Module- II

Syllabus
Chemical microscope, Spectroscopy – basics – Fourier
Transform-Infrared (FT-IR), Ultraviolet-Visible (UV-Vis),
Raman, mass spectrometry.
Elemental analysis – X-ray fluorescence (XRF), powder X-ray
techniques (PXRD). Gas Chromatography (GC), and High
Performance Liquid Chromatography (HPLC) – SEM- EDAX.
Tutorial: Instrumentation analysis of forensic samples
Light travels in straight line but this concept could not explain
phenomenon like Interference, Refraction, Diffraction etc.

To explain these phenomenon light is suppose to travel in waves.

All the properties of light can be explained by corpuscular theory

and wave theory.

Radiant energy is the energy transmitted from one body to

another in the form of radiations.

Radiant energy has wave nature and associated with electric as

well as magnetic fields, these radiations are called
electromagnetic radiation.

Radiant energy is emitted by fluctuation of magnetic and electric

field.
Various forms of electromagnetic radiations are Infra-red, Ultra-violet,
X-rays, Radio waves, Microwaves etc.

Characteristics of Electromagnetic radiations

Electromagnetic consist of discrete packages of energy which are
called as photons. Photons consist of oscillating electrical and
magnetic field.

Electromagnetic radiations are

produced by the oscillation of electric
and magnetic field residing on the
atom and they are perpendicular to
each other.

Electromagnetic radiations are characterised by wavelength or

frequency or wave number.
Frequency (ν): – It is defined as the number of times electrical field
radiation oscillates in one second. The unit for frequency is Hertz (Hz).
1 Hz = 1 cycle per second.

Wavelength (λ): – It is the distance between two nearest parts of the

wave in the same phase i.e. distance between two nearest crest or
troughs.

Energy of electromagnetic radiation

is directly proportional to its
frequency and no medium is
required for their propagation. They
can travel in vacuum.

The relationship between wavelength & frequency can be written as:

ν=c/λ
As photon is subjected to energy, so E=hν=hc/λ
When an atom/molecule undergoes transition from lower energy
level to higher energy level with the absorption of photon results in
absorption spectrum.

When an atom/molecule falls from excited energy level to ground

state with the emission of photon results in emission spectrum.

Spectroscopy is a branch of science which deals with the

study of interaction of matter with light or it deals with
interaction of electromagnetic radiation with matter with
light emission and absorption spectra.

When different types of electromagnetic radiations

interact with matter it give different types of
spectroscopies. Like UV-Visible Spectroscopy, IR
Spectroscopy, X-Ray Diffraction, Mass Spectroscopy,
Atomic Absorption Spectroscopy, NMR Spectroscopy.
Electromagnetic Spectrum
 Ultraviolet-Visible Spectroscopy or Electronic
Spectroscopy
Ultraviolet and visible (UV-Vis) absorption spectroscopy is the
measurement of the attenuation (reduction in the strength of signal)
of a beam of light after it passes through a sample or after reflection
from a sample surface. Absorption measurements can be at a single
wavelength or over an extended spectral range.

Ultraviolet and visible (UV-Vis) spectroscopy is useful to measure the

number of conjugated double bonds and aromatic conjugation within
various molecules.

It also distinguishes between conjugated and non-conjugated system,

homoannular and hetroannular conjugated dienes etc.
Ultraviolet and visible (UV-Vis) spectroscopy is helpful in
➢ Detection of functional group
➢ Detection of impurities
➢ Qualitative Analysis
➢ Quantitative Analysis
➢ Single compound without chromophore
➢ Drugs with chromophoric reagent
➢ Detection of conjugation of the compound

UV radiation region extends from 150nm to 400nm & visible region

extends from 400nm to 800nm.

UV region can be further subdivided into Near UV region & Far UV

region.

The near UV region extends from 200nm to 400 nm and the region
below 200nm is known as Far UV region or Vacuum UV region.
UV spectroscopy is not useful below 200nm because in this region
absorption occurs by Nitrogen & Oxygen. To study below 200nm the
whole path length is evacuated, hence the region is known as
Vacuum UV region.

