SRI KRISHNA COLLEGE OF ENGINEERING AND

TECHNOLOGY
(AN AUTONOMOUS INSTITUTION, AFFILIATED TO ANNA UNIVERSITY, ACCREDITED BY NAAC
WITH ‘A’ GRADE)
ACCREDITED BY NBA (CSE, IT, ECE, EEE, MECH, MCT & CIVIL)

SKCET CLASS LECTURE

SUBJECT : 23AS101 - APPLIED SCIENCE

(Common to B.E. CSD, CSE, CSE (CYBER SECURITY),
ECE, EEE, MCT & B.Tech. AI&DS, IT)

MODULE : ANALYTICAL TECHNIQUES

TOPIC : SPECTROSCOPY

FACULTY : Dr. M. JEYARAJ

www.skcet.ac.in
 Spectroscopy
One of the most powerful tools available for the study of

the atomic and molecular structure and is used in the

analysis of a most of the samples. Spectroscopy is used to

study the interactions of electromagnetic radiation with

matter.

During the interaction, the energy is absorbed or emitted

by the matter. The measurement of this radiation frequency

(absorbed or emitted) are made using spectroscopy.

TYPES OF SPECTROSCOPY
The study of spectroscopy can be carried out under the
following headings

1. Atomic spectroscopy.

2. Molecular spectroscopy.
1. Atomic spectroscopy
• It deals with the interaction of the electro
magnetic radiation with atoms.
• During which the atoms absorb radiation and
gets excited from the ground state electronic
energy level to another.
2. Molecular spectroscopy
• It deals with the interaction of electromagnetic
radiation with molecules.

• This results in transition between rotational,

vibrational and electronic energy levels.
 Difference between Atomic Spectroscopy
and Molecular Spectroscopy

Atomic Spectroscopy Molecular Spectroscopy

It occurs due to the interaction It occurs due to the interaction of

of atoms with electromagnetic molecules with electromagnetic
radiation radiation

Molecular spectra is a
Atomic spectra is a line spectra
complicated spectra

It is due to vibrational, rotational

It is due to electronic transition
and electronic transition in a
of an atom
molecule
How does a Spectrum arise?

. Absorption spectrum
Consider a molecule having only two energy levels E1
and E2. When a beam of electromagnetic radiation is
allowed to fall on a molecule in the ground state, the
molecule absorbs photon of energy hv and undergoes a
transition from the lower energy level to the higher energy
level.
 Absorption spectrum
• The measurement of this
decrease in the intensity of
radiation is the basis of
absorption spectroscopy.
The spectrum thus obtained
is called the absorption
spectrum.
2. Emission spectrum
If the molecule comes
down from the excited state
to the ground state with the
emission of photons of
energy hv, the spectrum
obtained is called emission
spectrum.
 PHOTOPHYSICAL LAW

Lambert’s Law
When a beam of monochromatic
radiation is passed through a
homogeneous absorbing medium the
rate of decrease of intensity of the
radiation dI with a thickness of
absorbing medium ‘dx’ is proportional
to the intensity of the incident radiation
‘I’.
Lambert’s Law
 Beer’s law (or) Beer-Lambert’s Law
When a beam of monochromatic radiation is passed
through a homogeneous absorbing medium the rate of
decrease of intensity of the radiation dI with a thickness of
absorbing medium ‘dx’ is proportional to the intensity of
the incident radiation ‘I’ as well as concentration of the
solution
Beer’s law (or) Beer-Lambert’s Law
 Application of Beer-Lambert’s law
Determination of unknown concentration
 Limitations of Beer-Lambert’s law
Beer-Lambert’s law is not obeyed if the radiation used is
not monochromatic.
It is applicable only for dilute solutions.
The temperature of the system should not be allowed to
vary to a large extent.
It is not applied to suspensions.
The deviation may occur, if the solution contains
impurities.
Deviation also occurs if the solution undergoes
polymerization (or) dissociation.
 ULTRAVIOLET (UV) AND VISIBLE
SPECTROSCOPY

Principle
Ultraviolet (UV) and visible spectra arise from the
transition of valence electrons within a molecule or ion
from a lower electronic energy level (ground state E0) to
higher electronic energy level (excited state E1).
 ULTRAVIOLET (UV) AND VISIBLE
SPECTROSCOPY

