UV / VISIBLE SPECTROSCOPY

September 18, 2019

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.

∆Erotational < ∆Evibrational < ∆Eelectronic

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
 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
I
2.303 log   Kl
I0
I K
log  l
I0 2.303
I0
Where, log  A Absorbance
I
K
E Absorption coefficient
2.303
A  E.l Lambert’s 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 the
intensity of the incident light as well as
concentration of the solution.

• Let I be the intensity of incident radiation.

x be the thickness of the solution.
C be the concentration of the solution.
Then
Beer’s Law
dI
  C .I
dx
So,  dI  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
I0
2.303 log  K .C.l
I
I0 K
log  C.l
I 2.303
Where, log I 0  A Absorbance
I
K Molar extinction
E
2.303 coefficient
A  E.C.l Beer’s Law
Beer’s Law
A  E.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 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

3 • n → σ* transition

4 • n → π* transition

5 • σ → π* transition

6 • π → σ* transition
 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
& • π → σ* transition 6
• 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.
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
e.g. O Acetone which has λmax = 279 nmO
C
H3C CH3
and that cyclohexane has λmax = 291 nm.

When double bonds are conjugated in 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
H2C H3C
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
O
Acetone has λmax = 279 nm
C
H2C CH2 H3C CH3
Crotonaldehyde has λmax = 290 nm
O

H2C C
CH3
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

OH

Phenol λmax = 270 nm

NH2

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

Alkaline
medium -
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 Cl
H
Acidic
medium

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 CH3
Pyridine 2-methyl pyridine
λmax = 257 nm λmax = 260 nm
ε = 2750 ε = 3560
 4 • Hypochromic Effect

• When absorption intensity (ε) of a compound is

decreased, it is known as hypochromic shift.

CH3

Naphthalene 2-methyl naphthalene

ε = 19000 ε = 10250
 Shifts and Effects
Hyperchromic shift

Blue Red
Absorbance ( A )

shift shift

Hypochromic shift

λmax
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 the important method to detect
impurities in organic solvents.
• Detection of isomers are possible.
• Determination of molecular weight using Beer’s
law.
REFERENCES
Reference Books
• Introduction to Spectroscopy
– Donald A. Pavia

• Elementary Organic Spectroscopy

– Y. R. Sharma

• Physical Chemistry
– Puri, Sharma & Pathaniya
Resources
• http://www2.chemistry.msu.edu/faculty/reu
sch/VirtTxtJml/Spectrpy/UV-
Vis/spectrum.htm

• http://en.wikipedia.org/wiki/Ultraviolet%E2
%80%93visible_spectroscopy

• http://teaching.shu.ac.uk/hwb/chemistry/tut
orials/molspec/uvvisab1.htm