Principle of Ultraviolet-Visible Spectroscopy

When an atom/molecule abosrbs UV radiation then the electrons in
outermost shell (ground state) are promoted to higher energy state.
In ground state the spin of electrons in molecule is paired (Opposite).

In excited energy state if the spin of electrons is paired then its called
excited singlet state. On the other hand if the spin of electrons is
parallel then its called excited triplet state.

Excited triplet state is lower in energy than excited singlet state, so

its more stable.
In UV spectroscopy the absorption of energy produces changes in the
valence electron of molecule and different types of electronic
transitions are possible.

Electronic Transitions
Possible Electronic Transitions
[Link]. Electronic Transition
1 σ → σ* transition
2 π → π* transition
3 n → σ* transition
4 n → π* transition
5 σ → π* transition
6 π → σ* transition

σ → σ* transition

σ electron from orbital is excited to corresponding anti-bonding

orbital σ* and energy required for such type of transition is large.
For example- In saturated hydrocarbon (like Methane, Ethane,
Propane) σ → σ* transition required higher energy and shows
absorbance maxima at 125 nm.
π → π* transition
π electron in a bonding orbital is excited to corresponding anti-
bonding orbital π*. Compounds containing multiple bonds like
alkenes, alkynes, carbonyl, nitriles, cyanide, azo, aromatic compounds
etc. undergo π → π* transitions.
For example- Alkenes generally absorb in the region 170 to 205 nm.

n → σ* transition

This type of transition takes place in saturated compounds

containing one hetroatom with lone pair of electrons like O, N, S and
halogens are capable of n → σ* transition. These transitions usually
requires less energy than σ → σ* transitions.
For example- Saturated Halide, Alcohols, Ether, Aldehyde, Ketone
and Amines. The number of organic functional groups with n → σ*
peaks in UV region is small (150 – 250 nm).
 n → π* transition
An electron from non-bonding orbital is promoted to anti-bonding π*
orbital. Compounds containing double bond involving hetero atoms
(RCHO, RCOR, C=O, C≡N, N=O) undergo such transitions and required
less energy.
n → π* transitions require minimum energy and show absorption at
longer wavelength around 300 nm.

σ → π* transition & π → σ* transition

These electronic transitions are forbidden transitions & are only

theoretically possible. Thus, n → π* & π → π* electronic transitions
show absorption in region above 200 nm which is accessible to UV-
visible spectrophotometer.
Terms used in UV-Visible Spectroscopy
Chromophore
The part of a molecule responsible for imparting colour, are called as
chromospheres or the functional groups containing multiple bonds
capable of absorbing radiations above 200 nm due to n → π* & π →
π* transitions.
For example- Ethylenes, Acetylenes, NO2, N=O, C=O, C=N, C≡N, C=C,
C=S, etc
Auxochrome

The functional groups attached to a chromophore which modifies the

ability of the chromophore to absorb light , altering the wavelength
or intensity of absorption.
The functional group with non-bonding electrons that does not
absorb radiation in near UV region but when attached to a
chromophore alters the wavelength & intensity of absorption.
Absorption & Intensity shift
Bathochromic or Red Shift

When absorption maxima (λmax) of a

compound shifts to longer
wavelength, it is known as
bathochromic shift or red shift. The
effect is due to presence of an
auxochrome or by the change of
solvent.

For example- An auxochrome group like –OH, -OCH3 causes

absorption of compound at longer wavelength.
Hypsochromic Shift or Blue Shift
When absorption maxima (λmax) of a compound shifts to shorter
wavelength, it is known as hypsochromic shift or blue shift. The effect
is due to presence of an group causes removal of conjugation or by
the change of solvent.

Hyperchromic Shift
When absorption intensity (ε) of a compound is increased, it is known
as hyperchromic shift. The introduction of auxochrome increases the
intensity of absorption.

