UV / VISIBLE

SPECTROSCOPY
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
I
Where, log  A Absorbance
I0
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.
Beer’s Law
dI
 C.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
I0
2.303 log  K .C.l
I
I0 K
log  C.l
I 2.303
I
Where, log 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
• 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.
• 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.
• 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).
• 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.
• 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 nm O
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
• 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.
- -
O + O O + O
N N

-
OH
Alkaline
medium -
OH O

p-nitrophenol
λmax = 255 nm λmax = 265 nm
• 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.

+ -
NH2 + NH3 Cl
H
Acidic
medium

Aniline
λmax = 280 nm λmax = 265 nm
• 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
• 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 )
INSTRUMENTATION
Components of spectrophotometer
 Source
 Monochromator
 Sample compartment
 Detector
 Recorder
INSTRUMENTATION

Fig.-block diagram of instrumentation of UV-spectrophotometer

 amplifier

Read out

Fig.- block diagrammatic representation of UV-spectrophotometer

RADIATION SOURCE
It is important that the power of the radiation source does not
change abruptly over its wavelength range. The electrical
excitation of deuterium or hydrogen at low pressure produces
a continuous UV spectrum.
Both Deuterium and Hydrogen lamps emit radiation in the
range 160 - 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
For ultra violet region-
Hydrogen discharge lamp
• consist of two electrode contain in deuterium filled silica
envelop.
UV-Vis spectrophotometer have both deuterium & tungsten
lamps.
 Selection of lamp is made by moving lamp mounting or
mirror to cause the light fall on Monochromator.
Deuterium lamps:-
• Radiation emitted is 3-5 times more than the hydrogen
discharge lamps.
Xenon discharge lamp:-
• Xenon stored under pressure in 10-30 atmosphere.
FILTERS OR MONOCHROMATORS
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.
Fig.-mechanism of working of prism.
SAMPLE CONTAINERS OR SAMPLE CELLS

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

• The cell holding the sample should be transparent to the

wavelength region to be recorded. Quartz or fused silica
cuvettes are required for spectroscopy in the UV region.
Silicate glasses can be used for the manufacture of
cuvettes for use between 350 and 2000nm. The
thickness of the cell is generally 1 cm. cells may be
rectangular in shape or cylindrical with flat ends.
DETECTORS
Three common types of detectors are used
I. Barrier layer cell
II. Photo cell detector
III. Photomultiplier , Photo voltaic cells
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.
CONTINUED
 Photomultiplier tube
It is generally used as detector in UV- spectrophotometer It is the
combination of photodiode & electron multiplier.
It consist of evacuated tube contains photo- cathode. 9-16 electrodes
known as dynodes.
Types of UV-visible spectrophotometer
1) Single beam spectrophotometer

2) Double beam spectrophotometer

Block diagram for a double-beam in-time scanning spectrophotometer .
WOODWARD-FEISER RULE
• It is used for calculating the absorption maxima
• Woodward (1941) gives certain rule for correlating λmax with the
molecular structure
This rule for calculating λmax in conjugated dienes, trienes, polyenes.

 Homoannular dienes:-
cyclic dienes having conjugated double bonds in the same ring.
e.g.

CH3

CH3
CONTINUED
 Hateroannuler dienes

e.g. Heteroannuler dienes

 Endocyclic double bonds
it is the double bond present in ring as shown.

Endocyclic double bond

Exocyclic double bonds

double bond in which one of the double bonded

atom is the part of ring system.

