Instrumentation Of Ultra

Violet Spectroscopy
Spectroscopy
Ultra Violet Visible Spectroscopy:
UV-Visible spectroscopy deals with the absorption of
electromagnetic radiations in the visible and UV
region.
It is also known as ‘Electronic Spectroscopy . Since it
involves the gain of energy by a molecule and transfer
of electrons from lower energy level to higher
energy level.
UV- visible region extends from 200nm – 800nm.
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Principle:
U.V visible radiations being of high energy than
infrared radiations cause the transition of valence electron
from ground state to excited state on their absorption by
a sample.
THEORY:
A molecule possess three types of energy;
Et = Electronic transitional energy
Er = Rotational energy
Ev = Vibrational energy
Thus total energy of molecule is
E = Et ,Er ,Ev
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Thus when an UV radiation strikes a molecule, it

first increases its rotational energy, than vibrational
energy and finally electronic energy. It means
electronic transition which are resposible for
absorption spectrum in U.V visible region may occur
from any level of vibrational and rotational energies
associated with one electronic energy level to any of
vibrational & rotational energy level.
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Electronic Transitions:
The absorption of U.V visible radiations by a sample
results in transfer of electrons from one energy state to
another energy state is known as Electronic
transition.
Types:
Four types of transition has been reported due to
absorption of radiations.
1)∂-∂* Transitions:
This occurs in saturated hydrocarbons which
contain only strongly bonded sigma electrons.
spectrum range , below 150nm
e,g; CH3- CH3
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2) n ∂* Transition:
This occurs in saturated molecules containing
heteroatom such as, O2, N2, X2.
Spectrum 150-250nm
e.g, CH3-OH
3) Л– Л* Transition:
This type of transition occur in molecule containing
double or triple bonds or aromatic rings.
Spectrum 160-250nm
e.g, CH2= CH2
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n—Л* transition:
This occur in molecules containing double or triple
bonds involving heteroatom.
Spectrum range, 275-245nm
e.g; R- CHO
Spectrophotometry
• Spectrophotometry is a special form of absorptiometry
which mainly deal with the ultra violet (185 – 400nm),
Visible (400- 760nm) and Infra red (0.76- 15um) region of
the electromagnetic spectrum.
• The UV- visible spectroscopy is mainly concerned with
• - quantitive analysis of compound
• - auxiliary tool for structural elucidation
• - determination of light absorptive capacity of a chemical
system.
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Instrumentation:
 Spectrophotometer can be divided into
a) Single beam Spectrophotometer
b) Double beam Spectrophotometer
Instrumentation:
Components of Spectrophotometer
Radiation Source
Monochromators
Cell
Test Sample
Detectors
Recording System
Radiation Source:
• In ultra violet spectrometers the most commonly used
radiation sources are,
• Hydrogen discharge lamp
• Deuterium lamp
• Xenon discharge lamp
• Tungsten lamp
• Mercury lamp
• When the pressure of the gas is low only line spectra
are emitted and if the pressure of gas is high, band
spectra and continuous spectra will be obtained .
.

The sources of radiation must the following

requirements
 It should be stable
 It should provide continuous radiation.
 It must be of the sufficient intensity for the
transmitted energy to be detected at the end of the
optical path.
Filters and Monochromators
Filters:
It is frequently necessary to filter (remove) wide bands
of radiation from a signal.
Filters isolate a wider band.
Filters are of two typesd
 - Absorption filters
 - Interference filters
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Absorption filters:
The absorption filters consist of colored glass or a
dye suspended in gelatin sand witched between
the two glass plates.
The coloured glass filter has the advantage of
greater thermal stability.
Each instrument is provided with a set of 12 filters
to cover the range upto 700µm.
A narrow spectral band can be obtained by
coupling cut off filters with other filters but this
combination decreases the intensity of light.
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Interference filters:
These filters rely on interference phenomenon at
desired wavelength thus permitting rejection of
unwanted radiation by selective reflection and
producing narrow band.
Monochromators:
Monochromator is used to disperse the
heterochromic radiation into its component
wavelength and permit the isolation of desired portion
of the spectrum.
It consist of an entrance slit, an exit slit and a
dispersing device either a prism or a grating.
Materials of construction should be selected with care
to suit the range in which it has to work.
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For example;
 Quartz for ultraviolet
 Normal glass for visual range &
 for infra red region.
Gratings are cheaper than prism.

