MODULE 2

SPECTROSCOPIC TECHNIQUES AND

APPLICATIONS
Introduction- Types of spectrum - electromagnetic spectrum -
molecular energy levels – Beer Lambert’s law (Numericals). UV-
Visible Spectroscopy – Principle - Types of electronic transitions -

Energy level diagram of ethane, butadiene, benzene and

hexatriene. Instrumentation of UV-Visible spectrometer and
applications. IR-Spectroscopy – Principle - Number of vibrational
modes - Vibrational energy states of a diatomic molecule and -
Determination of force constant of diatomic molecule (Numericals)
–Applications. 1H NMR spectroscopy – Principle - Relation between
field strength and frequency - chemical shift - spin-spin splitting
(spectral problems ) - coupling constant

(definition) - applications of NMR- including MRI (brief).

 INTRODUCTION
Spectroscopy deals with the study of the interaction of matter
with the electromagnetic spectrum.

3
 ELECTROMAGNETIC RADIATION
➢ Consists of electrical and magnetic waves perpendicular to
each other

➢ Displays the properties of both particles and waves

✓ Wavelength (λ, nm or Å)– Distance traveled in one cycle

✓ Frequency (ʋ, s-1)– Number of waves per second
✓ Wave number (ῡ, cm-1)– Number of waves per centimeter
✓ Same velocity 2.998 × 108 m/s (~3 × 108 m/s )
 ELECTROMAGNETIC RADIATION
The energy is proportional to the frequency
E = hʋ
Where h is the Planck’s constant (6.63 × 10-34 Js )
ʋ = c/λ
E = hc/λ
E = hcῡ
Calculate the frequency and wave number of light of λ 400 nm

 = 400 x 10 -9 m ῡ=1/ 
 = c/  = 1 / 400 x 10 -9 m
= 2.998 x 10 8 m s-1 / 400 x 10 -9 m = 1 / 400 x 10 -7 cm
= 7.495 x 1014 s-1 = 25,000 cm-1
 TYPES OF SPECTRA
Based on the nature of interaction
Absorption Spectra: An atom or molecule
undergoes transition from lower energy level
(Ei) to the higher energy level (Ef) with an
absorption of photon

UV- visible, IR, Microwave and NMR

Emission Spectra: An atom or molecule undergoes transition

from higher energy level (Ei) to the lower energy level (Ef) with
emission of photon

Hydrogen spectrum, fluorescence spectrum and AES

 TYPES OF SPECTRA
Based on the nature of interacting species

Atomic Spectra: Arises from the transition

of electrons between the atomic energy levels

Molecular Spectra: Arises from the transition

between the different molecular energy levels
 MOLECULAR ENERGY LEVELS
1.Electronic energy(Eel): Associated with the electrons present in a
molecule, which can occupy various molecular energy levels. The
molecular orbitals can be bonding or antibonding.

The energy gap between electronic energy levels falls in

UV-visible region.

2.Vibrational energy(Evib): Atoms in a molecule can vibrate by

keeping the position of center of mass constant, the energy
associated is called vibrational energy.

The energy gap between vibrational levels falls in the Infrared

region.
 MOLECULAR ENERGY LEVELS
3. Rotational energy (Erot): If a molecule rotate in space about an
axis passing through its centre of mass, it is said to possess
rotational energy. Rotational energy is possessed only by
molecules of a gas or a liquid.

The energy gap falls in the microwave region.

4. Translational energy (Etrans): If the position of centre of mass

changes with time the molecule is said to possess the translational
energy.

Translational energy of a molecule is usually not quantised.

MOLECULAR ENERGY LEVELS
 BEER-LAMBERT'S LAW
When a parallel beam of monochromatic electromagnetic
radiation is passed through an absorbing solution of given
concentration (c) , the rate of decrease in intensity (-dI) of
radiation with thickness of the solution (dx) is directly
proportional to the intensity of incident radiation (I) at that point
as well as concentration (c ) of the solution.

