substance—a critical step in tracking down the source
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of a specific batch of drugs.

Take heroin, for example. Heroin exists in four forms: free base,
hydrochloride monohydrate, tartrate and citrate. Using XRD for
forensic characterization, scientists can distinguish the form of heroin
by determining the structure and matching the profile to a database of
different heroin samples.

Similarly, cocaine exists in three forms: free base, hydrochloride and

crack cocaine. Using XRD, scientists can identify the form of the
cocaine they have seized. They can also identify the purity of the drug
sample as well as the percentage of lactose, caffeine and other cutting
agents mixed into the powder to increase the seller’s profitability.
Identifying the exact concentrations of each substance found within the
drug sample enables forensic scientists to characterize the exact batch,
which, in turn, helps them uncover the criminals who manufactured it.

To help with the forensic analysis of drugs, Thermo Fisher Scientific

offers the ARL EQUINOX series of X-ray diffractometers. The
portfolio includes easy-to-use benchtop systems that can be transported
between laboratories for routine analyses. It also includes more
advanced floor models that offer research-grade results even when
analyzing minute drug quantities. The ARL EQUINOX comes with a
unique, curved position sensitive detector that measures all diffraction
peaks simultaneously across a wide angular range for fast and accurate
results. Analyses typically take just a few minutes on most samples
regardless of the resolution requirements.

Using forensic XRD, scientists can easily identify the type, form and
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composition of drug samples, while tracking down the source of these

drugs. With the ability to quickly and accurately analyze even trace
amounts, criminal investigators are better positioned to curtail the flow
of illegal drugs both within and across national borders.

XRF

XRF (X-ray fluorescence) is a non-destructive analytical technique

used to determine the elemental composition of materials. XRF
analyzers determine the chemistry of a sample by measuring the
fluorescent (or secondary) X-ray emitted from a sample when it is
excited by a primary X-ray source. Each of the elements present in a
sample produces a set of characteristic fluorescent X-rays (“a
fingerprint”) that is unique for that specific element, which is why XRF
spectroscopy is an excellent technology for qualitative and quantitative
analysis of material composition.

WORKING

A solid or a liquid sample is irradiated with high energy X-rays from a

controlled X-ray tube. When an atom in the sample is struck with an X-
ray of sufficient energy (greater than the atom’s K or L shell binding
energy), an electron from one of the atom’s inner orbital shells is
dislodged.

The atom regains stability, filling the vacancy left in the inner orbital
shell with an electron from one of the atom’s higher energy orbital
shells.

The electron drops to the lower energy state by releasing a fluorescent

X-ray. The energy of this X-ray is equal to the specific difference in
energy between two quantum states of the electron. The measurement
of this energy is the basis of XRF analysis.

APPLICATIONS
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X-ray fluorescence is a particularly beneficial tool to forensic scientists

for a number of reasons. It is a non-destructive technique and the sample
is left intact after analysis, particularly advantageous if the amount of
sample is limited or further analysis is required. Naturally in forensic
science there may be only a limited amount of sample recovered.
Compared to certain other analytical techniques it is fast, requiring very
little sample preparation as material can often be analysed as is (perhaps
requiring homogenisation first) and producing results in minutes, thus
multiple analyses can be carried out in a shorter space of time.
Furthermore, portable XRF devices have been developed which can
allow for analysis in-situ, potentially enabling instant results at the
incident scene and additionally beneficial in the analysis of samples
which cannot easily be removed from the scene. However a fairly large
sample size is typically required for XRF spectroscopy.

This instrument has a wide range of potential applications to forensic

science. It is particularly beneficial in the analysis of rocks, soils and
other similar substances, allowing the analyst to compare the
composition of these samples. This could be used in suggesting whether
two similar-appearing soils are likely to have originated from the same
source or if they are different, based on the various elements detected.

The technique can be used in the detection of counterfeit coins. Genuine

coins are made up of very specific amounts of each component, for
instance, the British one pound coin is composed of 70% copper, 24.5%
zinc and 5.5% copper. Any one pound coin that is not made up of this
combination of elements is likely to be a counterfeit, even if by
appearance it is identical to a genuine coin. This is not limited to the
analysis of potentially counterfeit coins, as counterfeit notes/bills can
also be subjected to XRF spectroscopy.

Inks and paints are particularly suitable for analysis by XRF techniques,
as they typically contain metallic pigments which can differ wildly
between different brands and batches. The forensic analysis of writing
and printer inks can be of particular importance in the investigation of
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questioned documents, for instance in aiming to establish whether two

documents may have been written using the same ink.

The identification of gunshot residues can also be achieved using this

technique. When a gun is discharged, minute particles from the firearm,
bullet and gunpowder are propelled from the weapon, often onto the
hands and clothing of the shooter. As the composition of gunshot
residue is reasonably well documented and composed of compounds
that are not typically present on a person’s hand, XRF can be applied to
a piece of clothing or even an individual’s body to ascertain if such
residues might be present, indicating whether or not they could have
fired a weapon recently.

The ability of XRF to produce an elemental fingerprint of a sample is

by no means limited to the aforementioned types of evidence, but can
be applied to a wide range of samples providing they can be subjected
to this technique. Unfortunately, this instrumentation is not necessarily
readily available for use in forensic science.

MASS SPECTROSCOPY

Mass spectrometry is an analytical tool useful for measuring the mass-

to-charge ratio (m/z) of one or more molecules present in a
sample. These measurements can often be used to calculate the exact
molecular weight of the sample components as well. Typically, mass
spectrometers can be used to identify unknown compounds via
molecular weight determination, to quantify known compounds, and to
determine structure and chemical properties of molecules.

How does a mass spectrometer perform such a feat? Every mass

spectrometer consists of at least these three components:

• Ionization Source

• Mass Analyzer
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• Ion Detection System

1. The Ionization Source

Molecules are converted to gas-phase ions so that they can be moved

about and manipulated by external electric and magnetic fields. In our
laboratory we use a technique called nanoelectrospray ionization,
which is somewhat similar to how cars are industrially painted. This
method allows for creating positively or negatively charged ions,
depending on the experimental requirements. Nanoelectrospray
ionization can directly couple the outlet of a small-scale
chromatography column directly to the inlet of a mass
spectrometer. The flow from the column is passed through a needle that
is 10-15 um at its tip.

2. The Mass Analyzer

Once ionized, the ions are sorted and separated according to mass-to-
charge (m/z) ratios. There are a number of mass analyzers currently
available, each of which has trade-offs relating to speed of operation,
resolution of separation, and other operational requirements. The
specific types in use at the Broad Institute are discussed in the next
section. The mass analyzer often works in concert with the ion
detection system.

3. Ion Detection System

The separated ions are then measured and sent to a data system where
the m/z ratios are stored together along with their relative
abundance. A mass spectrum is simply the m/z ratios of the ions
present in a sample plotted against their intensities. Each peak in a mass
spectrum shows a component of unique m/z in the sample, and heights
of the peaks connote the relative abundance of the various components
in the sample.

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APPLICATIONS

By ionizing a sample, a scientist can cause it to separate into its

individual ions. This allows him to analyze and categorize those ions to
determine the sample's composition. Mass spectrometry has become a
valuable tool in forensic science, where it can provide clues from the
barest traces left by a suspect.

Toxicology Analysis

One area where a mass spectrometer is handy is in cases involving

poisons or toxins. Forensic analysts can take samples of a subject's
tissues or bodily fluids and determine if any toxic substances are
present, and if so, in what concentration. This can give vital clues to
investigators as to how a victim died as well as help identify the time
and dosage of any poison or medication ingested. Investigators can also
determine if a victim was a regular user of any substances that might
have contributed to his or her death.

Trace Evidence

Mass spectrometry is also useful in analyzing trace evidence.

Investigators at a crime scene may find microscopic materials like
carpet fibers, glass splinters or paint flakes. Ordinarily, these substances
might be extremely difficult to use as a starting point to identify a
suspect. However, a mass spectrometer can determine the precise mix
of dyes used in carpet fibers, the makeup of materials that went into any
particular glass fragment and the precise set of polymers present in any
paint sample. This information can lead investigators to a particular
manufacturer, who may be able to narrow down where a given sample
came from, helping detectives to identify suspects and build a case.

