Chemistry Department of Basic Sciences & Humanities

Unit V- Instrumental Methods And Applications

Electromagnetic spectrum
1. The entire range over which electromagnetic radiation exists is known as electromagnetic
spectrum.
2. The electromagnetic spectrum covers all ranges of wavelength.

3. A spatial representation of an electromagnetic wave at one instant in time is as shown.

4. The wave is propagating to the right, in the + X direction, so the electric and magnetic
fields are perpendicular to the X-axis and to each other.
5. Electromagnetic spectrum diagram can be represented in terms of frequency as below.

(1) Radio waves:

a) These are the lowest frequency electromagnetic waves.
b) They can be produced by charges oscillating through wires or conductors in ordinary
electric circuits.

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c) These are used to produce extremely low frequency radio waves to communicate with
submerged submarines.
(2) Microwaves and Radars
a) These are produced by electronic circuits
b) But with antennas that are typically around one meter or less in size.
(3) Infrared waves
a) These are produced by electric charges oscillating in atoms and molecules.
b) These are associated with the changes in the vibration of the molecules.
c) This region is further divided to Near-Infrared, Infrared, Far-Infrared.
(4) Visible light
a) These are produced by vibrational oscillations of atoms and molecules.
b) When electrons make a transition from one energy level to another, they may emit or
absorb electromagnetic waves.
d) The different colors of visible light correspond to electromagnetic waves with different
frequencies.
e) The lowest frequency of visible light is red, then orange, yellow, green, blue and indigo,
with violet as the highest frequency.
(5) Ultraviolet light
a) These are produced by electronic transitions in atoms.
b) It may also be produced by very high temperature materials (such as the sun) through
vibrational motions of atoms contained in the material.
(6) X-rays
a) These are produced by certain high-energy electronic transitions in heavier atoms.
b) X rays may also be produced by the rapid deceleration of high-energy electrons that occurs
when they are suddenly brought to a halt by striking a metal target.
(7) Gamma rays
a) They have very high frequencies that they are produced in the very tiny nucleus of the
atom.
b) There, electric charges may be pictured as oscillating extremely rapidly and have very high
energy levels.
(8) Cosmic rays
a) These are very high frequency gamma rays, and are produced by sources outside the solar
system.
b) The exact origin of these highest-energy electromagnetic waves is not completely
understood.

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UV (laws of absorption, instrumentation, theory of electronic spectroscopy, Frank-

condon principle, chromophores and auxochromes, intensity shifts, applications)
Laws of absorption:
Lambert’s law
It states that when a beam of monochromatic light passes through a homogeneous solution the
rate of decrease of intensity of radiation with thickness of absorbing medium is proportional to
the intensity of the incident radiation.
Mathematically, -dl/dx α I
-dl/dx =kI
Where k = proportionality constant and I = Intensity of incident radiation

It can also be written as log(I0/I) = A

Where A = Absorbance or optical density = ɛ lɛ
= Molar absorption coefficient
l = path length or thickness
I0 = Intensity of radiation before entering the absorption medium
I = Intensity of radiation after travelling distance l, in the absorption medium
Beer’s law:
It states that when a parallel beam of monochromatic light passes perpendicularly through a
dilute solution, the Absorbance is proportional to the concentration of the solution.
Mathematically, A = log(I0/I) = k‟c
Where A = Absorbance or optical density
k‟ is a constant and c = concentration of solution.
Beer - Lambert’s law:
It states that when a beam of monochromatic light passes through a homogeneous solution the
decrease in intensity of radiation with thickness of absorbing medium is directly proportional
to the intensity of the incident radiation as well as concentration of solution.

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Mathematically, A = log(I0/I) = ɛ c l

A = εcl
A= Absorbance
ε = molar absorption coefficient or absorptivity
c = concentration
l = path length
Derivation:
- dI / dl α I c
- dI / dl = k I c
dI / I = - k c dl
On integration at limits; I0  I and 0  l

 dI/I = - k c  dl
ln (I / I0) = - k c l
ln (I0 / I) = k c l
2.303 log (I0 / I) = k c l
log (I0 / I) = k c l / 2.303
A = ε cl
Absorbance is the logarithm of the reciprocal of transmittance.
A = log (1/T) or A= log (Io/ It)
Transmittance (T) is the ratio of the intensity of the light transmitted by the sample (I t) to
the intensity of light incident on the sample (I o)
T= log I/ I0 ; Thus T= -εcl
Limitations of Beer – Lambert’s Law
1. The Beer-Lambert law maintains linearity under specific conditions only.
2. The law will make inaccurate measurements at high concentrations because the molecules
of the analyte exhibit stronger intermolecular and electrostatics interactions which is due to the
lesser amount of space between molecules. This can change the molar absorptivity of the
analyte.

