U V Visible Spectroscopy

UV / Visible Spectroscopy
Presented By
Nandakumar V.G
Mphil student
Dept.of Opto electronics.
Kerala University, Kariavattom
.

Contents.
• Introduction
• Electromagnetic Radiation
• Basic Principle
• Electronic Transitions
• Instrumentation
• Interpretations
• Advantages and Disadvantages
• Applications
• Conclusion.

Introduction
• The history of spectroscopy began with Isaac
Newton's optics experiments (1666–1672). Newton applied
the word "spectrum" to describe the rainbow of colors that
combine to form white light and that are revealed when the
white light is passed through a prism. During the early
1800s, Joseph von made experimental advances with
dispersive spectrometers that enabled spectroscopy to
become a more precise and quantitative scientific technique.
Since then, spectroscopy has played and continues to play a
significant role in chemistry, physics and astronomy.

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.

Four common spectroscopic techniques use
to determine structure:
• UV/Visible Spectroscopy
• Infrared Spectroscopy (IR)
• Nuclear Magnetic Resonance Spectroscopy (NMR)
• Mass Spectrometry (MS or Mass Spec)

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.

• 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.

• The relationship between wavelength &
frequency can be written as:
c = ν λ
• As photon is subjected to energy, so
E = h ν = h c / λ

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
• 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.

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

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

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.

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
• 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
I
dx
dI

So, KI
dx
dI

Integrate equation between limit
I = Io at x = 0 and
I = I at x=l,
We get,
Kl
I
I

0
ln

Lambert’s Law
Kl
I
I

0
log303.2
l
K
I
I
303.2
log
0

Where, AbsorbanceA
I
I
0
log
E
K

303.2
lEA .
Absorption coefficient
Lambert’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
IC
dx
dI
.
So, ICK
dx
dI
.'
Integrate equation between limit
I = Io at x = 0 and
I = I at x=l,
We get,
lCK
I
I
.'ln
0


Beer’s Law
lCK
I
I
..log303.2 0

lC
K
I
I
.
303.2
log 0

Where, AbsorbanceA
I
I
0
log
E
K

303.2
lCEA ..
Molar extinction
coefficient
Beer’s Law

Beer’s Law
lCEA ..
0I
I
T  OR A
I
I
T 
0
loglog
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.

• In order to obtain a UV-VIS spectrum the sample is ideally
irradiated with the electromagnetic radiation varied over a
range of wavelength.
• A monochromatic radiation i.e., a radiation of a single
wavelength is employed at a time.
• This process is called scanning.
• The amount of the radiation absorbed at each wavelength is
measured and plotted against the wavelength to obtain the
spectrum.
• Thus, a typical UV spectrum is a plot of wavelength or
frequency versus the intensity of absorption.
6/22/2015 UV-VISIBLE SPECTROSCOPY 36
CHARECTERISTICS OF SPECTRUM

• The UV spectra of substances are characterised by two major
parameters, namely, the position of the maximum of the
absorption band called λmax, and the intensity of the bands.
• The λmax refers to the wavelength of the most absorbed
radiation and is a measure of the difference in the electronic
energy levels involved in the transition.
• The intensity on the other hand is indicative of the probability
of the transition i.e., whether the transition is allowed or not.
• It is also is a measure of the concentration of the absorbing
species.
CHARECTERISTICS OF SPECTRUM

• The wavelength of the radiation absorbed by an
organic molecule is determined by the difference in
energy between the ground state and the various
excited electronic states of the molecule.
• In organic molecules that the constituent atoms are
bonded through σ and π bonds.
• In addition, these have nonbonding electrons on the
atoms like, N,O,S and halogens etc.
• There are a number of transitions possible involving
the bonding and the nonbonding electrons.
ORGANIC ABSORPTION

The possible electronic transitions can
graphically shown as:

• σ → σ* transition1
• π → π* transition2
• n → σ* transition3
• n → π* transition4
• σ → π* transition5
• π → σ* transition6
The possible electronic transitions are

• 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.
• σ → σ* transition1

• 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.
• π → π* transition2

• 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).
• n → σ* transition3

• 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.
• n → π* transition4

•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.
• σ → π* transition5
• π → σ* transition 6&

Components of spectrophotometer
 Source
 Monochromator
 Sample compartment
 Detector
 Recorder
INSTRUMENTATION

Block Diagram
RADIANT
SOURCE
WAVELENGTH
SELECTOR
SOLVENT
PHOTO-
DETECTOR
READOUT
SAMPLE
Fig.-block diagram of instrumentation of UV-spectrophotometer

Tungsten filament lamps and Hydrogen-Deuterium lamps are
most widely used and suitable light source as they cover the
whole UV region. Tungsten filament lamps are rich in red
radiations; more specifically they emit the radiations of 375
nm, while the intensity of Hydrogen-Deuterium lamps falls
below 375 nm.
Problem-
• Due to evaporation of tungsten life period decreases.
• It is overcome by using tungsten-halogen lamp.
• Halogen gas prevents evaporation of tungsten.
RADIATION SOURCE