Arson Investigations

Arson investigations can also benefit from the use of mass

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spectrometry. While an arson investigator might be able to identify the

use of an accelerant through burn patterns or lingering odors, a mass
spectrometer can break down any residue and provide an accurate report
of its molecular makeup. This can help identify any unique or exotic
compounds that may be present. Discovering a similar mix used at
multiple crime scenes may be useful for identifying the work of a serial
arsonist.

Explosive Residue

Another area where spectrographic analysis is extremely useful is in

analyzing explosive residue. When a bomb detonates, it may not leave
behind much in the way of physical evidence -- perhaps only small
fragments and chemical residues. However, commercial explosive
manufacturers each utilize their own unique mix of chemicals, and a
spectrometer can analyze this residue to identify the particular makeup
of the explosive involved. Even in cases where a bomber used a
homemade mix, the analysis may identify the type of materials used and
give investigators a push in the right direction to identify the source.

THE IMMUNOASSAY

PRINCIPLE
An assay is a general term for an analytical laboratory procedure
designed to detect the presence of and/or the quantity of a drug in a
biological fluid such as urine or serum (the fluid component of the blood
obtained after removal of blood cells and fibrin clot). – An
immunoassay, therefore, is an analytical procedure which has as its
basis the principles of immunology-specifically the binding of drugs to
antibodies.
This binding of antibodies to drugs forms the basis for immunoassay. -
In the development of an immunoassay, the first step is to inject an
animal (host) with the drug that we ultimately wish to analyze. The host
immune system, recognizing the drug as a 'foreigner', generates
antibodies to this drug, and these antibodies can then be harvested from
the serum of the animal. - In the test-tube environment of the laboratory
(in vitro), these antibodies can be recombined with the appropriate drug.
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Just as it did inside the body (in vivo), the antibody will recognize the
drug based on the lock and key fit and will spontaneously bind to it. The
second step in the development of an immunoassay is to synthesize a
'labelled' drug.
This involves the chemical addition of a 'marker' to the drug. - This
marker can be small, such as an atom of radioactive iodine, or it can be
large, such as an enzyme, which is a fairly large protein. - Irrespective
of its size, this marker is added on such a way that it doesn't interfere
with the lock-and-key recognition between the antibody and the drug. –
All immunoassays work in the same basic fashion.
- They differ in the types of labels that are added to the labeled drug and
in the analytical methods by which the amount of binding of labeled
drug to the antibody is measured. The principle behind the
Immunoassay test is the use of an antibody that will specifically bind to
the antigen of interest. The antibodies used in the Immunoassay must
have a high affinity for the antigen. The antibodies used in the
Immunoassay can either be monoclonal or polyclonal antibodies.

The difference between the two antibodies are:

• Monoclonal antibodies only bind to very specific antigens and
therefore give more accurate and specific results, but these antibodies
tend to be more expensive.

• Polyclonal antibodies are inexpensive but can recognise multiple

epitopes on an antigen or they can recognize multiple antigens and these
antibodies tend to be less specific.

Types of immunoassays
The principle of an immune assay is based on competitions between a
fixed amount of labeled analyte and a variable amount of unlabeled
sample analyte for binding a limited number of binding sites of an
antibody specifically raised against the analyte. These methods can be
used to detect a target molecule quantitatively, semiquantitatively, or
qualitatively.
Several different types of immunoassay are routinely performed in the
laboratory. Although they differ in the types of reagents and
instrumentation used, they are all based on the same scientific principle
(the binding of drugs to antibodies). The three types of immunoassay
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that are commonly used for drug testing are the radioimmunoassay
(RIA), enzyme multiplied immunoassay (EMIT), and fluorescence
polarization immunoassay (FPIA). The immunoassay is based on the
competitive or non-competitive binding of the antigen with the antibody.
Competitive immunoassay
• (Competition between tagged and un-tagged antigen for the antibody)
Competitive Immunoassays are always designed so that there are fewer
antibody-binding sites present in the reaction mixture than there are
molecules of (labeled plus unlabeled) drug. . Because the label and
unlabeled drug appear the same to the anti-body, they will compete
equally for the limited number of available binding sites on the
antibody.
By measuring the amount of labeled drug bound to the antibody, the
analyst can calculate the amount of unlabeled drug in the biological
specimen. Commercially available immunoassay kits contain the
antibody (which the company has prepared described above) and the
labeled drug (which has been chemically synthesized) necessary to
perform the assay. In the laboratory, a fixed amount of antibody and a
fixed amount of labeled drug are placed into a reaction vessel (test tube).
If these were the only two ingredients, all the binding sites on the
antibody would react with (bind) to the labeled drug.
• A third ingredient added to the assay is, however the unlabeled drug
(i.e., the urine, saliva, or serum specimen containing the drug that is
being measured). Because the label on the labeled drug is placed in a
position that doesn't interfere with binding to the antibody (i.e., it is
'hidden'), the antibody cannot distinguish between the labeled and
unlabeled drug.

• Mix the three components together.

• Allow the antigens to compete for the limited antibody

. Antibody will bind with tagged or untagged antigen

• Separation step: antibody-antigen complexes are separated from
free antigens . Tagged antibody-antigen complex is measured.

• The tagged antigen and antibody from the reagent kit are constant. •
The only variable is the concentration of the patient antigen (the thing
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we want to measure) A standard curve can be made with known antigen

concentrations giving the following general results:

• High concentrations of patient antigen mean that more of the antibody-

antigen complexes are untagged Low concentrations of patient antigen
mean that more of the antibody-antigen complexes are tagged There is
an inverse relationship between patient antigen concentration and tag
activity after the separation process.
• The activity of the tag is measured twice:

• Before separation step= Total tag activity

• After separation step = Bound tag activity (antibody-antigen

complex) • Note that the separation process removes all unbound
(free) tag from the testing

• %B=B/T X 100 The ratio of the bound activity to the Total activity
(B/T) decreases as the concentration of the patient's (untagged) antigen
increases. Using standard solutions of known antigen concentrations,
the %B is plotted against the concentrations of the standards.

Non-competitive immunoassays
Noncompetitive (sandwich) immunoassays generally provide the
highest level of assay sensitivity and specificity. This format is referred
to as a 'sandwich' assay because the analysis id bound (sandwiched)
between two highly specific antibody reagents. The reaction mixture
typically includes an excess of labeled antibody, so that all
drug/metabolite is bound.
• The amount of antibody-antigen complex is then measured to
determine the amount of drug present in the sample.
• The measurement of labeled analyte, usually antibody, is directly
proportional to the amount of antigen present in the sample.

Homogeneous and Heterogeneous Immunoassay methods

Immunoassay methods that require separation of bound Ab-Ag complex
are referred to as heterogeneous immunoassays. Those that do not
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require separation are referred to as homogeneous immunoassays.

Homogeneous methods have been generally applied to the measurement
of small analytes such as abused and therapeutic drugs. As
homogeneous methods do not need the separation of the bound Ab-Ag*
from the free Ag, they are generally much easier and rapid to perform.

IMMUNOASSAYS: TECHNIQUES
Radioimmunoassay (RIA)

Radioimmunoassays use a radioisotope as a label to quantify hormones,

drugs, and viral antigens. A known concentration of radiolabeled
antigen is incubated with a fixed concentration of antibody specific for
that antigen. This radiolabeled antigen antibody complex is then mixed
with a biological sample containing an unknown concentration of the
same antigen. In principle, if the concentration of sample antigen is
higher than the radiolabeled antigen, the sample antigen will bind the
limited number of antibody binding sites by replacing the radiolabeled
antigen. Next, the radioactivity of these free (not bound to an antibody)
radiolabeled antigens is measured; which is directly proportional to the
concentration of unlabeled antigen present in the biological sample.

However, the short half-life of radioisotopes, as well as health hazards

associated with their use, considerably limits the application of this
method in many laboratories. Enzyme multiplied immunoassay In the
Enzyme multiplied Immunoassay (EMIT), the drug in the sample and
the drug labeled with G6PD compete for antibody binding sites. Binding
inhibits enzyme activity, while free enzyme remains active to interact
with. Enzyme activity/absorbance is directly proportional to drug
concentration.

