Instrumental Methods of Chemical Analysis

Outline
UV-Visible Spectroscopy:
a) Principle and theory
b)Applications

IR Spectroscopy:
a) Principle and theory
b)Applications

NMR Spectroscopy
a) Principle and theory
b)Applications
Spectroscopy

• It is the branch of science that deals with the study of

interaction of matter with light.

OR

• It is the branch of science that deals with the study of

interaction of electromagnetic radiation with matter.
Electromagnetic Radiation
• Electromagnetic radiation consist of discrete packages of
energy which are called as photons.

• A photon consists of an oscillating electric field (E) & an

oscillating magnetic field (M) which are perpendicular to
each other.
Electromagnetic Radiation
• 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.
Electromagnetic Radiation

• The relationship between wavelength & frequency can be

written as:
c=νλ

• As photon is subjected to energy, so

E=hν=hc/λ
Electromagnetic Radiation
Electromagnetic Spectrum

= 10-7 cm
Visible Spectrum
Principles of Spectroscopy
• The principle is based on the measurement of spectrum
of a sample containing atoms /molecules.

• Spectrum is a graph of intensity of absorbed or emitted

radiation by sample verses frequency (ν) or wavelength
(λ).

• Spectrometer is an instrument design to measure the

spectrum of a compound.
Principles of Spectroscopy
1. Absorption Spectroscopy:
• An analytical technique which concerns with the
measurement of absorption of electromagnetic radiation.

e.g. UV (185 - 400 nm) / Visible (400 - 800 nm) Spectroscopy,

IR Spectroscopy (0.76 - 15 μm)

2. Emission Spectroscopy:
• An analytical technique in which emission (of a particle or
radiation) is dispersed according to some property of the
emission & the amount of dispersion is measured.

• e.g. Mass Spectroscopy

 Interaction of EMR with matter
Energy Levels:

https://commons.wikimedia.org/wiki/File:Molecular_energy_levels_en.svg
http://hyperphysics.phyastr.gsu.edu/hbase/molecule/molelect.html
Interaction of EMR with matter
1. Electronic Energy Levels:
• At room temperature the molecules are in the lowest
energy levels E0.

• When the molecules absorb UV-visible light from EMR,

one of the outermost bond/lone pair electron is promoted
to higher energy state such as E1, E2, …En, etc is called as
electronic transition and the difference is as:

ΔE = hν = En - E0 where (n = 1, 2, 3, … etc)
ΔE = 35 to 71 kcal/mole
Interaction of EMR with matter
2. Vibrational Energy Levels:
• These are less energy level than electronic energy levels.

• The spacing between energy levels are relatively small i.e.

0.01 to 10 kcal/mole.

• e.g. when IR radiation is absorbed, molecules are excited

from one vibrational level to another or it vibrates with
higher amplitude.
Interaction of EMR with matter
3. Rotational Energy Levels:

• These energy levels are quantized & discrete.

• The spacing between energy levels are even smaller than

vibrational energy levels.

ΔErotational < ΔEvibrational < ΔEelectronic

PRINCIPLES OF UV – VISIBLE SPECTROSCOPY

• The UV radiation region extends from 10 nm to 400 nm

and the visible radiation region extends from 400 nm to
800 nm.

Near UV Region: 200 nm to 400 nm

Far UV Region: below 200 nm

• Far UV spectroscopy is studied under vacuum condition.

• The common solvent used for preparing sample to be

analyzed is either ethyl alcohol or hexane.
 Electronic Transitions
The possible electronic transitions can graphically shown as:

The possible electronic transitions are

1 • σ→ σ* transition
2 • π→ π* transition 5 • σ→ π* transition
3 • n → σ* transition 6 • π→ σ* transition
4 • n → π* transition
 Electronic Transitions
σ*

π*

σ
C-C C=C C=O C=C-C=C
σ 🡪 σ* π 🡪 π* π 🡪 π* π 🡪 π*
(150 nm) (170-190 nm) n 🡪 π* (185 nm) (400-700 nm)
n🡪 σ* (280 nm)
1. σ→ σ* transition

• σ electron from orbital is excited to corresponding anti-bonding orbital σ*.