Hypochromic Shift

When absorption intensity (ε) of a compound is decreased, it is

known as hypochromic shift.
Application of UV Spectroscopy
Qualitative & Quantitative Analysis:
– It is used for characterizing aromatic compounds and conjugated
olefins.
– It can be used to find out molar concentration of the solute under
study.
Detection of impurities: – It is one of the important method to detect
impurities in organic solvents.
Detection of isomers are possible.

Determination of molecular weight using Beer’s law.

UV/Vis spectroscopy is routinely used in analytical chemistry for the
quantitative determination of different analytes, such as transition
metal ions, conjugated organic compounds, and biological
macromolecules. Spectroscopic analysis is commonly carried out in
solutions but solids and gases may also be studied.
UV/Vis spectroscopy is useful tool for single component analysis of
sample with known or suspected composition such as
pharmaceuticals.

UV spectroscopy identify a class or group of compounds in sample.

Many drug groups produce characteristic UV spectra.

UV spectroscopy is used to identify coloured inks and fibres.

Instrumentation
 Infrared Spectroscopy
Infrared spectrum gives information about the structure of a
compound.

The absorption of IR radiation causes bonds in a molecule to stretch

and bend with respect to one another.

IR radiation lies between the visible and microwave portion of

electromagnetic spectrum.

IR spectroscopy is concerned with the study of absorption of infrared

radiations, which causes vibrational transition in the molecule. Hence
it also known as Vibrational Spectroscopy.

The IR spectra which is obtained from IR spectroscopy provide raw

data and its non-conclusive so, mathematical algorithm Fourier
transformation is used which converts raw data into actual spectrum.
The ordinary IR region extends from 2.5 to 15. The region from
0.8  to 2.5  is called Near IR region and from 15  to 200  is called
Far IR region.

The IR spectroscopy is employed to identify the two compounds or to

determine the structure of a new compound. This technique is also
useful to predict the presence of certain functional group which
absorbs definite frequencies.

Principle of Infrared Spectroscopy

Absorption in the Infrared region is due to the changes in the
vibrational and rotational levels.

When radiations with frequency range less than 100 cm-1 are
absorbed then molecular rotation takes place in the substance. When
more energetic radiations in the region 102 to 104 cm-1 are passed
through the sample then molecular vibrations also takes place.
In IR spectroscopy the absorbed radiations bring changes in the
vibrational energy which depends upon:-

Masses of atom present in the molecule

Strength of bond

The arrangement of atoms within the molecule

When IR radiation is passed through the sample then vibrational &

rotational energies of the molecule is increased and two types of
vibrations are found:-

▪ Stretching ▪ Bending

Stretching

Stretching: The distance between atoms increases or decreases but

the atoms remain in the same bond axis.
Stretching vibrations are of two types:
(a) Symmetric Stretching (b) Asymmetric Stretching

Symmetric Stretching: The movement of atom with respect to a

particular atom in a molecule is in same direction.

Asymmetric Stretching: The movement of atom with respect to a

particular atom in a molecule is in opposite direction.

Bending

In this type of vibration the position of the atom changes with respect
to the original bond axis. Bending vibrations of four type:-
Scissoring:- The two atoms approaches each other

Twisting :- One atom moves up and other moves down the plane
with respect to the central atom.
Rocking :- The movement of two atoms takes place in the same
direction

Wagging :- The two atoms moves up & below the plane with respect
to the central atom.
Finger Print Region in IR Spectrum
The important function of IR
spectroscopy is to determine the
structure of compounds.
The possibility that two
compounds having the same IR
spectrum is least so IR spectrum
is called “Finger Print” of a
molecule.

Two identical compounds may have same spectra when run in the
same medium under the similar conditions. The region below 1500
cm-1 is rich bending vibrations and stretching vibrations.

Bending vibrations are more than stretching vibrations as each

compound has its own absorption pattern in the region. This region is
called “Finger Print Region”.
Instrumentation
IR Spectroscopy exploits the property that alkali halides become
plastic when subjected to pressure and form a sheet that is
transparent in the infrared region. Potassium bromide (KBr) is the
commonest alkali halide used in the pellets.