Fluoroimmunoassays

Fluorescent probes are used to label antibodies. Biological samples

containing an antigen of interest is incubated with a fluorescent-labeled
antibody. The fluorescence intensity of the resulted antigenantibody
complex is measured to quantify the target antigen.
Chemiluminescence immunoassays

Luminescent molecules when return from an excited state to a ground

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state, emit visible or nearvisible light (luminescence), which can be

detected by luminescent signal measuring devices.

In chemiluminescence immunoassays, luminophore markers, such as

acridinium esters, can be used directly; or enzymatic markers including
alkaline phosphatase and horse radish peroxidase can be used indirectly
with adamantyl 1, 2-dioxetane aryl phosphate (AMPPD) and luminol
substrates, respectively. The major advantage of this method is that the
readout can be increased by using enhancers, which allow the reaction
to continue for a longer time without reducing the signal intensity.

Counting immunoassays

Counting immunoassays use particle counting technology. Polystyrene

beads (Latex particles) are coated with antibodies specific for an antigen
of interest. When incubated with a biological sample containing the
target antigen, these beads form immune complexes, which form visible
clumps (agglutination). Depending on the concentration of target
antigen, some beads remain unbound and can be counted using a cell
counter. The number of unbound beads inversely denotes the
concentration of target antigen. Monoclonal-Polyclonal Sandwich
Immunoassay In a typical microtiter plate sandwich immunoassay, a
monoclonal antibody is adsorbed onto a plastic microtiter plate.

When the test sample is added to the plate, the antibody on the plate will
bind the target antigen from the sample, and retain it in the plate. When
a polyclonal antibody is added in the next step, it also binds to the target
antigen (already bound to the monoclonal antibody on the plate),
thereby forming an antigen 'sandwich' between the two different
antibodies. This binding reaction can then be measured by radio
isotopes, as in a radio-immunoassay format (RIA), or by enzymes, as in
a enzyme immunoassay format (EIA or ELISA) attached to the
polyclonal antibody. The radio-isotope or enzyme generates a color
signal proportional to the amount of target antigen present in the original
sample added to the plate. Depending on the immunoassay format, the
degree of color can be detected and measured with the naked eye (as
with a home pregnancy test), a scintillation counter (for an RIA), or with
a spectrophotometric plate reader (for an EIA).
Step 1: Monoclonal antibodies adsorbed onto the well of a plastic
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microtiter plate with coating buffer (no sample added).

Step 2: Addition of a sample (such as human blood, diluted
appropriately) to the well of the microtiter plate. The target antigen
binds to the antibody adsorbed on the plate, retaining the antigen in the
well.
Step 3: Binding of an enzyme-conjugated polyclonal antibody to the
target antigen (bound to the monoclonal antibody on the plate), thereby
forming an antigen 'sandwich' between the two different antibodies.
Step 4: Addition of a colorimetric substrate for detection of the enzyme-
conjugated polyclonal antibodies will generate a color signal
proportional to the amount of target antigen present in the original
sample added to the plate.
Antigen-Down (Immunity Test) Immunoassay In an antigen-down
immunoassay, the analyte is coated onto a 96-well microtiter plate
(rather than an antibody) and used to bind antibodies found in a sample.
When the sample is added (such as human serum), the antigen on the
plate is bound by antibodies (IgE for example) from the sample, which
are then retained in the well. A species-specific antibody (anti-human
IgE for example) labeled with HRP is added next, which, binds to the
antibody bound to the antigen on the plate.
The higher the signal, the more antibodies there are in the sample.
Antigen-down assays can be configured as rapid tests and are often used
to diagnose allergy conditions - routinely a patient's blood is tested
against different allergens to see if the person has antibodies to that
allergen.
In addition to the Monoclonal-Polyclonal (Mo-Po) Antibody Sandwich
format, many immunoassays are structured in a competitive inhibition
format. Competitive inhibition assays are often used to measure small
analytes because competitive inhibition assays only require the binding
of one antibody rather than two, as in standard ELISA formats. Because
of the high probability for steric hindrance occurring when two
antibodies attempt to bind to a small molecule at the same time, a
sandwich assay format may not be feasible. Therefore a competitive
inhibition assay would be preferable.

In a sequential competitive inhibition assay, the sample and conjugated

analyte are added in steps like a sandwich assay, while in a classic
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competitive inhibition assay, these reagents are incubated together at the

same time. In a sequential competitive inhibition assay format, a
monoclonal antibody is coated onto a 96-well microtiter plate.
When the sample is added, the MoAb captures free analyte out of the
sample. In the next step, a known amount of analyte labeled with either
biotin or HRP is added. The labeled analyte will then also attempt to
bind to the MoAb adsorbed onto the plate, however, the labeled analyte
is inhibited from binding to the MoAb by the presence of previously
bound analyte from the sample. This means that the labeled analyte will
not be bound by the monoclonal on the plate if the monoclonal has
already bound unlabeled analyte from the sample.
The amount of unlabeled analyte in the sample is inversely proportional
to the signal generated by the labeled analyte. The lower the signal, the
more unlabeled analyte there is in the sample. A standard curve can be
constructed using serial dilutions of an unlabeled analyte standard.
Subsequent sample values can then be read off the standard curve as is
done in the sandwich ELISA formats. The classic competitive inhibition
assay format requires the simultaneous addition of labeled (conjugated
analyte) and unlabeled analyte (from the sample).
Both labeled and unlabeled analyte then compete simultaneously for the
binding site on the monoclonal capture antibody on the plate. Like the
sequential competitive inhibition format, the colored signal is inversely
proportional to the concentration of unlabeled target analyte in the
sample. Detection of labeled analyte may be made by using a peroxidase
substrate such as TMB, which can be read on a microtiter plate reader.
Rapid Immunoassay

In addition to microtiter plates, immunoassays are also configured as

rapid tests, such as a home pregnancy test. Like microtiter plate assays,
rapid tests use antibodies to react with antigens and can be developed as
MoAb PoAb sandwich formats, competitive inhibition formats, and
antigen-down formats. With a rapid test, the antibody and antigen
reagents are bound to porous membranes, which react with positive
samples while channeling excess fluids to a non-reactive part of the
membrane. Rapid immunoassays commonly come in 2 configurations:
a lateral flow test where the sample is simply placed in a well and the
results are read immediately; and a flow through system, which requires
placing the sample in a well, washing the well, and then finally adding
an analyte-colloidal gold conjugate and the result is read after a few
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minutes. One sample is tested per strip or cassette.

Because rapid tests are faster than microtiter plate assays, require little
sample processing, are often cheaper, and generate yes/no answers
without using an instrument, they often used in the field by non-
laboratory people testing whole samples. However, rapid immunoassays
are not as sensitive nor can they be used to accurately quantitate an
analyte. (Self-monitoring of blood glucose levels by diabetics is
considered quantitative rapid testing, however, immunoassay
technology is not used for these tests.) All rapid immunoassay tests can
be converted to a microtiter plate assay, but not all microtiter plate
assays can be converted to a rapid test.
The surface plasmon resonance (SPR)-based label-free immunoassay is
one such example that comes with many special features, such as
superfast analysis (within minutes), minimal requirement of sample and
reagents, label-free microfluidic immunoassay protocol, and fully
automated system with a highthroughput.
In addition, smartphone-based immunoassay formats, multiplex bead-
based assays, lateral flow immunoassay formats are among the latest
developments that have facilitated monitoring and management of
health-related complications in real-time.
APPLICATION

Immunoassays are popular tests used in medical diagnostics and

pharmacology studies. They are also important in forensic science. This
seminar will focus on two forensic applications of immunoassays: their
ability to detect trace amounts of drug in money and fingerprints, and
their ability to detect explosive residues.

When illegal drugs such as cocaine are found on money, they may help
link physical evidence (money) to drug trafficking. If cocaine is found
in a person’s fingerprints, it can show that the person was either using
cocaine or in direct contact with it. Money and latent fingermarks are
typically analyzed in the forensic lab with expensive chromatographic
methods and mass spectrometry. Though these methods are quite
accurate, they can be time-consuming and costly. Development of a
competitive enzyme immunoassay has shown promise in quantifying
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cocaine from both banknotes and fingermarks. 2 2,4,6-trinitrotoluene,

better known as TNT, is a popular explosive because it is easy to
manufacture and has strong explosive properties. If located in sites
where explosions have occurred, TNT can help forensic investigators
study the source of the explosion. Though liquid chromatography and
gas chromatography techniques are typically employed to detect TNT,
development of immunoassay tests for TNT detection have become
popular. Two tests in particular have been evaluated for their ability to
detect TNT. These include a chemiluminescent enzyme-linked
immunosorbent assay (CL-ELISA), and a colorimetric lateral flow
assay.