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3. Not only does a high concentration change molar absorptivity, but it also changes the
refractive index of the solution causing departures from the Beer-Lambert law.
Applications of Beer – Lambert’s Law
Beer-Lamberts law is applied to the analysis of a mixture by spectrophotometry, without the
need for extensive pre-processing of the sample. Ex: for determination of bilirubin in blood
plasma samples. It can be used to determine concentrations of a particular substance, or
determine the molar absorptivity of a substance.

Representation of Absorption spectrum: When electromagnetic radiation interacts with the

molecules, then some of the wavelengths (or frequency or wave numbers) of radiation are
absorbed; while the rest of its transmitted. Consequently, we can plot either
(i) Absorbance(A) versus λ or ν
(ii) Percent Transmittance (% T) versus λ or ν
Spectrophotometer is a device which detects the percentage of transmittance of light
radiation when light of certain intensity and frequency range is passed through the
sample.
A spectrophotometer consists of two instruments, namely the spectrometer for
producing light of any selected colour (wavelength) and photometer for measuring the intensity
of light. For solutions where the intensity of light is directly proportional to the concentration,
we use spectrophotometer. If the instrument is designed to measure the spectrum in absolute
units rather than relative units, then it is typically called a spectrophotometer. The majority of
spectrophotometers are used in spectral regions near the visible spectrum.
Instrumentation of Ultra Violet and Visible spectrophotometer:
1. In visible UV spectrophotometer, a beam of light is split into two halves.
2. One half of the beam (the sample beam) is directed through a transparent cell containing
solution of the compound being analyzed and one half (the reference beam) is directed
through an identical cell that contains only the solvent.
3. The instrument is designed that it compares the intensities of the two beams at each
wavelength of the region.
4. If the compound absorbs light at a particular wave length, then intensity of the sample
beam (It) will be less than that of the reference beam (Io).
5. The instrument gives output graph-a plot of the wavelength versus the absorbance (A)
of the light at each wavelength of the entire region.

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Block diagram of visible UV spectrophotometer

Components:
1. Light Source:
The tungsten filament lamp is commonly employed as a source of visible light. This type of
lamp is used in the wavelength range of 350 - 2500 nm. The energy emitted by a tungsten
filament lamp is proportional to the fourth power of the operating voltage. This means that for
the energy output to be stable, the voltage to the lamp must be very stable indeed.
2. Monochromator:
Polychromatic radiation (radiation of more than one wavelength) enters the monochromator
through the entrance slit. The beam is collimated, and then strikes the dispersing element at an
angle. The beam is split into its component wavelengths by the grating or prism. By moving
the dispersing element or the exit slit, radiation of only a particular wavelength leaves the
monochromator through the exit slit.
3. Sample and Reference Cells (Cuvettes):
The containers for the sample and reference solution must be transparent to the radiation which
will pass through them. Quartz or fused silica cuvettes are required for spectroscopy in the UV
region. These cells are also transparent in the visible region. Silicate glasses can be used for
the manufacture of cuvettes for use between 350 and 2000 nm.
4. Detectors:
The photomultiplier tube is a commonly used detector in UV-Vis spectroscopy. It consists of a
photo emissive cathode (a cathode which emits electrons when struck by photons of radiation),
several dynodes (which emit several electrons for each electron striking them) and an anode.

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5. Recorder: we can record the spectrum in computer display or printer.