Potassium bromide is transparent from the near ultraviolet to long-

wave infrared wavelengths (0.25-25 µm) and has no significant optical
absorption lines in its high transmission region. In infrared
spectroscopy, samples are analysed by grinding with powdered
potassium bromide and pressing into a disc.

To obtain an IR spectrum of a solid, a sample is combined with Nujol

(paraffin based solution/mineral oils) in a mortar and pestle or some
other device to make a mull (a very thick suspension), and is usually
sandwiched between potassium- or sodium chloride plates before
being placed in the spectrometer.
The liquid membrane method involves dripping several drops of the
“neat” sample onto an NaCl or KBr aperture plate and sandwiching it
under another aperture plate, such that no gas bubbles are trapped.
The thickness is adjusted according to the sample absorbance by
inserting spacers between the aperture plates or by appropriately
tightening the screws (without breaking the aperture plates). This type
of cell is called a "liquid cell."

Application of Infrared Spectroscopy

IR can be used in many forensic applications, such as the identification
of various inks, sweat prints, hair, and other fibres, and toxic industrial
materials and chemicals
Moisture- and protein-sensitive diffuse reflectance near-infrared
spectroscopy is capable to establish the time of death from skeletal
remains & it works on the principle that at death, a person’s bones
start to lose water, and the proteins begin to decompose
Infrared has been used to measure blood alcohol content; to analyse
drug, fibre and paint samples; and to visualize wounds such as bruises
or bite marks on tissue.
It also used to detect blood and explosives. Near-IR and Fourier
transform IR have been tapped for pharmaceutical forensics as well as
ink and fibre analysis.

IR spectroscopy can be used to identify the paint left from car in an

accident. Investigators can find out the composition of paint and
figure out which vehicle have that specific paint.
Raman Spectroscopy: A Scattering Technique
Raman spectroscopy was discovered by C. V. Raman in 1928
It is a spectroscopic technique used to observe vibration , rotational,
and other low-frequency modes in a system and uses laser light source
to irradiate the sample & generates infinitely small amount of Raman
scattered light.
Raman spectroscopy is commonly used in chemistry to provide a
fingerprint by which molecules can be identified.

Raman spectroscopy (RS) is a versatile method for analysis of a wide

range of forensic samples. It can be used for both qualitative as well
as quantitative purpose.

Qualitative analysis can be performed by measuring the frequency of

scattered radiations while quantitative analysis can be performed by
measuring the intensity of scattered radiations.
Raman effect
If any substance gaseous, liquid or even solid is exposed to radiation of
definite frequency then light scattered at right angle contains
frequency different from the incident radiation and it is the
characteristic of the substance under examination. Thus the
phenomenon due to which the scattering light has a slightly different
frequency from that of incident radiation and there is change in the
atomic oscillations within the molecule is called Raman effect.

Principle of Raman Spectroscopy

A Raman spectrum is presented as an intensity-versus-wavelength

shift. Raman spectra can be recorded over a range of 4000–10 cm−1.

In Raman spectroscopy, sample is illuminated with a monochromatic

laser beam which interacts with the molecules of sample and
originates a scattered light.
Raman spectra arise due to inelastic collision (Reduction in Kinetic
Energy) between incident monochromatic radiation and molecules of
sample. When a monochromatic radiation strikes at sample, it scatters
in all directions after its interaction with sample molecules.

Much of this scattered radiation has a frequency which is equal to

frequency of incident radiation and constitutes Rayleigh scattering.

Only a small fraction of scattered radiation has a frequency different

from frequency of incident radiation and constitutes Raman scattering.
When the frequency of incident radiation is higher than frequency of
scattered radiation i.e. vi>vs, Stokes lines appear in Raman spectrum.

When the frequency of incident radiation is lower than frequency of

scattered radiation i.e. vi<vs, anti-Stokes lines appear in Raman
spectrum. Scattered radiation is usually measured at right angle to
incident radiation.
The scattered light having a frequency different from that of incident
light (inelastic scattering) is used to construct a Raman spectrum.

If v1 and v2 are the frequencies of incident and scattered

radiation/light respectively then the difference v = v1 - v2 is known
as Raman frequency.