Chromatography techniques

Introduction

Liquid chromatography is one of the most commonly employed

analytical techniques for the separation, identification, and
quantification of different constituents present in a mixture. The
solvents, commonly called the mobile phase, are moved through the
stationary phase, where the separation takes place. Different kinds of
drugs, explosives, pesticides, toxins, etc., are analyzed using liquid
chromatography High performance liquid chromatography (HPLC)
(previously called high-pressure liquid chromatography) is the advanced
version of traditional chromatographic techniques such as paper
chromatography, thin layer chromatography, etc. It employs high
pressure to push the mobile phase through the stationary phase made up
of small particles that are densely packed inside a column. The merits of
HPLC over other techniques include its amenability for myriad samples
such as biomolecules, ions, and labile organic compounds; great
sensitivity, precision, and resolution; autosampling (through an
autosampler); and sample recovery post analysis. However, there are
some drawbacks also associated with this technique such as its costly
instrumentation and operation; extensive sample processing; time
consuming; the requirement of strict experimental conditions such as a
dust-free environment and constant temperature; and the requirement
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ofa skilled operator With advancements in different components of the

HPLC instrument, most of the aforementioned drawbacks have been
overcome. HPLC with new technological support in its pump, detector,
autosampler, etc., has taken over the market, and this new HPLC is
termed ultrahigh-performance liquid chromatography (UPLC) This
analytical technique has gained the attention of forensic investigators,
and it has been used, especially in forensic toxicology, for the analysis
of different toxicological evidence. Forensic toxicology is the division
of forensic science that helps in the legal or medical examination of drug
abuse,

HPLC

The major principle behind any liquid chromatography (LC) is the

interaction between the sample present in the mobile phase and the small
particles present in the stationary phase. Similarly, in HPLC, whenever
any mixture with myriad constituents is passed through the column with
a stationary phase, different types of physical as well as chemical
interactions occur between the analyte and the column packing material.
Principally, LC and HPLC work in a similar manner except that HPLC
is more efficient, sensitive, and easy to operate compared to LC. In
HPLC, different components of the mixture have different affinities
toward the column packing material (stationary phase). The components
with higher affinity move slower while the components with lesser
affinity travel faster in the column. Because ofthe different affinities
ofeach component, they get separated and elute from the column at
different times. The time duration for which the component is present
inside the column is termed the retention time for that component. At the
exit, the amount of these components is measured through a detector that
gives a graphical output, known as a chromatogram

Classification

HPLC techniques can be classified on the basis of separation mode, the

principle of separation, the operating scale, the type of elution, and the
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analysis type. These classifications on the basis of the aforementioned

parameters are as follows:

On the basis of separation mode

• Normal phase: In this case, the mobile phase is nonpolar in nature while
the stationary phase is its opposite, that is, polar.

• Reverse phase: Here, the stationary phase is nonpolar while the mobile
phase is polar. This technique is more common for drug analysis because
most of the drugs are hydrophilic in nature.

On the basis of principle of separation

• Ion exchange: In this type, polar compounds and ions get separated on
the basis of their charge. Resin is used in the stationary phase, which has
anions and cations attached on it. The oppositely charged ions in the
analyte interact electrostatically with the resin and get separated.

• Gel permeation: The analytes get separated on the basis of their size.
There is no interaction between the analyte and the stationary phase.

• Affinity: The interaction between the stationary phase and the analyte
is highly specific and selective. The technique is used to selectively
separate any desirable compound from a mixture. Usually, proteins are
separated using this method by using its antibody in the stationary phase.

• Adsorption: Here, the analyte molecules directly interact with the

surface ofthe stationary phase. Depending on their affinity with the
stationary phase, they elute at different times and thereby get separated.

• Chiral: The technique is use to separate stereoisomers. Enantiomers,

which are stereoisomers with nonsuperimposable mirror images, cannot
be separated using normal chromatography. To separate them, either the
stationary phase or the mobile phase needs to be chiral in nature.

On the basis of scale of operation

• Preparative: Discrete constituents of a compound can be recovered.

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• Analytical: Different constituents of the compound cannot be

recovered.132 Handbook of analytical techniques for forensic samples

On the basis of type of elution

• Isocratic: The composition of the mobile phase remains the same

throughout the process. The technique is suitable only for simple

separations. Upon increasing the complexity, the peaks become broad
and flat.

• Gradient: During the separation process, the composition of the mobile

phase keeps on changing. The technique helps in reducing the retention
time for the components that get eluted last, thereby forming sharp and
narrow peaks and thus assisting in complex separations.

On the basis of type of analysis • Qualitative: The technique is useful

only for the separation of compounds and analyzing the nature of the
chemical constituents of a mixture.

• Quantitative: The technique in addition to the separation of compounds

also gives data about their quantity and proportion in the mixture.

Instrumentation: The basic instrumentation of any HPLC includes a

solvent delivery system, pumps, a sample injector, a guard column, an
analytical column, a detector, and a data processing unit (computer)

Solvent delivery system The delivery system ofa solvent or the mobile
phase mainly consists of reservoirs and degassers. In the case of isocratic
elution, the full mobile phase is present in the reservoir while in case of
gradient elution, the reservoirs contain different solvents that constitute
the mobile phase. The mixing of these components takes place in a
mixing chamber, which is usually present before pumps. The
characteristics of solvents that can be used as the mobile phase in HPLC
are that they are noncorrosive; pure, cheap, and UV transparent; less
viscous; able to solubilize different analytes; nonflammable; and less
toxic. An online vacuum degasser is used for degassing in the case of
gradient elution in order to achieve a proper blending of the components
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of the mobile phase. If an online degasser is not available, a sonicator is

used for degassing.

Pumps

Pumps are used to push the mobile phase through the column at the
desired pressure and flow rate, which is usually expressed in mL/min.
Typically, the flow rate during operation is kept at 1 or 2 mL/min, and
the pressure is kept in the range of6000–9000 psi. The type of elution
(isocratic or gradient) is decided by the delivery of the mobile phase
through the pump. The different types of pumps used in an HPLC
instrument include:

• Syringe pump: This pump delivers a fixed volume (mostly between

250 and 500 mL) of the mobile phase and needs to be refilled again and
again, which makes it suitable only for columns with a small bore size.
The pump is driven by a mechanical lead screw that helps in delivering
the mobile phase at a perpetual rate.

• Reciprocating piston pump: The piston on this pump is connected to a

motor and moves back and forth inside a hydraulic compartment of
around 30–400 μL. The back stroke pulls the mobile phase from the
reservoir while the separation valve is shut. The following forward
stroke pushes the solvent out from the column. Alterations in the stroke
frequency or stroke volume vary the rate of flow for the mobile phase.

• Constant pressure pump: The flow rate remains in continuous phase in

this pump, which drives the mobile phase through the column by using
the pressure from a gas cylinder. The gas source is normally at low
pressure in order to achieve high pressure in the solvent.

Sample injector

The sample injector helps to introduce the sample into the flow stream
of the solvent (mobile phase). The sample is injected just before the
column with the help ofa Teflon and stainless steel valve, which is fitted
with a loop that has two positions: load and inject. In the load position,

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the sample is introduced into the sample loop using a syringe, during
which the injector is not in the flow path of the mobile phase. In the
inject position, the injector comes in the flow path of the mobile phase
and delivers the sample just ahead of the column. depicts these two
positions. The typical volume of a sample injection (manually) is 10–20
μL. The advanced versions of HPLC come equipped with autosamplers
that can continuously inject the sample with a volume ranging from 1
μL to 1 mL.

Guard column It is a small column, fitted just before the analytical

column. The purpose of using this column is to protect the analytical
column from any kind of contamination from foreign particles. It is also
packed with the same material as the analytical column. These columns
should not degrade the analytical performance of the instrument and
should not cause any significant variation in the pressure of the mobile
phase.

Analytical column

The analytical column is considered the heart ofthe HPLC instrument.