Example: 4-methyl-3-penten-2-one

Theory of electronic spectroscopy: When a molecule absorbs ultraviolet or visible light, its
electrons are promoted from the ground state to a higher energy state. The electrons in the
bonding molecular orbitals, for example, σ orbital, π orbital or non-bonding orbitals are
promoted to anti-bonding molecular orbitals like σ* and π* (star designates the anti-bonding
orbital).
The following types of electronic transitions are possible.
σ→σ* transitions: The σ bonded electrons are held firmly in the molecule. Hence, the
transitions from σ→σ* require large amount of energy. These transitions require high energy
radiations and therefore, occur in the Far ultraviolet region (-150 nm; high energy). Since
oxygen of air absorbs strongly in this region, air has to be evacuated (vacuum ultraviolet
spectroscopy). σ→σ*transitions occur in hydrocarbons like methane, propane and are less
informative.
n→σ* transitions: This type of transitions occurs in saturated compounds with one hetero
atom having unshared pairs of electrons (n electrons) like alcohols, ethers, amines, ketones,
aldehydes, etc. The energy required for these transitions is lesser than the energy required for
σ→σ* transitions. Eg: water absorbs at 167 nm, and methyl alcohol at 174 nm
π→π* transitions: These transitions take place in compounds containing double and triple
bonds. The excitation of π electrons requires lesser energy; hence they occur at longer
wavelength. In unconjugated alkenes, absorption bands appear around 170-190 nm.
n→π* transitions: In these transitions the electrons of the unshared electron pair on the hetero
atom is excited to π* anti-bonding orbital. As non-bonding electrons are loosely held these
require least energy and occur at larger wave lengths. They occur in unsaturated compounds
with hetero atoms having unshared pairs of electrons like C=O, C=S.

Relative energies of molecular orbitals:

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When excitation occurs, an electron from one of the filled σ, π or n molecular orbitals get
excited to vacant σ* or π* (anti bonding) molecular orbitals. Corresponding to the possible
excitations, there is various absorptions as shown in figure below

The decreasing order of energy of absorption is as follows

-σ* > σ-π*̴ ≈ π- σ* > π- π* ≈ n- σ* > n- π*(transition for absorption in 200-800nm region)

Chromophores:
The part of the molecule having n or π electrons is essentially responsible for absorption and
these fragments are called chromophores.
Ex: -C=C-, -C≡C-, -C≡N, -N=N-, C=O
Auxochromes: It is defined as any group which does not itself act as a chromophore but whose
presence brings about a shift of the absorption band towards the red end of the spectrum.
Polar groups such as -OH, -OR, -NH2, -SH and –X (halogen) having unshared pair of
electrons, when conjugated with chromophore modify the position of absorption band relative
to that of parent chromophore.

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Chromophore Example Type of excitation

-O- CH3OH n- σ*
-S- C6H13SH n- σ*
-Br CH3Br n- σ*
-N- (CH3)3N n- σ*
-C=C- H2C=CH2 π- π*
-C≡C- HC≡CH π- π*
π- π*
C=O (CH3)2CO
n- π *
π- π*
NO2 CH3 NO2
n- π *

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Applications of UV-Vis spectrophotometer:

1. Qualitative Analysis:
Ultraviolet absorption is particularly useful for identifying functional
groups and the structures of molecules containing unsaturated bonds (π
electrons) such as butadiene and lone pair electrons such as pyridine. It does
not indicate molecule at weight or give useful information on saturated
bonds (σ bonds)

H H
H H

C C C C
N
H H

2. Quantitative Analysis:
Ultraviolet absorption is used routinely for the quantitative determination
of unsaturated compounds. It is not generally for trace quantitative analysis.
3. Extent of conjugation: Conjugation lowers the energy required for
π→π* transition. The greater the extent of conjugation, the longer is the
absorption wavelength.
4. Determination of geometrical isomers: Effective π→π* transitions
take place when the molecule is coplanar. The steric strain in cis-isomer
prevents coplanarity. Thus, cis isomers absorb at a shorter wavelength when
compared to the trans-isomer.
Identification of unknown compounds: Unknown compounds can be identified by
comparing its spectra with the spectra of known compounds. Identical spectra reveal identical
structure and if the spectra are different, then the structure.

Infrared (IR) Spectroscopy

Infrared (IR) Spectra:
All type of molecules cannot interact with IR radiation. Only those
molecules which exhibit change in the dipole moment during a
vibration can exhibit infrared (IR) spectra
Homo-nuclear diatomic molecules (e.g. H2, O2, N2) do not show
change in dipole moment during vibration. Consequently, these do not
exhibit infrared spectra.
The quantum theory predicts that permitted vibrational energies
(Evib) are governed by the expression Evib= (ν + ½) hν0,
where ν is the vibrational quantum number, with possible
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values of 0, 1, 2, etc. and ν0 is the fundamental frequency.