-dI/dx α Ic
-dI/dx = kIc Differential form
-dI/I = kcdx
 BEER-LAMBERT'S LAW

ln I0/I = kcx
ɛ =k/2.303; molar extinction coefficient
2.303 log I0/I = kcx
or molar absorptivity coefficient
log I0/I = ɛcx
Transmittance, T = I/I0
Integrated form A = -log T = ɛcx
Absorbance, A = log I0/I -ln I/I0 = kcx
Simplified form A = ɛcx Exponential form I = I0e-kcx
 BEER-LAMBERT'S LAW
Unit of Molar Absorptivity Coefficient, ɛ: A = ɛcx,
thus ɛ = A/cx

A = absorbance, c = concentration in moles/L or

moles/dm3
x = length of light path through the cuvette in deci
meter (dm). Hence unit of ɛ = mol-1dm2
BEER-LAMBERT'S LAW: NUMERICALS
Limitations:
❖It cannot be applied for concentrated solutions or turbid
solutions.

❖only monochromatic light can be used.

A = -log T = ɛcx A = ɛcx; ɛ = A/cx

T = 20 % = 0.2 x = 2 cm = 0.2 dm
A = -log 0.2 ɛ = 0.6989/0.01 × 0.2
= 0.6989 = 349.4 mol-1dm2

14
 UV-VISIBLE SPECTROSCOPY
Principle: Ultraviolet-Visible spectroscopy (UV = 200-400 nm,
Visible = 400-800 nm) corresponds to electronic excitations
between molecular energy levels.

*

*
n



C-C C=C C=O
 → *  → * n → *
 UV-VISIBLE SPECTROSCOPY
 * transitions

✓Saturated hydrocarbons undergo this transition

✓Occurs below 150 nm

✓Ordinary UV machines take spectra only from 200-700 nm

✓So this transitioncannot be detected using ordinary UV-Visible

spectrometer

✓Eg. Methane, ethane etc.

✓They are called UV transparent

 UV-VISIBLE SPECTROSCOPY
π π* transitions
✓Unsaturated hydrocarbons undergo this transition
✓Ethylene ~ 169 nm: UV transparent
✓In conjugated double bond systems transitions occurs from HOMO
(Highest Occupied Molecular Orbital) to LUMO (Lowest Unoccupied
Molecular Orbital)
H H H H
C C
C C
H H
H H
*
*
LUMO LUMO
*
*
169 nm
HOMO
HOMO 




 UV-VISIBLE SPECTROSCOPY
1,3-butadiene 1,3,5-hexatriene

Benzene
❑As the number of double bonds in conjugation increases the
wavelength of absorption also increases
❑There is an increment of 30 nm
with every increment of a double bond in conjugation

https://www.masterorganicchemistry.com/2016/09/08/conjugation_and_color/ 19
 UV-VISIBLE SPECTROSCOPY
n π* transitions
✓ Compounds with both π electrons and non bonding electrons
✓ Low intensity transition since forbidden
✓ Aldehydes and ketones

n  * transitions
✓ Saturated Compounds with non bonding electrons
✓ Low intensity transition since forbidden
✓ Saturated alcohols, amines etc
CHROMOPHORE :
Any functional group that absorbs electromagnetic radiation
C=C, C≡C, C=O, NO2, C=C-C=C, C=C-C≡C, C=C-C=O, aryl group
AUXOCHROME:
Any functional group that could enhance the absorption property
of a chromophore, without itself being a chromophore
-OH, -OR, -NH 2 , -NR 2 , Etc

BAT H O C H RO M I C SHIFT (RED SHIFT):

– Shift of Absorption maximum to longer wavelengths
HYPSOCHROMIC SHIFT (BLUE SHIFT):
– Shift of Absorption maximum to shorter wavelengths

HYPERCHROMIC EFFECT:
– Leading to increased intensity of absorption
H Y PO C H RO M I C EFFECT:
– Leading to decreased intensity of absorption
UV-VISIBLE SPECTROSCOPY: INSTRUMENTATION
(D2 discharge lamp)