Different types of physical and chemical parameters govern the
separation of components through the stationary phase present inside the
column. Mostly, columns are made up of stainless steel, which allows
them to withstand high pressure ofthe mobile phase created by pumps.
Other materials include glass and polyether ether ketone (PEEK). The
particles used inside the column are referred to as packing material.
Depending on the type ofHPLC, the bonding groups in the packing
materials change. For normal HPLC, polar groups such as silica and
aminopropyl are used; for reverse phase HPLC, nonpolar groups such as
C18 (octadecyl), C8 (octyl), etc., are used. Among these bonding groups,
silica is the most common. However, a drawback of silica-based packing
material is that the column is suitable for separations only in the pH
range of2–8. Below pH 2, the SidObonds gets hydrolyzed, and above
pH 8, the structure of silica becomes susceptible to dissolution.
Depending upon the internal diameter ofthe column, it can be ofdifferent
types such as preparative (>25 mm), semiprep (10 mm), minibore (3.0
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mm), narrow bore (2.0 mm), microbore (1 mm), micro-LC (<0.5 mm),
and nano-LC (<0.1 mm). However, these diameters are not universal,
and may fluctuate with different manufacturers.

Detector The detector is used to identify and quantify the analytes that
get eluted from the column. The detector collects the data, converts it
into an electrical signal, and sends it as an output signal to the recorder
or the computer. The typical characteristics ofan HPLC detector include
high sensitivity, stability, and reproducibility; less response time;
nondestructive; and a similar response toward different analytes.
Various types of detectors used in HPLC are discussed below: •
Absorbance or UV-visible detector: It has a typical Z shape, and
measures the absorbance of the eluents from the column. It is of three
types: Fixed wavelength, variable wavelength, and diode array. In a
fixed wavelength detector, the wavelength is around 254 nm, and the
source is a mercury vapor lamp. In a variable wavelength detector, a
complete spectrum of the UV-visible range is available for analysis
(190–900 nm). In a diode array detector (DAD), the absorbance of the
analyte is simultaneously measured at various wavelengths.

• Fluorescence detector: The detector has the same design as a typical

fluorometer. Molecules with a fluorescence property can be very easily

detected using this detector. The sensitivity ofthese detectors is

dependent on the fluorescent property of the eluent.

• Refractive index detector: The ability of the eluent to bend or refract

light is measured through this detector. The degree of deflection can be
correlated to the concentration of the analyte present in the mobile phase.
A deflection refractometer and a Fresnel refractometer are the two main
kinds of refractive index detectors.

• Electrochemical detector: The current produced due to the redox

reaction going on at the electrode because of the analyte is measured
through this detector. The intensity of the current produced is in direct
proportionality with the concentration of the analyte.

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• Conductivity detector: The ability of the analyte to conduct or resist

current when it is placed between two electrodes to form a Wheatstone
bridge is measured through this detector. The resistance offered is in
proportionality with the concentration of the analyte.

• Evaporative light-scattering detector (ELSD): The scattering of light

by the particles present in the mobile phase postevaporation is measured
in this detector. The mobile phase gets evaporated upon passing through
a nebulizer, and the remaining analyte is thrown with a laser beam. The
scattering of light is measured at a right angle.

• Chiral detector: Depending upon the technique, it is one oftwo types:

circular dichroism (CD) and optical rotary dispersion (ORD). CD
detectors differentiate between enantiomers by quantifying the
difference between the absorption of circularly polarized light. ORD
detectors calculate the difference in the refractive index of the chiral
compounds.

Data processing unit The data are obtained from the detector and
utilized to decide the retention time and amount of analyte for the
qualitative and quantitative analysis, respectively, by the data processing
unit. The concentration of each component or analyte is measured by
calculating the area of the peak corresponding to that analyte

Application

HPLC finds extensive application in forensic toxicology, especially in

the detection of drugs, toxins, and pesticides. It can be used to
analyzeHPLC for the toxicological analysis of forensic samples

compounds that are nonvolatile, have a higher molecular weight, and are
thermally sensitive. A validated HPLC method is capable of analyzing a
compound with high sensitivity, reproducibility, automation, and
robustness. It can also be applied to a wide range ofsamples in different
matrices. Every class of analyte has a specific technique of sample

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preparation; analyzing a compound is highly dependent on the type of

sample preparation, the mobile phase, the column, and the detector.
Based on the sample, the HPLC conditions can be varied and modified
accordingly. In this section, the use of HPLC for the analysis of various
drugs, pesticides, and plant toxins will be discussed with special mention
to sample preparation. outlines the various HPLC parameters for the
analysis of opiates, pesticides, and plant toxins.

HPLC analysis of drugs In toxicology, drugs are the most widely used
compounds that contribute to a person’s death. Forensic drug analysis
using HPLC involves the identification and quantification of illicit
drugs. In this section, the HPLC analysis of cannabis and opiates will be
discussed in brief. Cannabis has long been used as a medicinal and
recreational drug and for various industrial purposes such as the
production of fiber and oil. The active constituent of cannabis is Δ9-
tetrahydrocannabinol or THC, which is known to produce euphoria and
relaxation as well as chronic dependence and addiction. Apart from
THC, cannabidiol is also an active constituent of this drug. Cannabis can
also be detected through gas chromatography (GC) and mass
spectrometry techniques, albeit with certain limitations. A major
drawback with using GC for the detection of THC and cannabidiol is
that upon heating during injection at the thermal port, these compounds
often get oxidized from their acidic form to neutral forms, thus resulting
in their decarboxylation. Therefore, they can be detected using HPLC.
In a study, THC, cannabidiol, Δ9-tetrahydrocannabinolic acid (THCA),
and cannabidiolic acid (CBDA), which are some of the common
constituents found in cannabis, were detected through HPLC-DAD. In
the study, sample preparation was carried out by drying the plant and
grinding it evenly, followed by the extraction ofthe components using a
mixture of methanol and hexane. The column used was TCC-3000RS
with a gradient elution of water and acetonitrile adjusted with 0.1%
formic acid. The compounds showed peaks at retention times of 6–12 at
a wavelength of 210 nm In yet another study, a more complex sample.

Gas chromatography

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Gas chromatography (GC) is a powerful analytical tool used for the

analysis ofvarious forensic evidence that can be made volatile. It offers
rapid analysis and can efficiently separate the analytes present in the
sample. This technique can also be efficiently coupled with mass
spectrometry and thus can be used to accurately identify the molecular
composition of the sample. Chromatography itself was first created by
Ramsey and Tswett in 1905

and 1906, respectively, for which they received the Nobel prize. The first
GC was developed by James and Martin in 1951 and 1952. While the
first ones created were very fragile and poorly constructed, they did the
job of separating the analytes in the sample. Over the years, this
instrument has undergone a revolution, and now almost every forensic
lab contains a GC equipment. In forensic science, evidence such as
drugs, toxins, and explosives is commonly encountered (Pandey et al.,
2017; Rawtani et al., 2019). Also, any forensic evidence that can be
easily solubilized in a solvent and volatilized can be analyzed using this
technique. Some equipment also has an inbuilt library of molecules
through which the analysis and identification of the evidence can be
easily done. In this chapter, a brief overview of the principle and theory
of GC, its instrumentation, and its applications in the analysis of various
forensic evidence such as alcohol, organic compounds, drugs,
explosives, ignitable materials, fingerprints, inks, paints, and toxins has
been discussed.

