1. UV-VISIBLE SPECTROSCOPY
Mr. Gaurav B Rathod
B .Pharm Final Year
GUIDED BY – Prof DR .Ranjit Tijare Sir
Principal Ishwar Deshmukh Institute
OF Pharmacy
2. CONTENTs
Introduction to Spectroscopy
Electromagnetic Radiation
(EMR)
Principle of UV Spectroscopy
Chromophores And
Auxochromes
Factors affecting Absorption
Spectra
Application of UV
spectroscopy
Instrumentation
3. Spectroscopy
Using electromagnetic radiation as a probe to
obtain information about atoms and molecules that
are too small to see.
Electromagnetic radiation is propagated at the
speed of light through a vacuum as an oscillating
wave.
4. Electrom
agnetic
Waves
Waves of energy emitted from
any accelerating charges
Any object that is above
absolute zero emits
electromagnetic waves
The entire range of
possibilities is called the
“Electromagnetic Spectrum”
5. Principle of UV Light
Absorption
Ultraviolet light: wavelengths between 190 and 400
nm
Visible light: wavelengths between 400 and 800 nm
Ultraviolet/visible spectroscopy involves the
absorption of ultraviolet light by a molecule causing
the promotion of an electron from a ground electronic
state to an excited electronic state.
6. Beer-
Lambert’s
law
In Beer-Lambert’s law the fraction of incident radiation absorbed is
proportional to the number of absorbing molecules in its path.
log I₀/I = εcl
Where ; I₀=Intensity of incident light
C=concentration of solute molecule
l=path length of the sample
ε=molar extension coefficient of substance whose light is investigated
log I₀/I = Absorbance
log I/I₀ =Transmittance
8. σ to σ∗
An electron in bonding σ orbital is excited to the
corresponding antibonding orbitals.
The energy required is large
For example : methane
9. n to σ∗
Saturated compounds
containing atoms with lone
pairs are capable of n to σ∗
transitions
These transition usually
need less energy than σ to
σ∗ transitions.
The number of organic
functional group with n to
σ∗ peaks in uv region is
small.
10. n to π∗ &
The most absorption spectroscopy of organic compound is based on transitions of n or π electrons to
the π∗ excited state.
These transitions need an unsaturated group in the molecule to provide the π electrons.
π to π∗ transition occurs with compound containing double bond or triple bond whereas n to π∗
transition occurs with compound containing double bond including hetero atom
π to π∗
11. Chromoph
ores
Molecules having
unsaturated bonds or
free nonbonding
electrons that can
absorb radiation of
relatively low energy
and which imparts
colour to an organic
compound are called
chromophores.
Examples include
alkenes, alkynes,
ketones, aldehydes,
phenyl and other
aromatic species, etc.
12. Auxochrom
e
Auxochrome is a functional
group that does not absorb
in UV region but
has the effect of shifting
chromophore peaks to
longer wavelength as well
As increasing their
intensity.
13.
14. Factors affecting UV
pH effect
In certain compounds generally
acids and bases pH changes affects
primary and secondary bands.
As pH decreases wavelength also
decreases.
15. Solvents:
Polar solvents such as water, alcohols,
esters, and ketones tend to obliterate
spectral fine structure arising from
vibrational effects; spectra that approach
those of the gas phase are more likely to
be observed in nonpolar solvents such as
hydrocarbons.
Generally polar solvents shifts n to
π∗ transition towards a shorter
wavelength.
16. APPLICATION OF UV-VISIBLE
SPECTROSCOPY
1)It is used in determination of molecular
weight of molecules.
2)It is used in determination of impurities
present in the sample.
3)The unknown concentration of the
solution can be determine using this
spectroscopy.
4)It is used in characterisation of aromatic
compound and detection of conjugation.
5)Useful in finding out dissociation
17. Instrumentation of UV spectroscopy
1. Source of light
a. Hydrogen discharge lamp
b. Xenon discharge lamp
c. Mercury arc lamp
2. Monochromators
a. Gratings
3. Sample holders/cuvettes
18. 4. Detectors
a. Barrier layer cell/Photovoltaic cell
b. Phototubes/ Photo emissive tube
c. Photomultiplier tube
5. Recorder
a. Single beam UV spectrophotometer
b. Double beam UV spectrophotometer
19. SOURCE FOR UV RADIATION
HYDROGEN DISCHARGE LAMP:
• In Hydrogen discharge lamp pair of electrodes is enclosed
in a glass tube (provided with silica or quartz window for UV
radiation to pass trough) filled with hydrogen gas.
They are stable in nature.
It gives radiation from 120-350 nm.