(Tungsten lamp)
UV-VISIBLE SPECTROSCOPY: INSTRUMENTATION

24
UV Visible Spectrophotometer

Quartz Cuvette
 UV-VISIBLE SPECTROSCOPY: APPLICATIONS
1. For the detection of aromatic compounds, conjugated dienes etc.
2. For the characterization of dyes and colorants.
3. Detection of impurities. For e.g. benzene is a common impurity in
cyclohexane which can be detected by the absorption at 255nm.
4. Determination of unknown concentration. Done by comparing the
absorbance with the absorbance of the standard solution.
5. Study of kinetics of chemical reactions. If a reaction involve shift of
absorption frequency or wavelength ,the rate of such reaction can
be easily monitored.
6. Widely used in medical lab for quantitative estimation of blood
sugar, cholesterol etc. These tests are done by measuring the

absorbance after developing colour with suitable reagents.

25
 IR (VIBRATIONAL)SPECTROSCOPY
Principle: Involves the transitions between vibrational energy
levels of a molecule, having change in dipole moment with
vibration of a bond.
These transitions are brought about by absorbing IR radiations
falling in the range 500 to 4000 cm-1

IR spectrum is the plot of

wave number (X-axis) and
% transmittance (Y- axis)
 IR SPECTROSCOPY
M E C H A N I S M OF INTERACTION

➢During the vibration of a molecule if there is a change in dipole

moment, it will lead to the generation of an oscillating electric field and
interacts with the electric field component of electromagnetic radiation.
➢When a photon of frequency come in resonance with the frequency of
vibration of a molecule, the absorption of photon takes place and the
molecule starts oscillating with the frequency of radiation.
 IR SPECTROSCOPY
❑For a molecule to absorb IR, there must be a change in the dipole
moment of the molecule. The alternating electrical field of the
radiation interacts with fluctuations in the dipole moment of the
molecule.

❑If the frequency of the radiation matches the vibrational frequency of

the molecule then radiation will be absorbed, causing a change in the
amplitude of molecular vibration.

❑A polar bond is usually IR-active. A nonpolar bond in a symmetrical

molecule will absorb weakly or not at all.
 IR SPECTROSCOPY
NUMBER OF VIBRATIONAL MODES IN A MOLECULE

➢Each atom has 3 degrees of freedom.

➢A molecule with N atoms has 3N degrees of freedom (composed of
translations, rotations and vibrations).
➢The translational movement uses three of 3N degrees of freedom
➢The rotation of non –linear molecule is three and linear molecule is
two,
➢no rotation about bond axis
➢The number of vibration al modes :
3N - 6 for non-linear molecules and 3N - 5 for linear molecules
Number of stretching vibration = N – 1
 IR SPECTROSCOPY

Stretching vibrations Bending vibrations

In-plane Out -plane In-plane

Asymmetric () Symmetric () Scissoring (s) Twisting () Wagging (ω) Rocking (ρ )
 NUMBER OF VIBRATIONAL M O D E S IN CO 2
❖ Linear molecule: 3N-5 vibrational modes (3 × 3) -5 = 4
❖ 2 stretching and 2 bending vibrations
Symm.Stretching is IR inactive since no change in d.m.

Asymm. Str. IR active since change in d.m. 2349 cm-1

Bending modes are degenerate; 667 cm-1

IR active since change in dm; 667 cm-1

 NUMBER OF VIBRATIONAL M O D E S IN CO 2

❖ Non linear molecule: 3N-6 vibrational modes

(3 × 3) -6= 3

❖ 2 stretching and 2 bending vibrations

IR active since change IR active since change IR active since change

in dm; 3652 cm-1 in dm; 3756 cm-1 in dm;1596 cm-1
 IR SPECTROSCOPY

Molecules c) CO and d) CO2

Those molecules which have change in dipole
moment during vibration are IR active.
Instrumentation
Perkin ElmerTM
Spectrum One

BRUKE TENSORTM
Series
 Molecular vibrations

https://www.chemtube3d.com/
 IR SPECTROSCOPY
Vibrational energy states of a diatomic molecule

υ0 – fundamental frequency
k – force constant

µ – reduced mass, m 1m 2/ m 1+m 2

Stretch the bond by a length `x' from the mean position

Potential energy;

Energy of a SHO;

in Joules
 IR SPECTROSCOPY
Vibrational energy states of a diatomic
molecule

'v‘: vibrational quantum number (0,1,2,3.....)