Principal: Principle and theory of gas chromatography

Gas chromatography, just like every other chromatography technique,

separates analytes through adsorption by a stationary phase, followed by
exposure to the mobile phase that in this case is a carrier gas. More
information on the type of stationary phases and the carrier gases used
is given in the next When the sample is introduced to the stationary
phase, it gets adsorbed on it. Based on the affinity of the analytes present
in the sample toward the stationary phase or the mobile phase, they get
separated into separate components. These analytes are further detected
by a suitable detector. The affinity ofthe sample toward the stationary
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phase is represented by the partition coefficient and provides a number

for the total amount ofanalytes adsorbed on the stationary phase. In GC,
the flow of the carrier gas is kept constant and the flow rate itself is
measured in terms ofmL/min. This flow rate is highly dependent on the
column volume and varies with different volumes; it is known as
volumetric flow. Another parameter that represents the flow of the gas
is the average linear velocity, which is a representative of the velocity of
the gas within the column according to the pressure inside it. This
parameter is more useful than the volumetric flow rate because it is
independent of the volume of the column and once calculated for a
sample, it can be applied to any instrument in any laboratory as it is not
affected by the column size. Other highly crucial parameters for analysis
using GC are the retention

time and factor. In GC, the mobile phase carries the samples that get
adsorbed on the stationary phase, thus making them travel at separate
times. The retention time is therefore the amount of time the sample
spends on the stationary phase and thereby in the column. This retention
time is an excellent parameter that indicates the time spent by the sample
in the column and the velocity of the mobile phase. Every compound
therefore has its characteristic retention time and is represented as a peak
at that particular retention time. Apart from retention time, the retention
factor is yet another parameter that indicates the retention capacity of the
sample. It is a ratio of the time spent by the analyte in the stationary
phase to the time spent in the mobile phase, and it is calculated from the
retention time. Because this technique too allows the separation of the
sample through two different phases, therefore there may be an
overlapping of two peaks. This overlapping can be prevented by
increasing the resolution in which either the mobile phase or the
stationary phase can be modified. Appropriate sample extraction
techniques can also increase the sample concentration. Also, by focusing
on factors such as the distance between two peaks and the area or width
of each peak, it is possible to optimize the resolution of a particular
sample. While resolution deals with how efficiently a chromatographic
method can be developed to separate two compounds and thus prevent

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the overlapping of their peaks, another factor known as selectivity gives

an idea of how the selectively distinguishes the two compounds and is
represented through the alpha value

Instrumentation of gas chromatography

GC typically consists of a sample injector, carrier gas, flow controller,

column, detector, and data acquisition system. The sample injector is
responsible for the inlet of samples into the stream of the carrier gas. It
is usually built to handle a myriad ofsamples such as solids, liquids, and
gases and their conversion to a volatile form so that they can be
introduced into the carrier gas. The sample inlets vary according to the
columns used. For instance, a flash vaporizer is used in case of a packed
column and split or splitless inlets are used in case of a capillary column.
More information on the different types of columns used is discussed
further. Gas samples are often obtained as mixtures of gases and liquids.
These samples are further converted to liquids before injection into the
carrier gas. Typically, syringes and valves that Gas chromatography in
forensic science are specific to gas sampling are used. Meanwhile, liquid
sampling is also done using syringes that are capable of injecting very
small amounts of samples. Extreme small amounts of liquid samples are
inserted because upon vapor-ization, they tend to expand and may
therefore pose problems in the col-umns. Solid samples are the most
challenging types of samples because of the challenges in volatilizing
them. One ofthe most common ways of pre-paring a solid sample is by
solubilizing it in a suitable solvent. Once solubi-lized, it is treated just
like any liquid sample. Here too, syringes are used to inject the samples.
However, these days, autosamplers are used in which the amount of
sample drawn from the prefitted vial is automatically done according to
the volume given in the software of the system.

The carrier gas as mentioned before serves as the mobile phase and
carries the samples from the sample inlet to the column and the detector.
The type of carrier gas used depends on the type ofdetector used because
this gas also serves as a matrix for the detector. For instance, a flame
ionization detector requires helium or nitrogen gas. Factors such as the
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purity, flow control, measurement, and compressibility of the carrier gas

are highly important. The carrier gas used must be free ofany impurities
because it can react with the stationary phase in the column, possibly
hindering and corrupting the sampling. Impurities here mean any foreign
substance apart from the gas. Therefore, even oxygen and water are
considered impurities in such cases. These impurities also tend to
produce their own peaks that may affect the movement of samples as
well as their resolution and width. The flow rate of the carrier gas is also
important as it can affect the column efficiency and the band broadening.
The flow of the carrier gas is controlled by using specific controls that
take care of the pressure, temperature, and flow rate.

The measurement is also typically done either by a soap bubble

flowmeter or an electronic device. The columns are the most crucial part
of GC and are considered the heart of the instrument. There are basically
two types of columns: the packed column and the capillary column. The
packed column consists of small particles typically of diatomaceous
earths that are usually modified with various organosilanes to enhance
the adsorption capacity of the stationary phase; they come in variable
heights, with the longest at 12 ft. Apart from this silica gel, alumina,
carbon adsorbents, and zeolites have also been used as stationary phases.
Meanwhile, the capillary column is a simple column with extreme
diameters, and it does not consist of any packing. These columns are
more efficient than the packed columns as they have a high
separation154 Handbook of analytical techniques for forensic samples
efficiency, as long columns can be easily used. These columns are also
coated by the stationary phases as mentioned above. Another equally
crucial part of the instrument is the detector. A good detector has
excellent sensitivity, signal-to-noise ratio, detectability, specificity,
linearity, and response time. Detectors commonly used for GC are
thermal conductivity detectors (TCD), flame ionization detectors (FID),
electron capture detectors (ECD), helium ionization detectors (HeD),
alkali flame ionization detectors (AFID), and flame photometric
detectors (FPD). In TCD, two filaments are used that are heated
sufficiently and the power to heat them is kept constant. Carrier gases

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such as hydrogen and helium are passed on these filaments. One filament
that is the reference filament receives only the carrier gas while the other
filament receives the carrier gas with the sample. Due to this difference,
there is a also a difference in the amount of power required to maintain
the temperature of the filament. This difference is considered and
processed further. Meanwhile, FID is highly suitable for the detection
oflow concentrations of analyte by detecting the ions that are formed
when an analyte is burned in a hydrogen flame. The greater the amount
of organic groups in the analyte, the greater the amount of ions
generated. ECD, as the name suggests, works by capturing electron
absorbing compounds such as halogens present in the carrier gas. The
greater the electronegativity, the greater the amount of electrons
captured, thus causing an increase in the current detected. HeD uses
metastable helium ions that produce ions of the analytes that hit the
metastable ions. The generated ions are captured, and an electric current
is generated that serves as the signal and is further processed. AFID is a
modification of the flame ionization detector in that the hydrogen flame
enters the FID perpendicularly to the FID jet. Due to this, there is a
constant supply of alkalis, thus limiting the detector fatigue time. Lastly,
FPD is commonly used to detect sulfur and phosphorous compounds by
measuring the fluorescence emissions of the molecules in organic
samples.

Application: Forensic sample analysis via gas chromatography

GC has been used widely in different laboratories and industries for the
detection and quantification of myriad analytes. In forensic science, GC
in combination with other techniques has helped in the investigation of
analytes such as alcohol and other volatile organic compounds (VOCs),
drugs,

Alcohol and other volatile organic compounds Alcohol and VOCs are
routinely analyzed in forensic laboratories in cases of abuse, poisoning,
leakage, etc. GC has assisted in this regard in many investigations, and
various research works have also been conducted. In a study by Davis
and Cortivo, biological specimens such as the blood, brain, liver, and
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kidney were analyzed to check for the presence of isopropanol using

headspace GC (HS-GC

Drugs Illicit drugs have been globally consumed, mainly by youngsters

for recreational purposes. Rave parties are one of the most common
locations for the possibility of such evidence. GC has been used by a
number of research teams for the detection of these drugs

HPTLC : High-performance thin-layer chromatography (HPTLC) is a

type of chromatography technique that is used for the analysis of
mixtures based on the migration of chemicals along the solid phase
under the influence family suitable mobile phase. This is a type of planar
chromatography that offers direct visual comparison of the separation of
mixture whose results can be developed into high-quality color photos
and can be easily produced as evidence and testimony in court. HPTLC
offers excellent automation, scanning, and minimum sample preparation
that can also be easily hyphenated with other techniques such as Fourier
transform infrared spectroscopy (FTIR). It is also a highly cost-effective
method that can offer high sample throughput as well

Principal

HPTLC is a type of planar chromatography that separates the samples

over a stationary phase under a suitable mobile phase and is developed
after modification in thin-layer chromatography. The differences
between TLC and HPTLC are highlighted in There are a few theoretical
considerations associated with this type of chromatography, namely
separation efficiency, partition coefficient, retention factor, spot factor,
capacity, resolution, and selectivity. During chromatography, ifa
compound has an increased affinity toward the stationary phase rather
than the mobile phase, then it tends to move slowly over the stationary
phase and separates at a different rate. This separation efficiency is
affected by factors such as the polarity, pH, and composition of the
mobile phase. Conventionally, the stationary phases in this technique are
made of silica gel that offers an extremely low contact angle for all
mobile phases. However, this water contact angle increases with the

