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
• 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 Radiation

Violet 400 - 420 nm Yellow 570 - 585 nm

Indigo 420 - 440 nm Orange 585 - 620 nm
Blue 440 - 490 nm Red 620 - 780 nm
Green 490 - 570 nm
Principles of
Spectroscopy
 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)
 Principles of Spectroscopy
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
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:
∆E = h ν = En - E0 where (n = 1, 2, 3, … etc)
∆E = 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.

∆E < ∆E < ∆E
rotational vibrational electronic
Lambert’s
Law
Lambert’s
Law
• When a monochromatic radiation is passed
through a solution, the decrease in the
intensity of radiation with thickness of the
solution is directly proportional to the
intensity of the incident light.

• Let I be the intensity of incident radiation.

x be the thickness of the solution.
Then
Lambert’s
Law
− dI α
I dx
− dI
So, = KI
dx
Integrate equation between limit
I = Io at x = 0 and I = I at x=l,
We get,

I
ln = − Kl
I0
Lambert’s
Law 2 .303 log I
= − Kl
I0
I K
log = − l
I0 2 .303
I0
Where, log = A Absorbance
I
K
= Absorption coefficient
2 ϵ
.303
A= Lambert’s
ϵ.l Law
Beer’s
Law
Beer’s
Law
• When a monochromatic radiation is passed
through a solution, the decrease in the
intensity of radiation with thickness of the
solution is directly proportional to intensity of
the incident light as well as the concentration
of the solution.
• Let I be the intensity of incident radiation, x
be the thickness of the solution and C be the
concentration of the solution. Then,
Beer’s
Law
− dI αC
.I dx
− dI
So, = K C .I
dx
Integrate equation between limit
I = Io at x = 0 and
I = I at x=l,
We get,
I
ln = − K C .l
I0
Beer’s
Law 2.303 log
I0
= K .C
I .l
I0 K
log = C .l
I 2 .303
I0
Where, log = A Absorbance
I
K Molar extinction
=
2 coefficient
ϵ
.303
A= ϵ .C Beer’s
.l Law
Beer’s
Law A= ϵ .C
.l
I I
T = OR − log T = log = A
I0 I0

From the equation, it is seen that the absorbance

which is also called as optical density (OD) of a solution
in a container of fixed path length is directly
proportional to the concentration of a solution.
PRINCIPLES OF UV - VISIBLE
SPECTROSCOPY
 Principle
• The UV radiation region extends from 100 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.
 Pigments and Quantum mechanics

High degree of conjugation!!

• Electrons have wave properties and they don’t jump off the

pigments when they reach its ends.

•These electrons resonances determine which frequencies of

light and thus which colors, are absorbed & emitted from
pigments
 Experimental Set Up

I0 I

Light Cuvette with

Monochromator Detector
source sample

•Various designs of spectrophotometers : Fixed and

variable wavelength; Single, split and double beam
•Cuvettes: Glass or plastic – visible, Quartz – visible, UV
and Acrylic – visible, UV
Monochromator
Monochromator
Instrumentation
Absorbance and the Beer – Lambert Law
According to the Beer-Lambert Law, absorbance
is proportional to concentration, and
Absorbance versus concentration plot is a
straight line.
Deviation from Lambert’s Beer Law
 Electronic levels and transitions

n→σ
σ → σ * Unoccupied
*
σ *

levels
*
π
n→π
*

n
π→π*
Occupied
levels
π
σ→π*
σ
σ→σ* Alkanes

σ→π* Carbonyl compounds

π →π* Alkenes, carbonyl compn, alkyne

etc.
n→σ* Oxygen, nitrogen, sulfur and
halogen compounds
n →π* Carbonyl compounds
 Typical absorptions of simple
chromophores

Class Transition Wavelength max

(nm)
R-OH n→σ* 180
R-O-R n→σ* 180
R-NH2 n→σ* 190
R-SH n→σ* 210
R2C=CR2 π →π* 175
R-C≡C-R π →π* 170
 Typical absorptions of simple
chromophores
Class transition Wavelength max
(nm)
R-CHO π →π* 190
n →π* 290
R2CO π →π* 180
n →π* 280
RCOOH n →π* 205
R-COOR’ n →π* 205
Terms used in
UV / Visible
Spectroscopy
 Chromophore
The part of a molecule responsible for imparting
color, 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.

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

 Chromophore
To interpretate UV – visible spectrum
following points should be noted:

1. Non-conjugated alkenes show an intense

absorption below 200 nm & are therefore
inaccessible to UV spectrophotometer.

2. Non-conjugated carbonyl group compound

give a weak absorption band in the 200 - 300
nm region.
 Chromophore
O
e.g. Acetone
C
which has λmax = 279 nmO
H 3C CH3

and that cyclohexane has λmax = 291 nm.

When double bonds are conjugatedin

a compound λmax is shifted to longer wavelength.

e.g. 1,5 - hexadiene has λmax = 178 nm 2,4

- hexadiene has λmax = 227 nm
CH2 CH3
H 2C H 3C
 Chromophore
3. Conjugation of C=C and carbonyl group shifts
the λmax of both groups to longer wavelength.

e.g. Ethylene has λmax = 171 nm Acetone

O
has λmax = 279 nm H 2C CH 2
C
H3C CH3

Crotonaldehyde has λmax = 290 nm

O

C
H2
C CH
 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.
OR
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.
 Auxochrome
e.g. Benzene λmax = 255 nm

O
H

Phenol λmax = 270 nm

NH
2
Aniline λmax = 280 nm
Absorption &
Intensity
Shifts
 1 • 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.
 1 • Bathochromic Shift (Red Shift)
• In alkaline medium, p-nitrophenol shows red
shift. Because negatively charged oxygen
delocalizes more effectively than the unshared
pair of electron.
-
O O -
+ O O +
N N

-
OH

A lkaline

m edium -
OH O

p-nitrophenol
λmax = 255 nm λmax = 265 nm
 2 • 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.
 2 • Hypsochromic Shift (Blue Shift)

• Aniline shows blue

shift in acidic medium, it loses conjugation.
+ -
NH2 + NH 3
H Cl

A cidic

m edium

Aniline
λmax = 280 nm λmax = 265 nm
 3 • 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.

N N CH 3

Pyridine 2-methyl pyridine

λmax = 257 nm λmax = 260 nm
 4 • Hypochromic Effect

• When absorption intensity (ε) of a compound is

decreased, it is known as hypochromic shift.

CH 3

Naphthalene 2-methyl naphthalene ε

ε = 19000 = 10250
 Shifts and Effects
Hyperchromic shift

Blue Red
Absorbance ( A )

shift shift

Hypochromic shift

λ
max

Wavelength ( λ
APPLICATIONS OF UV / VISIBLE
SPECTROSCOPY
 Applications
• 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 theimportantmethod to detect
impurities in organic solvents.
• Detection of isomers are possible.
• Determination of molecular weight using
Beer’s law.