When v = 0; E = ½ hυ0 ........................ Zero

point energy.

Even at absolute zero (thermal death) there will

be a vibration to satisfy Heisenberg's
Uncertainty principle.
 IR SPECTROSCOPY
Vibrational energy states of a diatomic
molecule
 IR SPECTROSCOPY
Vibrational energy states of a diatomic
molecule
 IR SPECTROSCOPY: APPLICATIONS
1. Determination of force constant of a diatomic molecule.

2. Identification of functional groups in organic molecules. The

presence of functional groups can be readily detected from the
stretching vibrations of the bonds present in the functional group.
 IR SPECTROSCOPY: APPLICATIONS
3.Identification of unknown compounds. This is generally done by
comparison of IR spectrum of an unknown compound with that of a
known compound. The comparison is especially made at the
fingerprint region 700-1500 cm-1 which involves complex bending
and fluxional modes of vibration.

4.Determination of purity.
5.To distinguish between intra molecular and intermolecular hydrogen
bonding.
 NMR SPECTROSCOPY
Nuclear Magnetic Resonance spectroscopy depends on the
absorption of energy when the nucleus of an atom is excited from its
lowest energy nuclear spin state to the next higher one.

The nuclear energy levels are produced by keeping the nuclei in a

magnetic field.

The energy required for transitions falls in radio frequency region

(60 -500 MHz)

Nucleus that possess overall/effective spin will be NMR active.

NMRSPECTROSCOPY
1. If mass number is even
(a)If both number of protons and number of
neutrons are even.
Protons and neutrons separately pair-up inside
nucleus, then the nucleus has no net spin (I=0)
such nuclei are NMR inactive.

(b)If both number of protons and number of

neutrons are odd. Protons and neutrons
separately pair-up inside nucleus leaving a half
integral spin in each kind of particles they
combine parallel giving integral spins for the
nucleus (I = 1, 2, 3) and are NMR active.
NMRSPECTROSCOPY
2. If mass number is odd
a) If number of protons odd and number
of neutrons even .
Net neutrons spin is zero but, proton
pairing leaves odd half integral spins

I = 1/2 ; 3/2 ; 5/2; such nuclei are NMR

active

(b) If number of protons even and number

of neutrons odd.
Net proton spin is zero but, neutrons
pairing leaves odd half integral spins

I = 1/2; such nuclei are NMR active

 NMRSPECTROSCOPY

Nucleus of spin 'I' will have (2I+1) possible orientations.

I = 1/2 will have 2 [(2 × ½) + 1 = 2] possible orientations.

In the absence of an external magnetic field, these orientations are of

equal energy. If a magnetic field is applied, the energy levels split.
NMRSPECTROSCOPY
 NMRSPECTROSCOPY
When a nucleus is spinning on its axis in the presence of a magnetic
field B, this axis of spin will precess around the magnetic field. The
frequency of precession is termed as Larmor frequency. When this
frequency becomes identical to the radio frequency, resonance occurs
and flipping of proton takes place with the absorption of radiation
and spin orientation of nucleus become

-1/2.
NMRSPECTROSCOPY

56
46
NMRSPECTROSCOPY

57
47
 NMRSPECTROSCOPY
CHEMICAL SHIFT
When a molecule is placed in a magnetic field its electrons are caused
to circulate and thus they produce secondary magnetic field. This
induced magnetic field may oppose or reinforce the applied field.