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increase in the number of alkyl chains in the stationary phases. Another

factor that effects the separation efficiency of the compounds is the
particulate nature of the stationary phase. The mobile phase tends to
travel at a higher velocity and to a long distance in cases of stationary
phases with fine particle coating. However, this rate significantly slows
in the case of a coarse particle layer. In HPTLC, HPTLC there is a
significantly shorter migration distance. Due to this, the number of
theoretical plates, which is basically a hypothetical zone, is considered
as the equilibrium stage between the liquid and the stationary phases.
The greater the number of theoretical plates, the greater the efficiency of
separation. The separation of the samples on the stationary phase also
depends on the partition coefficient that serves as an indicator of the
migration rate of the samples. During migration, a certain part of the
compound gets absorbed by the stationary phase while a certain part
remains in the mobile phase, due to which there is a variation in the
migration times of the compound. This is presented in terms of the
partition coefficient that is further represented as a ratio of the
concentration of the sample in the stationary (Cs) and mobile phase (Cm)
at equilibrium,

HPTLC instrumentation

Typically, in HPTLC, a series of steps is followed to develop the

method. It first starts with the preparation of the standard and the sample.
After that, the stationary phase and the mobile phase are prepared and
conditioned. Once they are prepared, the sample and the standard are
applied on the stationary phase and kept in a chamber saturated with the
fumes of the mobile phases. This phase is known as chromatographic
development and is highly crucial to allow the separation ofthe analytes
in the sample and the standard. Once the chromatographic development
is done, the separated spots are later detected and scanned. Additionally,
a camera is also fixed to ensure the photographic development of the
sample and standard separation. The HPTLC instrumentation is
therefore designed to support this methodology. The stationary phase is
made up of equal layers of pristine or modified silica gel. The HPTLC
techniques are usually divided into normal phase and reverse-phase
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techniques based on the type of stationary phase used. For instance, if

silica gel is used without any modification, then it is considered to be a
normal phase technique where a nonpolar mobile phase such as
chloroform and methanol are used. However, in reverse-phase HPLC,
the silica gel is modified with compounds that are lipophilic such
HPTLC as C18 or C-12 phenyl compounds. In this case, the mobile
phase used will be polar. Apart from silica gel, aluminum oxide,
magnesium silicate, and amino or thiol modified silica gels are also used
as adsorbents for the stationary phase. It is important that the plates are
carefully stored and protected from direct sunlight or dust to prevent
contamination. The handling of the plates is also extremely important as
it can cause unnecessary contamination. Prior to analysis, layer
prewashing with a suitable solvent is done to remove any impurity and
make the stationary phase more reactive to adsorption. Preparation of
the sample and the standard is highly important in this technique, as too
high a concentration of the sample or the presence of any additional
analytes in the sample may affect the migration of the analytes on the
plate. Sample preparation processes such as grinding, sonication,
extraction, and centrifugation are the basic sample preparation
techniques. Another crucial aspect is the solvent in which the sample
will be dissolved. Typically, solvents such as methanol, ethanol, or
chloroform are used that are nonpolar and volatile. In TLC, the sample
is loaded manually but in HPTLC, special automated applicators exist
that load the correct optimal amount of the sample in the form of narrow
bands. The applicator consists of a syringe connected to a motor and the
sample is loaded in the syringe. Duetosuchan applicator, problems such
as overloading ofthe samples are minimized. Once the sample is
developed, the plate must be chromatographically developed by placing
it in a twin trough chamber. In this chamber, the mobile phase is first
poured, and the chamber is tightly closed in order to ensure that the
chamber is saturated with the vapors of the mobile phase. Once
saturated, the plate is kept inside and developed for 10–25 min, during
which the migration of the analytes occurs. During the migration of the
analytes, specific zones are created that vary in color and other
properties such as fluorescence or UV-absorbing. Once the
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chromatographic development is done, the zones are detected through

special viewing chambers that allow visualization within a specific
wavelength. UV, fluorescence, colorimetric zones, and bioluminescence
can be easily detected through this method. Currently, the detection is
done with the help of a densitometer or a scanner in which the light
source is fixed and emitted from a narrow rectangular slit. The scanner
then processes the zones and detects the absorbance or fluorescence of
the zones. Once the scanning is done, photographic documentation of
the developed film is done, which is usually done under UV light of 254
and 366 nm wavelengths and white light.

Application: HPTLC analysis of forensic samples HPTLC has been

widely used in industries such as pharmaceuticals, food, textiles, etc., for
the separation of various kinds of analytes on chromatographic plates.
Forensic experts also require separation, identification, and
quantification of analytes such as drugs, explosives, inks, dyes in fibers,
and toxins. HPTLC has been utilized by forensic experts and researchers
for such purposes. This section sheds light on the research works that
employed HPTLC for the analysis of the aforementioned forensic
samples

TLC : Thin Layer Chromatography is a technique used to isolate non-

volatile mixtures. The experiment is conducted on a sheet of aluminium
foil, plastic, or glass which is coated with a thin layer of adsorbent
material. The material usually used is aluminium oxide, cellulose, or
silica gel.

On completion of the separation, each component appears as spots

separated vertically. Each spot has a retention factor (Rf) expressed as:

Rf = dist. travelled by sample / dist. travelled by solvent

The factors affecting retardation factor are the solvent system, amount
of material spotted, absorbent and temperature. TLC is one of the fastest,
least expensive, simplest and easiest chromatography technique

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Thin Layer Chromatography Principle

Like other chromatographic techniques, thin-layer chromatography

(TLC) depends on the separation principle. The separation relies on the
relative affinity of compounds towards both the phases. The compounds
in the mobile phase move over the surface of the stationary phase. The
movement occurs in such a way that the compounds which have a higher
affinity to the stationary phase move slowly while the other compounds
travel fast. Therefore, the separation of the mixture is attained. On
completion of the separation process, the individual components from
the mixture appear as spots at respective levels on the plates. Their
character and nature are identified by suitable detection techniques.

Thin Layer Chromatography Applications

• The qualitative testing of Various medicines such as sedatives, local

anaesthetics, anticonvulsant tranquilisers, analgesics, antihistamines,
steroids, hypnotics is done by TLC.

• TLC is extremely useful in Biochemical analysis such as separation or

isolation of biochemical metabolites from its blood plasma, urine, body
fluids, serum, etc.

• Thin layer chromatography can be used to identify natural products like

essential oils or volatile oil, fixed oil, glycosides, waxes, alkaloids, etc

Hyphenated techniques :

Gas chromatography-mass spectrometry The main principle behind

GC is the volatilization of the sample in a special heated chamber
followed by the separation of the analytes present in the sample and later
detection using a detector. Here, in order to separate the samples, a
special carrier gas, which is usually an inert gas such as hydrogen or
helium, serves as the mobile phase carrying the samples over a
specialized column coated with the stationary phase. Just like any other
chromatography technique, the affinity of the analytes toward the mobile
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phase or the stationary phase determines their separation rates. The

instrumentation of GC typically consists of a headspace or an injector
that is responsible for the sample injection, followed by the GC column
that consists of nonpolar stationary phases such as dimethyl siloxanes.
The stationary phases are also made of different thicknesses, with the
thin stationary phases suitable for analytes requiring higher temperature
for volatilization and the thick stationary phases for compounds
requiring lower temperatures for volatilization. Once separated, the
detectors are used to detect the analytes. There are several detectors such
as flame ionization detectors (FID), thermal conductivity detectors
(TCD), thermionic specific detectors, and electron capture detectors
(ECD). These detectors have different modes of working and are used
according to the type of sample to be analyzed. In GC-MS, a special
device responsible for the transport of the samples from the GC to the
MS is fitted. This device ensures the integrity of the sample and
maintains its phase prior to ionization at the MS. Here too, an inert
carrier gas is used to transport the samples from one instrument to the
other. The devices used can be capillary columns, packed columns, or
jet separators, which are basically a series oftwo vacuums present under
high vacuum. Precautions are taken to ensure that the columns used are
completely inert in order to avoid any interaction of the samples with
them. Once the samples enter the mass spectrometer, they undergo
ionization followed by their separation by the mass analyzer and their
detection.