In Raman spectra, raman lines

appears on either side of the line
of incident radiation with higher
as well as lower frequencies. The
Raman line on lower frequency
side are called Strokes lines (i.e. vi
> vs ) and those on the higher
frequency side are termed as anti
strokes lines (i.e. vs < vi )
Stokes shifted Raman bands involve
the transitions from lower to higher
energy vibrational levels and
therefore, Stokes bands are more
intense than anti-Stokes bands and
hence are measured in conventional
Raman spectroscopy.

Anti-Stokes bands are measured with fluorescing samples because

fluorescence causes interference with Stokes bands.

The magnitude of Raman shifts does not depend on wavelength of

incident radiation but Raman scattering depends on intensity of
incident radiation.

A change in polarizability (distortion in electron cloud) during

molecular vibration is an essential requirement to obtain Raman
spectrum of sample.
Raman scattering arises from molecular vibration causing a change in
polarizability. This means that intense Raman scattering occurs from
symmetric vibrations which induce a large distortion of the electron
cloud around the molecule.

A peak appearing in the Raman spectrum will be derived from a

specific molecular vibration or lattice vibration. Peak position shows
the specific vibrational mode of each molecular functional group
included in the material.

The same vibrational modes for each functional group will show a
shift in peak position due to the nearby environment surrounding the
functional group, thus the Raman spectrum shows the "molecular
fingerprint" of the target.
Application of Raman Spectroscopy
Raman spectroscopy is useful in the forensic analysis of different
types of inks in questioned documents.
Characterizing trace amounts of body fluids using Raman
spectroscopy.

Infrared and Raman spectroscopy can be used for the identification

of explosives.

Raman spectrophotometric method is used to analysed eight

barbiturates (Sedatives, Sleep inducing drugs) (phenobarbital,
pentobarbital, barbital, secobarbital, ambarbital, hexobarbital,
butabarbital, mephobarbital) and three sodium salt analogs.

Raman spectroscopy is used for the determination of drug content of

street sample of cocaine, amphetamine, ecstasy, ketamine
(Psychomotor stimulants) and deposited in latent fingerprints.
Fourier transform-Raman and infrared spectroscopy is used to
characterize benzodiazepines (A class of psychoactive drugs, used for
the treatment of anxiety and panic, seizures (fits) and insomnia or
trouble sleeping) (delorazepam, fludiazepam, flurazepam,
tetrazepam).

Raman spectroscopy is used for the rapid identification of drug

particles on nail surface and under a coating of nail varnish without
any interference from nail or nail varnish and clothing.
 Mass Spectrometry (MS)

Mass Number
The mass number (A) also known as atomic mass number or nucleon
number is total number of protons and neutrons in the atomic
nucleus.

Difference between Mass Number & Atomic Weight

Mass number is the number of protons and neutrons in an atom, and
it tells us about the mass of the atom in atomic mass units (amu).

Atomic weight is the average mass of all the isotopes of a certain

type. It is a weighted average that takes into account the abundances
of all of the different isotopes.
Mass spectrometry is a powerful analytical technique used to
quantify known materials, to identify unknown compounds within a
sample, and to elucidate the structure and chemical properties of
different molecules.

A mass spectrometer generates multiple ions from the sample under

investigation, it then separates them according to their specific mass-
to-charge ratio (m/z), and then records the relative abundance of
each ion type.

This technique basically studies the effect of ionizing energy on

molecules. It depends upon chemical reactions in the gas phase in
which sample molecules are consumed during the formation of ionic
and neutral species.
Principle
A mass spectrometer generates multiple ions from the sample under
investigation, it then separates them according to their specific mass-
to-charge ratio (m/z), and then records the relative abundance of
each ion type.

The first step in the mass

spectrometric analysis of
compounds is the
production of gas phase ions
of the compound, basically
by electron ionization. This
molecular ion undergoes
fragmentation i.e. A sample,
which may be solid, liquid,
or gas, is ionized by
bombarding it with
electrons.
This may cause some of the sample's molecules to break into charged
fragments. Each primary product ion derived from the molecular ion,
in turn, undergoes fragmentation, and so on.