• The energy required is large for this transition.

• e.g. Methane (CH4) has C-H bond only and can undergo σ → σ* transition
and shows absorbance maxima at 125 nm.

2. 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

aromatic compounds, etc undergo π → π* transitions.

• e.g. Alkenes generally absorb in the region 170 to 205 nm.

3. n → σ* transition
• Saturated compounds containing atoms 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.

• The number of organic functional groups with n → σ* peaks in UV region is

small (150 – 250 nm).

4. n → π* transition

• An electron from non-bonding orbital is promoted to anti-bonding π*

orbital.

• Compounds containing double bond involving hetero atoms (C=O, C≡N, N=O)
undergo such transitions.

• n → π* transitions require minimum energy and show absorption at longer

wavelength around 300 nm.
5. σ→ π* transition &
6. 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.

•The UV spectrum is of only a few broad of absorption.

The
wavelength
at which a
substance
has its
strongest
photon
absorption
Electronic Transitions
Electronic Transitions
 Terms used in UV / Visible Spectroscopy
Chromophore
Any isolated covalently bonded group that shows a characteristic
absorption in the UV or in the visible region.

e.g. NO2, N=O, C=O, C=N, C≡N, C=C, C=S, etc

Eg: -NO2 (Nitro-), -N=N (diazo-), -N= (Nitroso), -N= (azoxo)

o o
These are of two types 1. chromophore in which the
group contains Π electrons and they undergo π→ π*
transition.
2. Chromophore which contains Π and n electrons,
these undergo π→ π* and n→ π* transition

Benzene : colourless

Nitrobenzene : yellow colour

A chromophore is the part of a molecule responsible for its
color.
The color that is seen by our eyes is the one not absorbed within
a certain wavelength spectrum of visible light.
The chromophore is a region in the molecule where the energy
difference between two separate molecular orbitals falls within
the range of the visible spectrum. Visible light that hits the
chromophore can thus be absorbed by exciting an electron from
its ground state into an excited state.
In biological molecules that serve to capture or detect light
energy, the chromophore is the moiety that causes a
conformational change of the molecule when hit by light.

Healthy plants are perceived as green

because chlorophyll absorbs mainly the blue
and red wavelengths but green light,
reflected by plant structures like cell walls, is
less absorbed.

https://en.wikipedia.org/wiki/Chromophore#:~:text=A%20chromophore%20is%20the%20part,wavelength%20spectrum%20of%20visible%20light.
The eleven conjugated double bonds that form the
chromophore of the β-carotene molecule are highlighted in red.
The long chain of alternating
double bonds (conjugated) is
responsible for the orange
color of beta-carotene. The
conjugated chain in
carotenoids means that they
absorb in the visible region -
green/blue part of the
spectrum. So β-carotene
appears orange, because the
red/yellow colors are
reflected back to us.
Examples
Auxochrome
An auxochrome is a group of atoms attached to a chromophore which
modifies the ability of that chromophore to absorb light. They
themselves fail to produce the colour; but when present along with the
chromophores in an organic compound intensifies the colour of the
chromogen.

OR

An auxochrome is a functional group of atoms with one or more lone

pairs of electrons when attached to a chromophore, alters both the
wavelength and intensity of absorption.

Examples include the hydroxyl group (−OH), the amino group (−NH 2),
the aldehyde group (−CHO), and the methyl mercaptan group (−SCH3).
Auxochrome
e.g.
Benzene λmax = 255 nm

Phenol λmax = 270 nm

Aniline λmax = 280 nm

 Absorption & Intensity Shifts
General – Substituents may have any of four effects on a chromophore

i. Bathochromic shift (red shift) – a shift to longer λ; lower energy

ii. Hypsochromic shift (blue shift) – shift to shorter λ; higher energy

iii. Hyperchromic effect – an increase in intensity

iv. Hypochromic effect – a decrease in intensity

 Bathochromic Shift (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.