This transition occurs due to the absorption of UV

(wavelength 100-400 nm) or visible (wavelength 400-750
nm) region of the electronic spectrum by a molecule (or)
ion.
The actual amount of energy required depends on the
difference in energy between the ground state and the
excited state of the electrons.
E1 – E0= hν
 Types of electrons involved in organic molecule
S.No Electron Examples Energy to require Present In
1 σ Saturated long Absorption appears Single
electrons chain below 200 nm bonded
Hydrocarbons (large energy) compounds
Eg Methane
125 nm
2 π Unsaturated Absorption appears Double bond
electrons hydrocarbons UV to visible and triple
Alkenes, Alkynes bonded
and aromatic compounds
compounds
3 n Organic UV radiation Unshared or
electrons compounds non bonded
containing N, O electrons
and Halogens
Three types of electrons present in the HCHO
(Formaldehyde)
The energy values for different transitions are in the
following order.
n → π∗ < π → π∗ < n → σ∗ << σ → σ∗
1. σ → σ∗ electrons
The electrons are involved is single bond there is no lone
pair of electrons
The energy required for the transition is very large.
The absorption band appears in the far UV region.
Example: Saturated Hydrocarbons
CH4, CH3-CH3, CH3-CH2-CH3 (alkanes)
CH4 for σ → σ∗ λmax = 121 nm
CH3-CH3 for σ → σ∗ λmax = 135 nm
2. π → π∗ transitions
This transitions occurs due to the transitions of an
electron from bonding π orbital to anti bonding orbital π*.
This transitions occur in any molecule having π electron
system.
Example: Ethylene CH2=CH2
It shows intense band at 174 nm (allowed transitions) and
weak band at 200 nm. Both are due to π → π∗
3. n → π∗ transitions
n → π∗ transitions are shown by unsaturated
molecules containing hetero atoms like N, O & S. It
occurs due to the transition of non-bonding lone pair of
electrons to the anti bonding orbitals. This transition
shows a weak band, and occurs in longer wavelength
with low intensity.
Example
4. n →σ* Transitions
Saturated compounds containing atoms with lone pairs
(non-bonding electrons) are capable of n→σ* transitions.
These transitions usually need less energy than
σ →σ* transitions. They can be initiated by light whose
wavelength is in the range 150 - 250 nm. The number of
organic functional groups with n →σ* peaks in the UV
region is small.
 Important term used in UV Spectroscopy

Chromophores (colour producing groups)

The presence of one or more unsaturated linkages (π
electrons) in a compound is responsible for the colour of
the compound. This linkages are referred to as
Chromophores.
Examples

Chromophore undergoes π→π∗ transitions in shorter

wavelength regions of UV radiations.
Auxochrome (colour intensifying group)
It refers to an atom or a group which does not gives rise to
absorption band on its own. But when conjugate to
chromophore will cause red shift.
Example -OH, -NH2, -Cl, -Br, -I etc
Chromophore Auxochrome

Benzene (λmax = 225nm) Aniline

λmax = 280 nm
 Difference between Chromophore and Auxochrome

S.No Chromophore Auxochrome

1 This group is responsible It does not impart colour , but

for the colour of the when conjugate with
compound
chromophore produce colour

2 It does not form salt But it forms salt

3 It contains at least one It contains lone pair of

multiple bond electrons
Example –NO2, -NO, Example
-N=N -OH, -NH2, -NR2
 Some important definition related its
change in wavelength and intensity

1 Bathochromic Shift Shift to higher wavelength

(red shift) (lower frequencies)
2 Hypsochromic Shift Shift to lower wavelength
(blue shift) (Higher frequencies)
3 Hyperchromic effect An increase in intensity

4 Hypochromic effect A decrease in intensity

Intensity
 INSTRUMENTATION
I. Components
1. Radiations source
Invisible – UV spectrometers, the most commonly used
radiation sources are hydrogen (or) deuterium lamps.
2. Monochromators
The monochromator is used to disperse the radiation
according to the wavelength. It allows the light of the
required wave length to pass through, but absorbs the light
of other wave length.
3. Cells (sample cell and reference cell)
The cells, containing samples or reference for analysis,
should fulfill the following conditions.
They must be uniform in construction.
The material of construction should be inert to solvents.
They must transmit the light of the wavelength used.