These ions are then separated according to their mass-to-charge

ratio, typically by accelerating them and subjecting them to an
electric or magnetic field: ions of the same mass-to-charge ratio will
undergo the same amount of deflection and are detected in
proportion to their abundance.

Mass spectra is a plot of ion abundance versus mass-to-charge ratio.

Ions provide information concerning the nature and the structure of
their precursor (ancestor) molecule.

In the spectrum of a pure compound, the molecular ion, if present,

appears at the highest value of m/z (followed by ions containing
heavier isotopes) and gives the molecular mass of the compound.
Vaporization-Ionization-Acceleration-Deflection-Detection
Application of Mass Spectrometry
Isotope dating and tracing: Isotope ratios are used to determine the
age of materials. For example as in Carbon Dating.
Toxicology Analysis: Mass spectrometry is useful in cases involving
poison/toxins. Tissue or bodily fluids samples are collected and the
presence of toxic substance is determined, and if present, in what
concentration.

Trace Evidence: Mass spectrometry is also useful in analysing trace

evidence. Investigators at a crime scene may find microscopic
materials like carpet fiber, glass splinters or paint flakes.

A mass spectrometer can determine the precise mix of dyes used in

carpet fibres, the makeup of materials that went into any particular
glass fragment and the precise set of polymers present in any paint
sample.
Arson Investigation: An arson investigator might be able to identify
the use of an accelerant through burn patterns or lingering odours, 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.

Analysis of Explosives: When a bomb detonates, it may not leave

behind much in the way of physical evidence, only small fragments
and chemical residues can be retrieved from the crime scene.
Commercial explosive manufacturers each utilize their own unique
mix of chemicals and a spectrometer can analyse 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.
 X-ray fluorescence
XRF (X-ray fluorescence) is a non-destructive analytical technique used
to determine the elemental composition of materials. XRF analysers
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.

Principle of X-ray fluorescence (XRF)

When an element is placed in a beam of x-rays, the x-rays are
absorbed. The absorbing atoms become ionized (e.g. due to the x-ray
beam ejects the electron in the inner shell).
An electron from higher energy
shell (e.g., the L shell) then fall
into the position vacated by
dislodged inner electron and emit
x-rays or characteristic
wavelength. This process is called
x-ray fluorescence.

The wavelength of fluorescence is characteristic of the element being

excited, measurement of this wavelength enable us to identify the
fluorescing element.
The measurement of the fluorescence intensity makes possible the
quantitative determination of an element.

Most atoms have several electron orbitals (K shell, L shell, M shell).

When x-ray energy causes electrons to transfer in and out of these
shell levels, XRF peaks with varying intensities are created and will be
present in the spectrum.
A graphical representation of X-ray intensity peaks as a function of
energy peaks. The peak energy identifies the element, and the peak
height/intensity is indicative of its concentration.

Application of X-ray fluorescence (XRF)

X-ray fluorescence spectroscopy (XRF) is a method for measuring the
thickness of coatings and for analysing materials.

It can be used for the qualitative and quantitative determination of

the elemental composition of a material sample as well as for
measuring coatings, coating systems and analysis of lead based paints.

An X-ray fluorescence (XRF) is used for non-destructive chemical

analyses of rocks, minerals, sediments, ceramics and fluids.
X-ray fluorescence (XRF) indicates protein distribution & provides a
diagnostic link for the medical practitioner.

Study of paintings and sculptures etc.

Determination of Chloride, Strontium in blood serum and bone tissue.
Elemental analysis of tissues, bone and body fluids.

Used in the determination of pesticides on fruits and herbal drugs.

Determination of trace elements in plants and foods.

Measurement of heavy metals in soil, sediments, water & aerosols.