LC-MS The hyphenation of GC-MS is relatively easy as the sample is

already in the vapor form, due to which the ionization at the MS becomes
easier. Also, the interface of GC-MS is relatively simple due to this
factor. However, with liquid chromatography, there are certain
challenges to develop an interface as the sample is in a liquid phase.
Over the years, there have been several developments to develop a
suitable interface for these techniques and now LC-MS is one of the
most widely used analytical techniques. Advantages such as accurate
separation by the LC and its appropriate identification using the MS have
influenced the development of such an interface. HPLC and ultra-HPLC

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(UHPLC) are the most commonly used LC techniques that have been
hyphenated with MS. These instruments are commonly composed of a
mobile phase reservoir and a pump to ensure the movement of the
mobile phase from the reservoir to the column that consists of the
stationary phase. A sample inlet injects the sample into the column and
the analytes are later detected by the detector. Usually, in LC-MS, the
ionization source is responsible for the ionization of the liquid sample.
One ofthe earliest ionization sources was the moving belt interface in
which the analytes and the mobile phase were present in a moving belt
that moved continuously. The mobile phase was later removed through
infrared heating followed by ionization using the fast atom
bombardment (FAB) technique. However, these had certain
disadvantages such as the no uniformity of the sample. Later, soft
ionization techniques such as electrospray ionization (ESI) and
atmospheric pressure chemical ionization (APCI) were developed that
are still most commonly used today.

LC-IR IR or Fourier transform IR (FTIR) spectroscopy is a powerful

vibrational spectroscopy technique capable of analyzing the
composition of a sample. In this technique, an IR source is responsible
for emitting IR radiation that hits the sample and excites the electrons
present in the sample, due to which there are molecular vibrations. The
vibrations are specific to a particular functional group, due to which they
can be easily used to identify the nature and composition of different
samples. LC itself is an efficient tool that separates the samples
efficiently. However, developing an LC-FTIR interface is a challenging
task due to the presence of the mobile phase in HPLC that may also
contribute to its own vibrational information. Therefore, in order to
overcome this challenge, specialized flow cells are created that allow the
direct coupling of the LC with the FTIR. Typically, the most basic
approach for the LC-IR interface is the use ofa flow cell in which the
sample from the Hyphenated techniques for forensic sample

LC along with the sample are directly added. Once the sample is
detected, the spectral data from the mobile phase are subtracted, thus
allowing the information of only the sample to be retained. However,
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this method is vey much prone to errors, as any error during the
subtraction may cause the development of erroneous spectra. Also, this
technique is not suitable in cases where there is gradient elution of the
sample in LC. Apart from the flow cell approach, a solvent removal
method has also been developed in which the mobile phase is removed.
The separated analytes are fixed on a substrate, after which the analytes
are subjected to sample testing. This method is similar to allowing the
flow of salt water on a heated belt, due to which the water evaporates
and the salt is left behind. In this case, it is important that the mobile
phase used is completely free of impurities in order to eliminate any
contamination of the analytes present on the substrate

IMS-MS : IMS is a technique that separates ions on the basis ofthe time
the sample ions take to move from the source to the detector. Here, an
inlet to inject the vaporized form of the sample is present; after that, the
group of ions also known as a swarm enters a specific region known as
the drift region where the movement of the ions is controlled by a voltage
gradient. The ions move with a drift speed from the ionization source to
the detector and the time taken for this movement is known as the drift
time. Upon reaching the detector, they generate an electric current that
is further amplified for analysis. The IMS results are interpreted through
a mobility spectrum that is basically a plot between the intensity of the
signals generated and the drift time. Typically, the parameters that are
looked out for in the mobility Hyphenated techniques for forensic
sample analysis 195

spectrum are the mobility coefficient through which the properties or the
characteristic ions can be determined, the peak shape through which the
behavior ofthe ions in the drift region is known, and the secondary
spectral details that offer information on the ion fragmentation and give
an idea of the chemical class (Eiceman and Karpas, 2005)

Application : Hyphenated technique-mediated analysis of forensic

samples

A number of hyphenated techniques, are present to assist analysts and

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researchers around the world in characterizing different kinds

ofsamples. Forensic studies have also been done for many years using
hyphenated techniques for the analysis ofsamples such as drugs,
explosives, fibers, food products, inks, and toxins

Electrophoresis : Electrophoresis is a technique used to separate

macromolecules in a fluid or gel based on their charge, binding affinity,
and size under an electric field. In the year 1807, Ferdinand Frederic
Reuss was the first person to observe electrophoresis. He was from
Moscow State University. Anaphoresis is the electrophoresis of negative
charge particles or anions whereas cataphoresis is electrophoresis of
positive charge ions or cations. Electrophoresis has a wide application
in separating and analysing biomolecules such as proteins, plasmids,
RNA, DNA, nucleic acids.

Electrophoresis Principle and its types:

Charged macromolecules are placed in the electric field move towards

the negative or positive pole based on their charge. Nucleic acid has a
negative charge and therefore it migrates towards the anode.

This technique is divided into two types viz slab electrophoresis and
capillary electrophoresis.

Types of Electrophoresis:

1. Capillary electrophoresis

1. Gel electrophoresis

2. Paper electrophoresis

2. Slab electrophoresis

1. Zone electrophoresis

2. Immunoelectrophoresis
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3. Isoelectrofocusing

Immunoelectrophoresis :

• Immunoelectrophoresis refers to precipitation in agar under an electric

field.

• It is a process of a combination of immuno-diffusion and

electrophoresis.

• An antigen mixture is first separated into its component parts by

electrophoresis and then tested by double immuno-diffusion.

• Antigens are placed into wells cut in a gel (without antibody) and
electrophoresed. A trough is then cut in the gel into which antibodies are
placed.

• The antibodies diffuse laterally to meet diffusing antigens, and lattice

formation and precipitation occur permitting determination of the nature
of the antigens.

• The term “immunoelectrophoresis” was first coined by Grabar and

Williams in 1953.

Principle : When an electric current is applied to a slide layered with

gel, the antigen mixture placed in wells is separated into individual
antigen components according to their charge and size. Following
electrophoresis, the separated antigens are reacted with specific antisera
placed in troughs parallel to the electrophoretic migration and diffusion
is allowed to occur. Antiserum present in the trough moves toward the
antigen components resulting in the formation of separate precipitin
lines in 18-24 hrs, each indicating reaction between individual proteins
with its antibody.

Procedures:

1. Agarose gel is prepared on a glass slide put in a horizontal position.

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2. Using the sample template, wells are borne on the application zone
carefully.

3. The sample is diluted 2:3 with protein diluent solution (20μl antigen
solution +10 μl diluent).

4. Using a 5 μl pipette, 5 μl of control and sample is applied across each

corresponding slit (Control slit and Sample slit).

5. The gel is placed into the electrophoresis chamber with the samples on
the cathodic side, and electrophoresis runs for 20 mins/ 100 volts.

6. After electrophoresis completes, 20 μl of the corresponding antiserum is

added to troughs in a moist chamber and incubated for 18- 20 hours at
room temperature in a horizontal position.

7. The agarose gel is placed on a horizontal position and dried with blotter
sheets.

8. The gel in saline solution is soaked for 10 minutes and the drying and
washing repeated twice again.

9. The gel is dried at a temperature less than 70°C and may be stained with
protein staining solution for about 3 minutes followed by decolorizing
the gel for 5 minutes in distaining solution baths.

10. The gel is dried and results evaluated.

Application:

1. The test helps in the identification and approximate quantization of

various proteins present in the serum. Immunoelectrophoresis created a
breakthrough in protein identification and in immunology.

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2. Immunoelectrophoresis is used in patients with suspected monoclonal

and polyclonal gammopathies.

3. The method is used to detect normal as well as abnormal proteins, such

as myeloma proteins in human serum.

4. Used to analyze complex protein mixtures containing different antigens.

5. The medical diagnostic use is of value where certain proteins are

suspected of being absent (e.g., hypogammaglobulinemia) or
overproduced (e.g., multiple myeloma).

6. This method is useful to monitor antigen and antigen-antibody purity

and to identify a single antigen in a mixture of antigens.

7. Immunoelectrophoresis is an older method for qualitative analysis of M-

proteins in serum and urine.

8. Immunoelectrophoresis aids in the diagnosis and evaluation of the

therapeutic response in many disease states affecting the immune
system.

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