- Monochromators generally composed of prisms and slits.
The most of the spectrophotometers are double beam
spectrophotometers. The radiation emitted from the primary
source is dispersed with the help of rotating prisms. The various
wavelengths of the light source which are separated by the prism
are then selected by the slits such the rotation of the prism
results in a series of continuously increasing wavelength to pass
through the slits for recording purpose. The beam selected by
the slit is monochromatic and further divided into two beams
with the help of another prism.
.
Monochromator

All Monochromators contain the following component parts;
• An entrance slit
• A collimating lens
• A dispersing device (a prism or a grating)
• A focusing lens
• An exit slit

 Filters –
a)Glass filters- Made from pieces of colored glass which
transmit limited wave length range of spectrum. Wide band
width 150nm.
b)Gelatin filters- Consist of mixture of dyes placed in gelatin
& sandwiched between glass plates. Band width 25nm.
c)Inter ferometric filters- Band width 15nm
Prisms-
-Prism bends the monochromatic light.
-Amount of deviation depends on wavelength
-They produce non linear dispersion.

A variety of sample cells available for UV region. The choice of
sample cell is based on
a) the path length, shape, size
b) the transmission characteristics at the desired wavelength
c) the relative expense
• One of the two divided beams is passed through the sample
solution and second beam is passé through the reference
solution. Both sample and reference solution are contained in
the cells. These cells are made of either silica or quartz. Glass
can't be used for the cells as it also absorbs light in the UV
region. The thickness of the cell is generally 1 cm. cells may be
rectangular in shape or cylindrical with flat ends.
Sample and reference cells-

Generally two photocells serve
the purpose of detector in UV spectroscopy. One of the photocell receives
the beam from sample cell and second detector receives the beam from
the reference. The intensity of the radiation from the reference cell is
stronger than the beam of sample cell. This results in the generation of
pulsating or alternating currents in the photocells.
Three common types of detectors are used.
I. Barrier layer cell
II. Photo cell detector
III. Photomultiplier cells
DETECTORS

1. Barrier layer cells
It consist of flat Cu or Fe electrode on
which semiconductor such as selenium is
deposited. on the selenium a thin layer of silver
or gold is sputtered over the surface.

3. Photomultiplier
Photomultipliers have an internal amplification that
gives them great sensitivity and a wide spectral range. Light
causes emission of electrons from a photocathode which
accelerate past a series of dynodes maintained at
progressively increasing potentials.

Amplifier
• Amplifier- The alternating current
generated in the photocells is transferred to the amplifier.
The amplifier is coupled to a small servometer. Generally
current generated in the photocells is of very low intensity,
the main purpose of amplifier is to amplify the signals many
times so we can get clear and recordable signals.

Recording Device
•
• Recording devices- Most of the time amplifier is
coupled to a pen recorder which is connected to the
computer. Computer stores all the data generated and
produces the spectrum of the desired compound.

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. Acetone which has λmax = 279 nm
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
CH3
C
CH3
O
O
CH2
CH2
CH3
CH3

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 has λmax = 279 nm
Crotonaldehyde has λmax = 290 nm
CH3
C
CH3
O
CH2 CH2
C
CH3
O
CH2

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
Phenol λmax = 270 nm
Aniline λmax = 280 nm
OH
NH2

• 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.
• Bathochromic Shift (Red Shift)1

• In alkaline medium, p-nitrophenol shows red
shift. Because negatively charged oxygen
delocalizes more effectively than the unshared
pair of electron.
p-nitrophenol
λmax = 255 nm λmax = 265 nm
• Bathochromic Shift (Red Shift)1
OH
N
+ O
-
O
OH
-
Alkaline
medium
O
-
N
+ O
-
O

• 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.
• Hypsochromic Shift (Blue Shift)2

• Aniline shows blue shift in acidic medium, it
loses conjugation.
Aniline
• Hypsochromic Shift (Blue Shift)2
NH2
H
+
Acidic
medium
NH3
+
Cl
-

• 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
ε = 2750 ε = 3560
• Hyperchromic Effect3
N N CH3

• When absorption intensity (ε) of a compound is
decreased, it is known as hypochromic shift.
Naphthalene 2-methyl naphthalene
ε = 19000 ε = 10250
CH3
• Hypochromic Effect4

Wavelength ( λ )
Absorbance(A)
Shifts and Effects
Hyperchromic shift
Hypochromic shift
Red
shift
Blue
shift
λmax

Solution too concentrated Diluted five-fold

NH2
NO2
Solvent: Ethanol
Concentration: 15.4 mg L-1
Pathlength: 1 cm
UV-visible spectrum of 4-nitroanaline

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.
• Detection of unknown compound.

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

U V Visible Spectroscopy

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