Powder X-ray techniques (PXRD)

Powder diffraction is a scientific technique using X-Ray, neutron, or
electron diffraction on powder or microcrystalline samples for
structural characterization of materials.
An instrument dedicated to performing such powder measurements is
called a powder diffractometer.
X-rays are electromagnetic radiations with wavelength in the range
0.1-100 ºA (0.01nm – 10nm)
Max von Laue, in 1912, discovered X-ray diffraction i.e. crystalline
substances act as three-dimensional diffraction gratings for X-ray
wavelengths

X-ray powder diffraction (XRD) is a rapid analytical technique

primarily used for phase identification of a crystalline material and
can provide information on unit cell dimensions. The analysed
material is finely ground, homogenized, and average bulk composition
is determined.

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

produce electrons, accelerating the electron towards the target by
applying the voltage & bombarding the target material with electron.

When electrons have sufficient energy to dislodge inner shell electrons

of the target material, characteristic X-ray spectra are produced.
Crystal monochrometers, is required to produce monochromatic X-
rays needed for diffraction. These X-rays are collimated (made parallel
/ set at particular angle) and directed 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 (imposing) the
sample satisfies the Bragg Equation Bragg’s Law (nλ = 2d sin θ),
constructive interference occurs and a peak in intensity occurs.

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.
Where n = an integer (Order of reflection),  = Wavelength of the
incident X-ray, d = inter-planar spacing of the crystal,  = Angle of
incidence.

Bragg’s law relates the wavelength of electromagnetic radiation to the

diffraction angle and the lattice spacing in a crystalline sample.

The diffracted X-rays are then detected, processed and counted. By

scanning the sample through a range of 2θ angles, 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. This is achieved
by comparison of d-spacings with
standard reference patterns.
Application of Powder X-ray Diffraction
X-ray powder diffraction is most widely used for the identification of
unknown crystalline materials (e.g. minerals, inorganic compounds).
Determination of unknown solids is critical to studies in geology,
environmental science, material science, engineering and biology,
forensic science.

Characterization of crystalline materials

Identification of fine-grained minerals such as clays and mixed layer

Determination of unit cell dimensions

Measurement of sample purity

Determining the thickness, roughness and density of the film using

glancing incidence X-ray reflectivity measurements.
Make textural measurements, such as the orientation of grains, in a
polycrystalline sample.
Chromatography
‘Chromatography’ is an analytical technique for identification,
separation and purification of components of a mixture on the basis of
difference in their affinity for stationary and mobile phase.
Chromatography involves two mutually immiscible phase (Stationary &
Mobile phase) which are brought into contact. A sample to be
analysed is introduced into a mobile phase.
The sample is then carried along through a stationary phase packed in
the form of column or thin layer.

Different constituents of the sample undergo repeated partition

between mobile phase and stationary phase. This result in gradual
separation of constituents of the sample into bands in the mobile
phase.
The liquid stationary phase is coated onto an inert powdered or
granular solid support which is either spread on a supporting sheet in
the form of a thin layer or packed into a column.

During chromatographic separation the solute molecules are

continuously moving from mobile phase to stationary phase. The rate
of migration depends on the time solute spends in mobile phase. The
process transferring of solute from mobile phase to stationary phase is
known as “Sorption”.

Partition Ratio is the concentration of solute molecules in stationary

phase to the concentration of solute molecules in mobile phase .

Retention Factor (Rf)=

Distance travelled by the constituent substance on stationary phase
Distance travelled by the solvent on stationary phase
Rf value is the good indicator of whether an unknown compound and
known compound are similar, if not identical.

Types of Chromatography
Paper Chromatography Thin Layer Chromatography

Liquid Column Chromatography Size Exclusion Chromatography

Ion Exchange Chromatography Affinity Chromatography

High Performance Liquid Chromatography

Gas Chromatography
 Gas Chromatography (GC)
When any separation process involving a moving gaseous phase for
separation of compounds from mixture is known as Gas
Chromatography.

Principle

The mixture is introduced into the moving carrier gas (H2, He, N2, Ar,
CO) and its allowed to pass through column (Stationary Phase - Liquid
/ Solid) so that the component of mixture distributes themselves
between two phases.