Refracting Prism:
The Monochromators based upon the refracting
prism need to be encased in specially designed
within the spectrophotometer.
In this type of monochromator the white light duly
enters the monochromator through an entrance slit
before undergoing collimation
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The ray of light passes via a refraction prism which

eventually disperse the light into its component
wavelength.
Finally the ray of light is dully focused by another lens
near an exit slit that is located at the focal plane.
The refracting prism is carefully rotated by using a stage
and a stepper motor to select a desired radiation.
.

Diffraction Grating:
In this instance the white light is made to pass via an
entrance slit and is focused subsequently towards a
diffraction grating through a concave mirror.
Thus the diffraction grating disperses the light into its
component wavelength, and thereby result into the
reflection of the light upon a second concave mirror.
The emerged light may now be reflected duly and
focused critically by the concave mirror.
.

Test Sample;
The sample to be analyzed is placed between the
source and the monochromator or between the
detector and monochromator.
The walls of the cell in which the test sample is
placed should be transparent to the radiation used.
The solvents for the test solution must be one that
does not absorb the radiant energy within the
region of electromagnetic spectrum being studied.
.

Detectors:
Detectors are used to measure accurately the
absorption of an analyte via the intensity of the
transmitted light.
In order to detect radiations three types of
photosensitive devices are mainly used.
 Photovoltaic cell or Barrier layer cell
 Phototubes
 Photomultiplier cell
.

Photomultiplier Tubes:
A photomultiplier tube consists of a electrode covered
with a photo emissive material.
Most photomultiplier tubes have about to dynodes.
Each dynode is maintained at 75 to 100 V more positive
than the preceding dynode.
Upon striking the dynode each photoelectrons causes the
ejection of several additional electrons which in turn are
accelerated towards the next dynode.
.

Recording Device:
The signal from the detector is finally received by the
recording device.
The recording is done by a recording device.
This type of arrangement is only done recording UV
spectrophotometer.
 .

Applications:
Identification of compounds
Structure of Organic compounds
Qualitative Analysis
Purity determination
Determination of Impurities
Analysis of mixture of compounds
pH of Indicator
 .

Lambert’s Law:
When a beam of monochromatic light is passed through a
substance, the absorption of light is directly proportional
to the path length of the sample.
Thus this law states that proportion of light absorbed by a transparent
medium is independent of the intensity of incident light and that each
successive layer of medium absorb fraction of incident light depending
upon its thickness.
It= Io x e-αc
log Io/It = αb or A=αb (Io-I =A)
Where
Io = Intensity of incident light
It = Intensity of transmitted light
b= Thickness of layer
α = Absorbance
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Beer’s Law:
When a beam of monochromatic light is passed
through a solvent containing absorbing substance, the
absorption of light is directly proportional to the
molar concentration of the absorbing substance.
Thus this law states that amount of light absorbed is
proportional to the number of absorbing molecules
through which light passes.

log Io/It = αc or A = αc
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Chromophores:
Groups that cause light to be absorbed are called
chromophores.
Any functional group in a molecule, such as the
carbonyl group in aldehyde and ketones and double
bond in alkenes, which is responsible for the
absorption of electromagnetic radiation in the UV-Vis
region is called Chromophore.
The chromophores generally contain some degree of
unsaturation.
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Auxochrome:
 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 Aniline λmax = 280 nm NH2
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For example, substitution of alkoxy groups on C=C (methyl vinyl ether,

CH2 = CHOCH3) causes a shift to longer wavelength (red shift). Such
functional groups are called auxochromes. All the au
-xochromic groups contain nonbonding electrons which are in
conjugation with the chromophores. This conjugation shifts the λmax
to a longer wavelength.

 Bathochromic shift (Red shift):

It is a shift of λmax to longer wavelength
 Hypsochromic Shift (blue shift):
It is a shift of WAVELENGTH max to shorter wavelength.
 Hyperchromic shift:
It is accompanied by increase in intensity of absorption.
 Hypochromic shift:
It is accompanied by decrease in intensity of absorption.
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 Ultraviolet absorptions of some simple isolated Chromophore.