20. XENON DISCHARGE LAMP:
• It possesses two tungsten electrodes separated
by some distance.
• These are enclosed in a glass tube (for visible)
with quartz or fused silica and xenon gas at 10-
30 atmospheric pressure is filled under pressure.
This is a good source of continuous plus
additional intense radiation. Its intensity is
higher than the hydrogen discharge lamp.
21. MERCURY ARC LAMP
In mercury arc lamp, mercury vapor is stored
under high pressure and excitation of
mercury atoms is done by electric discharge.
DEMERIT:
Not suitable for continuous spectral studies
(because it doesn't give continuous
radiations).
22. MONOCHROMATORS
GRATING
Are most effective one in converting a polychromatic light to
monochromatic light. As a resolution of +/- 0.1nm could be achieved
by using gratings, they are commonly used in spectrophotometers.
> Gratings are of two types.
1. Diffraction grating.
2. Transmission gratings.
23. 1. Diffraction Grating
More refined dispersion of light is
obtained by means of diffraction
gratings.
These consist of large number of parallel
lines (grooves) about 15000-30000/inch
is ruled on highly polished surface of
glass, quartz or alkyl halides.
24. It is similar to diffraction grating but
refraction takes place instead of
reflection. Refraction produces
reinforcement. this occurs when radiation
transmitted through grating reinforces
with the partially refracted radiation.
2. Transmission grating
25. SAMPLE HOLDERS/CUVETTES
> The cells or cuvettes are used for handling liquid
samples.
> The cell may either be rectangular or cylindrical in
nature. For study in UV region; the cells are prepared from
quartz or fused silica whereas color corrected fused glass is
used for visible region.
The surfaces of absorption cells must be kept scrupulously
clean. No fingerprints or blotches should be present on
cells.
>Cleaning is carried out washing with distilled water or
with dilute alcohol, acetone.
26.
27. DETECTORS
> Device which converts light energy into electrical signals,
that are displayed on readout devices.
>The transmitted radiation falls on the detector which
determines the intensity of radiation absorbed by sample
The following types of detectors are employed in
instrumentation of absorption spectrophotometer
1. Barrier layer cell/Photovoltaic cell
2. Phototubes/Photo emissive tube
3. Photomultiplier tube
28. Barrier layer cell/Photovoltaic cell
The detector has a thin film metallic layer
coated with silver or gold and acts as an
electrode.
It also has a metal base plate which acts as
another electrode.
These two layers are separated by a
semiconductor layer of selenium.
>When light radiation falls on selenium layer,
electrons become mobile and are taken up by
transparent metal layer.
29. >This creates a potential
difference between two
electrodes & causes the flow of
current.
>When it is connected to
galvanometer, a flow of current
observed which is proportional
to the intensity and wavelength
of light falling on it.
30. 2.Photo Tubes/ Photoemissive Tubes
> Consists of a evacuated glass tube with a
photocathode and a collector anode.
The surface of photocathode is coated with a
layer of elements like cesium, silver oxide or
mixture of them.
> When radiant energy falls on photosensitive
cathode, electrons are emitted which are
attracted to anode causing current to flow.
>More sensitive compared to barrier layer cell
and therefore widely used.
31. 3. Photo Multiplier Tubes
The principle employed in this detector is that,
multiplication of photoelectrons by secondary emission
of electrons.
In a vacuum tube, a primary photo-cathode is fixed
which receives radiation from the sample.
• Some eight to ten dynodes are fixed each with
increasing potential of 75-100V higher than preceding
one.
Near the last dynode is fixed an anode or electron
collector electrode.
• Photo-multiplier is extremely sensitive to light and is
best suited
32. INSTRUMENTS
SINGLE BEAM SPECTROPHOTOMETER
Light from the source is carried through lens
and/or through aperture to pass through a
suitable filter.
• The type of filter to be used is governed by
the colour of the solution.
The sample solution to be analysed is placed
in cuvettes.
33.
34. After passing through the solution, the light
strikes the surface of detector (barrier-layer
cell or phototube) and produces electrical
current.
• The output of current is measured by the
deflection of needle of light-spot
galvanometer or micro ammeter. This meter
is calibrated in terms of transmittance as
well as optical density. The readings of
solution of both standard and unknown are
recorded in optical density units after
adjusáng instrument to a reagent blank.
35. Chopper is a device consisting of a circular disc. One
third of the disc is opaque and one third is
transparent, remaining one third is mirrored. It splits
the monochromatic beam of light into two beams of
equal intensities.
Double beam instrument is the one in which two
beams. are formed in the space by a U shaped mirror
called as beam splitter or beam chopper.
DOUBLE BEAM UV SPECTROPHOTOMETER