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4. Detectors
Three common types of detectors.
They are Barrier layer cell, Photomultiplier tube, Photocell.
The detector converts the radiation, falling on which, into
the current. The current is directly proportional to the
concentration of the solution.
5. Recording system
The signal from the detector is finally received by the
recording system. The recording is done by recorder pen.
II Working of Visible and UV spectrophotometer
 Working of Visible and UV Spectrophotometer

The radiation from the source is allowed to pass through

the monochromator unit.
The monochromator allows a narrow range of wavelength
to pass through an exit slit.
The beam of radiation coming out of the monochromator
is split into two equal beams.
One-half of the beam (the sample beam) is directed to pass
through a transparent cell containing a solution of the
compound to be analysed.
 Working of Visible and UV Spectrophotometer
The another half (the reference beam) is directed to pass
through an identical cell that contains only the solvent.
The instrument is designed in such a way that it can
compare the intensities of the two beams.
If the compound absorbs light at a particular wavelength,
then intensity of the sample beam (I) will be less than that
of the reference beam (Io).The instrument gives output
graph, which is a plot of wave length Vs absorbance of the
light. This graph is known as an absorption spectrum.
 Applications
1. Predicting relationship between different groups
Between two or more C–C multiple bonds(= (or) ≡ bonds).
Between C – C & C – O double bonds.
Between C – C double bonds and the aromatic benzene ring.
2. Qualitative analysis
Identification of aromatic compounds and conjugated olefins
–comparing sample with the same of known compounds
available in reference books.
3. Detection of impurities
UV absorption spectroscopy is the best method for detecting
impurities in organic compounds.
4. Quantitative analysis
UV absorption spectroscopy is used for the quantitative
determination of compounds, which absorbs UV light.
5. Determination of molecular weight
The molecular weight of a compound can be determined if it
can be converted into a suitable derivative, which gives an
absorption band.
6. Dissociation constants of Acid and Base
The dissociation constant (Pka) of an acid (HA) can be
determined by determining the ratio of [HA]/[A]
spectrophotometerically from the graph plotted between
absorbance Vs wavelength at different pH values. The
values substituted in the equation.
7. Study of tautomeric equilibrium
The percentage of various keto-enol forms present in
a tautomeric equilibrium can be determined by measuring
the strength of the respective absorption band using UV
spectroscopy.
Example
Ethyl acetoacetate

λmax = 275nm λmax = 244 nm

8. Determination of calcium in blood serum
Calcium in the blood can be determined by
converting the ‘Ca’ present in 1 ml of the serum as its
oxalate and dissolving it in H2SO4 and treating it with dilute
ceric sulphate solution. The absorption of the solution is
measured at 315 nm. Thus the amount of ‘Ca’ in the blood
serum can be calculated.
 INFRARED SPECTROSCOPY
Principle
IR spectra are produced by the absorption of energy by a
molecule in the infrared region and the transitions occur
between vibrational levels. So, IR spectroscopy is also
known as vibrational spectroscopy.
Range of Infrared Radiation
The range of the electromagnetic spectrum extending from
12500 to 50 cm− 1 (0.8 to 20 μ) is commonly referred to as the
infrared. This region is further divided into three sub-regions.
 λ = 0.8 2.5 15 20 μ