Shielding: If the induced magnetic field oppose the applied field,

the net field felt by a hydrogen nucleus in a molecule will be less than
the applied field, and the hydrogen nucleus is said to be shielded. A
more shielded proton absorbs RF radiation at lower frequency.

58
48
 NMRSPECTROSCOPY
CHEMICAL SHIFT
Deshielding: If the induced magnetic field reinforces the applied
field, the net field felt by a hydrogen nucleus in a molecule will be
more than the applied field, and the hydrogen nucleus is said to be
deshielded. A more deshielded proton absorbs RF radiation at
higher frequency.

59
49
 NMRSPECTROSCOPY
CHEMICAL SHIFT
❖The dependence of the resonance frequency of a nucleus that results
from its molecular environment is called its chemical shift.
❖Different protons in a molecule give signals at different frequency.
❖Instead of measuring chemical shifts in absolute terms, we measure
them with respect to a standard - tetramethylsilane (CH3)4Si,
abbreviated as TMS.
❖The protons of TMS are more shielded than most of the organic
compounds (and gives a, so all the signals of the a sample ordinarily
appear at higher frequency than those of the TMS which

is the reference. 50
50
 NMRSPECTROSCOPY
CHEMICAL SHIFT
❑The chemical shift may also be defined as the shift in radio
frequency from TMS per MHz radio frequency due to shielding or
deshielding of hydrogen nucleus in different structural environment.
❑Chemical shift (δ) is reported by converting them to parts per
million (ppm) from TMS

❑TMS is assigned a value of zero.

51
51
 NMRSPECTROSCOPY:
FACTORS AFFECTING CHEMICAL SHIFT
1. ELECTRONEGATIVITY:
a. Electronegative atom deshield the protons
and δ value will increase

b. Cumulative effect: As the number of electronegative atom

increases deshielding increases and δ value will also increase

62
52
 NMRSPECTROSCOPY: FACTORS
AFFECTING CHEMICAL SHIFT
1. ELECTRONEGATIVITY:
c. Distance from the electronegative atom: When the
electronegative atom is closer deshielding will be high and δ value
will increase

2. HYDROGEN BONDING:
Hydrogen bonding will deshield the protons
and δ value will increase

63
52
 NMRSPECTROSCOPY:
FACTORS AFFECTING CHEMICAL SHIFT
3. MAGNETIC ANISOTROPY: Non uniform magnetic field

4. DESHIELDING & SHEILDING:

54
64
NMRSPECTROSCOPY

54
65
NMRSPECTROSCOPY

54
66
 INTERPRETATION OF NMRSPECTRUM
1.The number of signals, tells us how many different kinds of protons
are present.
2.The position of the signals (δ values) gives information about the
nature of protonic environment.
3.The intensity of the signals are measured by the area under each
peak, which tells us the relative ratios of the different kinds of
protons.

67
57
 INTERPRETATION OF NMRSPECTRUM
Predict the number of signals

a) CH3-O-CH2-CN -2

b)CH3-CHCl2 - 2

c) CH3-CH2Br - 2
d)CH3-C-O-CH2-CH3 - 3 -
=

O
O
h)
O
O

O 4
68
57
 NMRSPECTROSCOPY: SPIN-SPIN SPLITTING
❖Every hydrogen in a molecule spins and generates their own
magnetic field.

❖The protons on neighbouring carbons (carbons adjacent to the

carbon whose protons are generating the NMR signal) will generate
magnetic fields whose magnetic moments will interact with the
magnetic moment of the external magnetic field. This results in the
splitting of the NMR signal.

❖Splitting of NMR signals due to coupling interactions with

neighbouring protons is called spin-spin coupling or spin-spin
splitting.

59
69
 NMRSPECTROSCOPY: SPIN-SPIN SPLITTING
❑The number of peaks into which the signal for a particular proton is
split is called its multiplicity.