CH2

CH2

Exocyclic double bond

WOODWARD’S-FIESER RULE FOR CONJUGATED DIENES
• a)Parent values-
1. acyclic & Heteroannuler conjugated dienes 215 nm
2.Homoannular conjugated dienes 253 nm
3.Acyclic trienes 245 nm

• b)Increments-
1.Each alkyl substituent or ring residue 5 nm
2.Exocyclic double bond 5 nm
3.Double bond extending conjugation 30 nm
4.auxochromes-
• -OR 6 nm
• -SR 30 nm
• -Cl , Br 5 nm
• -NR2 60 nm
• -OCOCH3 0 nm
1,4- dimethyl cyclohex-1,3,-diene

H3C CH3

Parent value for Homoannular dienes = 253 nm

Two alkyl substituent's 2 ˣ 5 = 10 nm
Two ring residues 2ˣ 5 = 10 nm

Calculated value = 273 nm

 SOLVENT EFFECTS - INTENSITY

Solvents can induce significant changes in the intensity of peaks.

Hyperchromic – Increase in absorption intensity.

Hypochromic – Decrease in absorption intensity.

Absorption characteristics of 2-methylpyridine

Solvent max max

Hexane 260 2000
Chloroform 263 4500
Ethanol 260 4000
Water 260 4000
Ethanol - HCl (1:1) 262 5200
 SOLVENT EFFECTS
• π -> π* transitions leads to more polar excited state that is more easily
stabilized by polar solvent associations (H-bonds). The π* state is more polar
and stabilized more in polar solvent relative to nonpolar one, thus in going
from nonpolar to polar solvent there is a red shift or bathochromic shift
(increase in λmax, decrease in ΔE).

• For n -> π* transition, the n state is much more easily stabilized by polar
solvent effects (H-bonds and association), so in going from nonpolar to polar
solvent there is a blue shift or hypsochromic shift (decrease in λ max, increase
in ΔE).
APPLICATIONS:

A. APPLICATIONS IN ORGANIC COMPOUNDS

1.It helps to show the relationship between different groups, it is useful to detect
the conjugation of the compounds
2.Detection of geometrical isomers, In case of geometrical isomers compounds,
that trans isomers exhibits λmax at slightly longer wavelength and have larger
extinction coefficient than the cis isomers .
3.Detection of functional groups, it is possible to detect the presence of certain
functional groups with the help of UV Spectrum.

GENERAL APPLICATIONS:

1.Qualitative analysis, UV absorption spectroscopy can characterizes those type

of compounds which absorb UV radiation. Identification is done by comparing the
absorption spectrum with the spectra of known compound.

2. It is useful in Quantitative analysis of the compounds.

3. Detection of impurities, UV absorption spectroscopy is the one of the best

method for detecting impurities in organic compounds.
Tautomeric equilibrium, UV spectroscopy can be used to determine the
percentage of various keto and enol forms present in tautomeric equilibrium.

5. Chemical kinetics, UV spectroscopy can be used to study the kinetics of

reactions.

6. Molecular weight determination, molecular weights of compounds can be

measured by spectroscopy.

7. Analysis of inorganic compounds.

8. Measuring concentration of solution, absorption band can also used to

determine the concentration of compounds in a solution.

9. Inorganic chemistry, absorption spectra have been used in connection with

many problems in inorganic chemistry.

10. It is useful to determine the structure of the chloral.

QUALITATIVE ANALYSIS
Pharmacopoeial identification of drug
(1) By using absorbance & wavelength
(2) By taking absorption ratio
(3) Limit test (b)Structural analysis

Quantitative analysis
Quantitative analysis A)By using beer’s law and using absorptivity value,
By using reference standard, Multiple standard method

B)Single compound analysis by direct analysis Using separation method

After extraction, after chromatographic separation Using column
chromatography , Using HPLC

Indirect analysis
a)Single compound without chromophore
b) Drugs with chromophoric reagent
1.For analyte which absorb weakly in UV region
2.For avoiding interference
3.Improve selectivity of assay
4. Determination of composition of complex Mole ratio method
Continuous variation method ( job curve method )
5 . Study of kinetics

Disadvantages:

 Samples should be in solution. Mixture of substances poses difficult to

analyse and requires prior separation.
Interference from the sample’s matrix makes the measurement difficult .
REFERENCE
S
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.ht
m

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

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