γ = 12500 4000 667 50 cm-1

Near infrared : The region is from 12500 to 4000cm-1

Infrared (or) ordinary IR : The region is from 4000 to 667
cm-1
Far infrared : The region is from 667 to 50 cm-1
 Molecular Vibrations and Origin of IR
Spectrum
1. Stretching vibrations: During stretching the distance
between two atoms decreases or increases, but
bond angle remains unaltered.
2. Bending (or) deformation vibrations: During bending
bond angle increases and decreases but bond distance
remains unaltered.
Note: Vibrational changes depend on the masses of the
atoms and their spatial arrangement in the molecule.
Finger Print Region
The vibrational spectral (IR spectra) region at 1400 − 700
cm− 1 gives very rich and intense absorption bands. This
region is termed as fingerprint region. The region 4000 −
1430 cm− 1 is known as a Group frequency region.
Uses of fingerprint region
1. IR spectra are often characterized as molecular
fingerprints, which detect the presence of functional
groups. Fingerprint region is also used to identify and
characterize the molecular just as a fingerprint can be used
to identify a person.
 Types of stretching and bending vibrations
The number of fundamental (or) normal vibrational
modes of a molecule can be calculated as follows.
1. For Non-linear molecule
A non-linear molecule containing ‘n’ atoms has (3n − 6)
fundamental vibrational modes.
H2O = (3 x 3- 6) = 3 fundamental vibrational modes
CH4 = (3 x 5 -6) = 9 fundamental vibrational modes
C6H6 = (3 x12- 6) = 30 fundamental vibrational modes.
Thus, for a vibration to be active in IR, the dipolemoment
of the molecule must Change
IR spectrum of Water
2. For linear molecules
A linear molecules containing ‘n’ atoms has (3n-5)
fundamental vibrational modes
Example
CO2 = (3x3 -5) = 4 fundamental vibrational modes
Carbon dioxide
Carbon dioxide is a linear triatomic molecule and has 3n −
5 = (3 × 3 − 5) = 4 fundamental vibrational modes.
Out of the four normal modes of vibration of CO2, only the
asymmetric stretching and bending vibrations ie., (ii),
(iii) and (iv) involve a change in dipole moment (all are
IR - active) and (i) no change in dipole moment (IR
inactive)
 Thus, though there are three active vibrations, two of
them ((iii) and (iv)) have the same frequency, so the IR
spectrum of CO2 exhibits only two bands ie., one at 526
cm− 1 and another at 2565 cm− 1.
 INSTRUMENTATION
1. Radiation Source
The main source of IR radiation.
Nichrome wire Nernst glower, which is a filament
containing oxides of Zr, Th, Ce, held together with a
binder. When they are heated electrically at 1200 to
2000°C, they glow and produce IR radiation.
2.Monochromator
It allows the light of the required wavelength to pass
through but absorbs the light of another wavelength.
 INSTRUMENTATION

3. Sample Cell
The cell, holding the test sample, must be transparent to
IR radiation.
4. Detector
IR detectors generally convert thermal radiant energy
into electrical energy. There are so many detectors, of
which the followings are important.
Types of detectors
• Photoconductivity cell.
• Thermocouple
• Pyro-electric detectors.
5. Recorder
The recorder records the signal coming out from the
detector.
 Working of IR Spectrophotometer
The radiation emitted by the source is split into two
identical beams having equal intensity. One of the beams
passes through the sample and the other through the
reference sample.
 Working of IR Spectrophotometer

When the sample cell contains the sample, the half-beam

travelling through it becomes less intense. When the two
half beams (one coming from the reference and the other
from the sample) recombine, they produce an oscillating
signal, which is measured by the detector. The signal from
the detector is passed to the recording unit and recorded.
 Applications of IR spectroscopy
1. Identity of the compound can be established
The IR spectrum of the compound is compared with that of
known compounds. From the resemblance of the two
spectra, the nature of the compound can be established.
This is because a particular group of atoms gives a
characteristic absorption band in the IR spectrum.
Example :
2. Detection of Functional groups
(i) In a given environment a certain functional group will
absorb IR energy of nearly same wavelength in all the
molecule.
Example
3. Testing Purity of the sample
Pure sample will give sharp and well resolved peak. But
impure will give broad and poorly resolved absorption
bands. Thus by comparing IR spectra of pure compound,
presence of impurity can be detected.
4. Study of progress of a chemical reaction
The progress of a chemical reaction can be easily
followed by examining the IR spectrum of test solution at
different time intervals.
5. Determination of shape (or) Symmetry of a molecule
Whether the molecule is linear (or) non-linear (bend
molecule) can be found out by IR spectra.
Example
IR spectra of NO2 gives three peaks at 750, 1323, and
1616 cm− 1. According to the following calculations,
For non-linear molecule = (3n − 6) = 3 peaks.
For linear molecule = (3n − 5) = 4 peaks.
6. To study tautomerism
Tautomeric equilibrium can be studied with the help of IR
spectroscopy.
Example
The common systems such as keto-enol, lacto-lactum,
mercapto- thioamide contain groups like C=O, -OH, -NH
or C=S. These groups show a characteristics absorption
band in the IR spectrum, which enable us to find at which
form predominates in the equilibrium.
 Industrial applications
(a) Determination of structure of chemical products
Example: Polymer structure
(b) Determination of molecular weight
Molecular weight, of a compound, can be determined by
measuring end group concentrations, using IR
spectroscopy.
(c) Crystallinity
The physical structure like crystallinity can be studied
through changes in IR spectra.
 FLAME PHOTOMETRY (OR) FLAME EMISSION
SPECTROSCOPY