• e.g. An auxochrome group like –OH, -OCH3 causes

absorption of compound at longer wavelength.

• In alkaline medium, p-nitrophenol shows red shift. Because

negatively charged oxygen delocalizes more effectively than
the unshared pair of electron.
 Hypsochromic Shift (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.

• Aniline shows blue shift in acidic medium, it loses

conjugation.
Hyperchromic Effect
• When absorption intensity (ε) of a compound is increased,
it is known as hyperchromic shift.

• If auxochrome introduces to the compound, the intensity

of absorption increases.

Pyridine 2-methyl pyridine

λmax = 257 nm λmax = 260 nm
Hypochromic Effect
• When absorption intensity (ε) of a compound is decreased,
it is known as hypochromic shift.

Naphthalene 2-methyl naphthalene

ε = 19000 ε = 10250
Selection rules in UV-visible spectroscopy
1. Not all transitions that are possible are observed

2. For an electron to transition, certain quantum mechanical

constraints apply – these are called “selection rules”
APPLICATIONS OF U.V. SPECTROSCOPY
Woodword-Fieser Rules
These rules are used in calculating in dienes. In 1945 Robert
Burns Woodward gave certain rules for correlating λmax with
molecular structure.
In 1959 Louis Frederick Fieser modified these rules with more
experimental data, and the modified rule is known as
Woodward-Fieser Rules. It is used to calculate the position and
λmax for a given structure by relating the position and degree of
substitution of chromophore.

According to these rules each type of a diene has a certain

fixed basic value and the value of λmax depends on
1. Number of alkyl substituents or ring residue on the double
bond.
2. Number of double bonds which extend conjugation
3. The presence of polar groups such as –Cl, -Br, -OR, -SR etc.
Various rules for calculating λmax in case of dienes & trienes
are summarized as
1. Parent/Base value for butadiene system or cyclic
conjugated diene = 217 nm
2. Parent value for cyclic triene = 245 nm
3. Parent value for homo annular diene = 253 nm
4. Base value for hetero annular diene = 214 nm

Increment for each substituent

Examples
UV/Vis Spectrophotometer
Born–Oppenheimer approximation
In quantum chemistry and molecular physics, the
Born–Oppenheimer(BO) approximation is the assumption that the
motion of atomic nuclei and electrons in a molecule can be treated
separately.

In molecular spectroscopy, using the BO approximation means the

total energy of the molecule (molecular energy) in the gas or liquid
phase is a sum of independent energies such as electronic,
vibrational and rotational energies. Translational energy of the
molecule can be neglected.

The electronic energy consists of kinetic energies, interelectronic

repulsions, internuclear repulsions, and electron–nuclear
attractions, which are the terms typically included when computing
the electronic structure of molecules.
 Franck–Condon principle

In 1925, before the development of the Schrödinger equation,

Franck put forward qualitative arguments to explain the
various types of intensity distributions found in vibronic
transitions.

His conclusions were based on the fact that an electronic

transition in a molecule takes place much more rapidly than a
vibrational motion of the nuclei that the instantaneous
internuclear distance and the velocity of the nuclei can be
considered remain unchanged during the electronic transition
(later used as Born-Oppenheimer Approximation).
Diagrams is showing the potential energy curve of the two
electronic states of the molecule, the transition represented by
the vertical lines, i.e. the most probable or most intense
transition will be those represented by the vertical lines.

Figure demonstrating the Franck principle: for re’ > re’’ (left) and re’ =
re’’ (right). The vibronic transition A → B is the most probable in both
cases.
Figure . Franck–Condon principle energy diagram. Since electronic transitions
are very fast compared with nuclear motions, vibrational levels are favored
when they correspond to a minimal change in the nuclear coordinates. The
potential wells are shown favoring transitions between v = 0 and v = 2.