Chromophore Examples Λmax (nm) Electronic Transition

 
 C =C Ethylene 171 π – π*
 
 -C ≡ C- Acetylene 173 π – π*
 
 C=O Acetone 190 π – π*
 
 C≡N Acetonitrile 167 π – π*
 
 N=N Azomethane 388 π – π*
 Benzene Benzene 254 π – π*
.

Lower Wavelength
Common UV Solvents and their Solvent Cutoffs
 .
Lower wavelength
cutoff (nm)
Acetone 330
Pyridine 306
Toluene 285
Carbon tetrachloride 265
Chloroform 245
Diethyl ether 215
Methanol 205
Cyclohexane 205
95% Ethanol 204
Hexane 195
Isooctane 195
Water 195
Acetonitrile 190
 .

INTRODUCTION
The word spectroscopy implies that we will use the
electromagnetic spectrum to gain information about
organic molecules. The modifier ultraviolet means
that the information will come from a specific region
of the electromagnetic spectrum called the ultraviolet
region (190 to 400 nm U.V. Region and 400 to 800 nm
Visible Region) .
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 Detection of Impurities
UV absorption spectroscopy is one of the best methods
for determination of impurities in organic molecules.
Additional peaks can be observed due to impurities in
the sample and it can be compared with that of standard
raw material. By also measuring the absorbance at
specific wavelength, the impurities can be detected.
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Structure elucidation of organic compounds.

UV spectroscopy is useful in the structure elucidation of

organic molecules, the presence or absence of
unsaturation, the presence of hetero atoms. From the
location of peaks and combination of peaks, it can be
concluded that whether the compound is saturated or
unsaturated, hetero atoms are present or not etc.
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QUANTITATIVE ANALYSIS
UV absorption spectroscopy can be used for the
quantitative determination of compounds that absorb
UV radiation. This determination is based on Beer’s law
which is as follows. A = log I0 / It = log 1/ T = – log T =
abc = εbcWhere :ε -is extinction co-efficient,c- is
concentration, andb- is the length of the cell that is used
in UV spectrophotometer.
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QUALITATIVE ANALYSIS
UV absorption spectroscopy can characterize those
types of compounds which absorbs UV radiation.
Identification is done by comparing the absorption
spectrum with the spectra of known compounds
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CHEMICAL KINETICS
Kinetics of reaction can also be studied using UV
spectroscopy. The UV radiation is passed through the
reaction cell and the absorbance changes can be
observed.
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DETECTION OF FUNCTIONAL
GROUPS This technique is used to detect the presence
or absence of functional group in the compound
Absence of a band at particular wavelength regarded as
an evidence for absence of particular group
 .

 QUANTITATIVE ANALYSIS OF
PHARMACEUTICALSUBSTANCES 
Many drugs are either in the form of raw material or in
the form of formulation. They can be assayed by
making a suitable solution of the drug in a solvent and
measuring the absorbance at specific wavelength. 
Diazepam tablet can be analyzed by 0.5% H2SO4 in
methanol at the wavelength 284 nm.
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EXAMINATION OF POLYNUCLEAR
HYDROCARBONS
Benzene and Polynuclear hydrocarbons have
characteristic spectra in ultraviolet and visible region.
Thus identification of Polynuclear hydrocarbons can
be made by comparison with the spectra of known
Polynuclear compounds. Polynuclear hydrocarbons
are the Hydrocarbon molecule with two or more
closed rings; examples are naphthalene, C10H8, with
two benzene rings side by side, or diphenyl, (C6H5)2,
with two bond- connected benzene rings. Also known
as polycyclic hydrocarbon.
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MOLECULAR WEIGHT DETERMINATION

Molecular weights of compounds can be measured
spectrophotometrically by preparing the suitable
derivatives of these compounds. For example, if we
want to determine the molecular weight of amine then
it is converted in to amine picrate. Then known
concentration of amine picrate is dissolved in a litre of
solution and its optical density is measured at λmax
380 nm.
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 After this the concentration of the solution in gm

moles per litre can be calculated by using the
following formula. "c" can be calculated using above
equation, the weight "w" of amine picrate is known.
From "c" and "w", molecular weight of amine picrate
can be calculated. And the molecular weight of picrate
can be calculated using the molecular weight of amine
picrate.
AS HPLC DETECTORA UV/Vis spectrophotometer
may be used as a detector for HPLC
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