Distribution is controlled by three physical transport phenomena:

✓ Flow
✓ Diffusion
✓ Partition of the solute between the mobile phase and stationary
phase
The process of distribution of the solute between two phases
continues till dynamic equilibrium is established. At this stage the
concentration of molecules of each solute in the stationary & mobile
phase is constant.

Partition Ratio = Concentration of solute molecules in stationary phase

Concentration of solute molecules in mobile phase

Partition Ratio depends upon:

✓ Nature of solute (Volatile / Non Volatile)

✓ Nature of solvent (Polar / Non Polar)
✓ Concentration of Liquid Phase (In case of GLC)
✓ Temperature
Application of Gas Chromatography
GC is widely used for analysis of body fluids for the presence of illegal
substances, to testing of fibre and blood from a crime scene, and to
detect residue from explosives.

GC is used to determine which fluids and compounds are present

inside a human body after death. This is vital in determining whether
or the person was intoxicated either from alcohol or drug abuse at the
time of death, or indeed whether there is any poison or other harmful
substance present in their body.

Gas chromatography can also be used to test samples found at a crime

scene, whether these be blood samples or fibre samples from clothing
or other materials.
Gas chromatography−differential mobility spectrometry (GC−DMS)
was investigated as a tool for analysis of ignitable liquids from fire
debris.
Scanning Electron Microscopy-Energy Dispersive X-ray
Analysis (SEM-EDAX)
Scanning Electron Microscopy (SEM) is used to determine the
morphological aspects of sample (shape, size of particles), with EDAX
we can determine information on the chemical composition of
sample. The instrument is the same for both analysis, so the
information can be complementary. The choice of one or another
depends on need.

EDAX develops the best solutions for micro- and nano-

characterization, where elemental and/or structural information is
required, making analysis easier and more accurate.

The scanning electron microscope (SEM) uses a focused beam of high-

energy electrons to generate a variety of signals at the surface of solid
specimens.
The signals that derive from electron-sample interactions reveal
information about the sample including external morphology
(texture), chemical composition, and crystalline structure and
orientation of materials making up the sample.

In most applications, data are collected over a selected area of the

surface of the sample, and a 2-dimensional image is generated that
displays spatial variations in these properties.
Areas ranging from approximately 1 cm to 5 microns in width can be
imaged in a scanning mode using conventional SEM techniques
(magnification ranging from 20X to approximately 30,000X, spatial
resolution of 50 to 100 nm).

The SEM is also capable of performing analyses of selected point

locations on the sample; this approach is especially useful in
qualitatively or semi-quantitatively determining chemical
compositions (using EDAX), crystalline structure, and crystal
orientations (using Electron backscattered Diffraction “EBSD”).
Principle- (SEM-EDAX)
Accelerated electrons in an SEM carry significant amounts of kinetic
energy, and this energy is dissipated as a variety of signals produced
by electron-sample interactions when the incident electrons are
decelerated in the solid sample.
These signals include secondary electrons (that produce SEM
images), backscattered electrons (BSE), diffracted backscattered
electrons (EBSD is used to determine crystal structures and
orientations of minerals), photons (Characteristic X-ray that are used
for elemental analysis and continuum X-rays), visible light
(Cathodoluminescence –CL) and heat.
X-ray generation is produced by inelastic collisions of the incident
electrons with electrons in discrete orbitals (shells) of atoms in the
sample. As the excited electrons return to lower energy states, they
yield X-rays that are of a fixed wavelength (i.e. related to the
difference in energy levels of electrons in different shells for a given
element).
Application of SEM-EDAX
Typical Applications
✓ Characterization of material structures
✓ Assessment of reaction interfaces, service environment and
degradation mechanisms
✓ Characterization of surface defects, stains and residues on metals,
glasses, ceramics and polymers
✓ Measurement of the thickness of layered structures, metallised
layers, oxide films, composite materials using cross sectional
imaging
✓ Particulate and contaminant analysis on and within materials.
Typical Industries using SEM / EDX
✓ Aerospace, Automotive
✓ Materials, Minerals
✓ Glass, Ceramics and Refractories
✓ Healthcare, Medical Devices
✓ Semiconductors, Electronics.