The lowest two energy ν = 0 and ν = 1 possess E vib values of ½ hν0 and
3/2hν0 respectively, and the difference between them is hν 0.
The appropriate frequency of the electromagnetic radiation
associated with this energy change is the fundamental frequency
(ν0).
It may be noted that in the ground vibrational state, the molecule has the
vibrational energy =
½hν0. This is called the zero-point energy, which signifies that a
molecule must always vibrate and can never be at rest.
When a molecule absorbs energy in the infra-red region of the electromagnetic spectrum, it can
change its vibrational and rotational levels and these energy changes account for the feature of IR
spectrum. Such a spectrum is much complicated due to rotational fine structure. But this complication
can be avoided by taking IR spectrum for liquid or in solution or solid sample. Under such condition,
the rotational spectrums become buried and consequently a broad peak is observed for vibrational
change only.

Selection rules and Fundamental modes of IR:

Polyatomic Molecular Vibration

The bond of a molecule experiences various types of vibrations and rotations. This
causes the atom not to be stationary and to fluctuate continuously. Vibrational
motions are defined by stretching and bending modes. These movements are easily
defined for diatomic or triatomic molecules. This is not the case for large molecules
due to several vibrational motions and interactions that will be experienced. When
there is a continuous change in the interatomic distance along the axis of the bond
between two atoms, this process is known as a stretching vibration. A change in the
angle occurring between two bonds is known as a bending vibration. Four bending
vibrations exist namely, wagging, twisting, rocking and scissoring. A CH 2 group is
used as an example to illustrate stretching and bending vibrations below.

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Instrumentation of IR spectrophotometer
1. The infrared spectrum of a sample is recorded by passing a
beam of infrared light through the sample.
2. When the frequency of the IR is the same as the vibrational
frequency of a bond, absorption occurs.
3. Examination of the transmitted light reveals how much energy
was absorbed at each frequency (or wavelength).
4. This can be achieved by scanning the wavelength range using a
monochromator.
5. Alternatively, the whole wavelength range is measured at once
using a Fourier transform instrument and then a transmittance or
absorbance spectrum is generated using a dedicated procedure.
6. Analysis of the position, shape and intensity of peaks in this
spectrum reveals details about the molecular structure of the
sample.
7. This technique works exclusively on samples with covalent bonds.

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8. Simple spectra are obtained from samples with few IR active

bonds and high levels of purity.
9. More complex molecular structures lead to more absorption
bands and more complex spectra.
10. The technique has been used for the characterization of very complex
mixtures.
Block diagram:

1. Light Source: The common radiation source for the IR

spectrometer is an inert solid heated electrically to 1000 to 1800 °C.
Three popular types of sources are Nernst glower (constructed of
rare-earth oxides), Globar (constructed of silicon carbide), and
Nichrome coil. They all produce continuous radiations, but with
different radiation energy profiles.
2. Filter (or) Monochromator: The Monochromator is a device used
to produce continuous monochromatic electromagnetic energy
bands of Infrared wavelength or frequency range.
3. Sample holder: Solid samples are ground with KBr and made into
a disc. The sample should be dry as water absorbs strongly in the IR
region. Solvents such as CHCl3, CCl4, CS2 etc., are used.
4. Detectors: Most detectors used in dispersive IR spectrometers can
be categorized into two classes: thermal detectors and photon
detectors. Thermal detectors include thermocouples, thermostats,
and pneumatic devices (Golay detectors).
5. Recorder: we can record the spectrum in computer display or printer.

Applications of Infrared Spectroscopy:

1. Qualitative Analysis:
a. IR Spectroscopy readily distinguishes between aliphatic and aromatic
compounds.
b. It identifies functional groups present in the molecule and gives
some indication of the presence of hetero elements - O, S, N, Halides
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etc. including their environment.

2. Quantitative Analysis:
a. IR Spectroscopy is used for quantitative analysis of organic
compounds particularly at higher concentration.
b. It is mostly for liquid samples and has less application to solid
sample analysis. This method requires skill in order to get good
results.
3. Identification of polymers and polymer blends.
4. Determination of degrees of crystallinity in polymers like LDPE and HDPE.
5. Other applications include Polymer analysis, Lubricant formulation and
fuel additives, Foods research Quality assurance and control,
Environmental and water quality analysis methods, Biochemical and
biomedical research, coatings and surfactants, etc.

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