❑The signal may be split into

✓ two peaks (a doublet)
✓ three peaks (a triplet)
✓ four peaks (a quartet)
✓even more (a multiplet)
❑The multiplicity of signal is n+1, where n is equal to the number of
equivalent protons that are vicinal.
❑Peak intensity ratio is given by the coefficients of the
binomial series (a + b)n or Pascal's triangle
NMRSPECTROSCOPY: SPIN-SPIN SPLITTING
 NMRSPECTROSCOPY
1H N M R SPECTRUM OF CH 3 -CHCl 2

72
62
 NMRSPECTROSCOPY
1H N M R SPECTRUM OF CH 3 -CH 2 Br

73
62
 NMRSPECTROSCOPY
1H N M R SPECTRUM OF CH 3 -CH 2 -CH 3

74
62
 NMRSPECTROSCOPY
1H N M R SPECTRUM OF ETHYL ACETATE

75
62
 NMRSPECTROSCOPY
1H N M R SPECTRUM OF METHYL BENZOATE

76
66
 NMRSPECTROSCOPY
1H N M R SPECTRUM OF DIETHYL SUCCINATE

77
66
 NMRSPECTROSCOPY
1H N M R SPECTRUM OF ETHANOL

78
66
 NMRSPECTROSCOPY
1H N M R SPECTRUM OF DIETHYL ETHER

COUPLING CONSTANT (J)

The spacing between the peaks of a multiplet is measured
in Hertz is called the coupling constant.

7969
 NMRSPECTROSCOPY

Solvents used are deuterated solvents like CDCl 3 , CD 3 OD etc

 APPLICATIONS OF NMRSPECTROSCOPY
1.NMR spectroscopy ( 1H and 13C) is a powerful tool used for the
structure elucidation of organic molecules

2.NMR spectroscopy is used to study keto-enol tautomerism

and hydrogen bonding

3.Two-dimensional NMR spectra provide more information

about a molecule than one- dimensional NMR spectra and are
especially useful in determining the structure and
stereochemistry of complex molecules

4.Magnetic Resonance Imaging (MRI)

 M A G N E T I C R E S O N A N C E I M A G I N G (MRI)

❖ Used to visualize internal structures of the body in detail

❖Body tissue contains lots of water, and hence protons (1H
nuclei), which get aligned in a large magnetic field
 M A G N E T I C R E S O N A N C E I M A G I N G (MRI)
❖When a person is inside the powerful magnetic field of the
scanner (0.2 to 3 Tesla), average magnetic moment of many
protons becomes aligned with the direction of the field.
❖A radio frequency current is turned on, producing resonance
frequency.
❖The radio waves are absorbed and thus flip the spins of the
protons in the magnetic field.
❖When the electromagnetic field is turned off, the spins of the
protons return to thermodynamic equilibrium called relaxation.
❖During this relaxation, a radio frequency signal is generated from
the body, which can be measured with receiver coils and

recorded and mapped 73

 M A G N E T I C R E S O N A N C E I M A G I N G (MRI)
❖Once the RF signal is removed, the nuclei realign themselves such
that their net magnetic moment is again aligning parallel. This
return to equilibrium is referred to as relaxation.
❖During relaxation, the nuclei lose energy by emitting their own RF
signal.
❖This signal is referred to as the free-induction decay (FID)
response signal. The FID response signal is measured by a
conductive field coil placed around the object being imaged.
❖This measurement is processed or reconstructed to obtain 3D
grey-scale MR images.
❖Relaxation times for molecules to regain their natural alignment
vary depending on the type of tissue being scanned. 74
 APPLICATIONS OF MRI
✓Diffusion MRI: It uses the diffusion of water molecules in
biological systems. Useful for diagnosis of neurological disorder
and help in better understanding of connectivity of central
nervous system.
✓Magnetic resonance angiography: It generates the pictures of
the arteries.
✓Functional MRI: It measures signal changes in brain that are
due to changing neural activity.
✓Used to measure the levels of different metabolites in body
tissues, particularly in brain.