Flame photometry is a method in which, the intensity of

the emitted light is measured when a atomized metal is
introduced into a flame. The wavelength of the colour
tells us what the element is, and the intensity of the colour
tells us how much of the element is present.
Theory (or) Principle
When a metallic salt solution is introduced into a flame,
the following processes will occur.
The solvent is evaporated, leaving behind the solid salt
particle.
The salt is vaporized into the gaseous state and dissociated
into atoms.
Some of the atoms from the ground state are excited to a
higher energy state by absorbing thermal energy from the
flame.
 INSTRUMENTATION

I. Components
1. Burner
The flame must possess the following characteristics.
It should evaporate the solvent from the sample solution.
It should dissociate the solid into atoms.
It should excite the atoms and cause them to emit radiant
energy.
 2. Mirror
The radiation from the flame is emitted in all directions
in space. In order to increase the amount of radiation
reaching the detector, a convex mirror is used which is set
behind the burner.
3. Slits
Entrance slits : It is kept between the flame and
monochromator. It permits only the radiation coming from
the flame and a mirror.
Exit slit: It is kept between the monochromator and
detector. It prevents the entry of interfering lines.
4. Monochromator (or) Prism (or) Grating (or) Filter
It allows the light of the required wavelength to pass
through but absorbs the light of other wavelengths.
5. Detector
The radiation coming out from the filter is allowed to fall
on the detector, which measures the intensity of the
radiation falling on it. Photomultiplier (or) photocell is
used as a detector, which converts the radiation into an
electrical current.
6. Amplifier & Recorder
The current coming out of the detector is weak, so it is
amplified and recorded.
 Working of Flame photometer

Air, at a given pressure, is passed into an atomiser. The

suction so-produced draws some solution of the sample
into the atomiser.
Air + sample solution is then mixed with fuel gas in the
mixing chamber. The Air + sample solution + fuel gas
mixture is then burnt in the burner.
The radiation, emitted by burner flame, is passed
successively through the lens, filter, detector, amplifier and
finally into a recorder.
Calibration Curve
 Applications of flame photometry
1. Estimation of sodium by flame photometry

The instrument is switched on. Air supply and gas supply

are regulated. First distilled water is sent and ignition is
started.
After the instrument is warmed up for 10 min, the
instrument is adjusted for zero reading in the display.
Since sodium produces a characteristic yellow emission at
589 nm, the instrument is set at λ = 589 nm and the
readings are noted.
 Applications of flame photometry

2. Qualitative Analysis
The elements of group I & II. (K, Na, Li, Ca, Mg, etc) can
be detected visually from the colour of the flame.
Non-radiating elements such as carbon, hydrogen and
halides cannot be detected using this method.
3. Quantitative analysis
The amount of the elements in a group I & II (alkali &
alkaline-earth metals) can be determined from the sample.
Certain transition elements, such as Cu, Fe & Mn can also
be determined using flame photometry.
Other applications
The measurement of these elements is very useful in
medicine, agriculture and plant science.
Flame photometry is extensively used in the analysis of
biological fluids and tissues.
In soil analysis, the elements like Na, K, Al, Ca, Fe, etc.,
are determined. Industrial and natural waters, petroleum
products, cement, glass, and metallurgical products can
also be analysed by this method.
Limitations of flame photometry
It cannot be used for the determination of all metal
atoms and inert gases.
Only liquid samples must be used.
It does not provide information about the molecular
form of the metal present in the original sample.

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