Instrumentation of uv visible spectroscopy

1. INSTRUMENTATION OF UV-
VISIBLE SPECTROSCOPY
AYESHA SHAFI

2. Instrumentation
• Spectrophotometer can be divided into
I. Single beam spectrophotometer
II. Double beam spectrophotometer
Single beam spectrophotometer:
Practically all UV visible spectrophotometer can possess
five essential components.
i. Source
ii. Monochromator
iii. Cell
iv. Detector
v. Recorder

3. Instrumentation

4. SOURCE
• In UV spectrophotometer, the source of radiant energy
used is the hydrogen discharge lamp. It is composed of a
tube fitted with two electrodes and hydrogen gas at low
pressure . when high voltage is applied across the
electrode, the electrons of hydrogen gas molecules are
excited to higher energy state. The return of excited
molecule to the ground state result in the emission of
continuous radiation in the region between 180-350nm.
• For visible spectrophotometer, source is usually a
tungsten filament lamp and the radiation lies in the
range of 350-2500µm.

5. Monochromator
• Devices that are employed to isolate the wavelength are called
as the monochromator.
• A typical monochromator usually consist of
i. Entrance slit
ii. Prism or diffraction grating
iii. Mirror
iv. Exit slit
v. Polychromatic light from the source enter the
monochromator system through entrance slit and collimated
by either a lens or a mirror. This collimated light is then
dispersed by the prism or grating.
• Depending upon the type of dispersion element, there are two
types of monochromators.
• Prism type
• Grating type

6. Monochromator…..
• Prism type:
In this type, a prism is used to get monochromatic light. The
prism breaks light into the narrow bands, when it is passed
through the prism.
For visible region: glass prism is used.
For UV region: quartz prism is used.
• Grating type:
Diffraction grating is of two types
a) Transmission grating
b) Reflection grating
Reflection grating is usually used for UV region which consists
of large number of equispaced lines ruled on a glass coated with
thin film of aluminium.
The desired portion of dispersed spectrum is then focussed by a
lens or mirror on the exit slit on the test sample.

7. Filter
• Filters used mostly in simple analysis while monochromators
are used for ultraviolet, visible and infrared radiation.
• Filters used mostly commonly are colored glass, gelatin filter
and interference filter.
Filter may be used to filter out the light of undesirable
wavelength. Filters are of following types;
• Glass filter (pieces of colored glasses that transmit limited
wavelength)
• Gelatin filter (consist of thin gelatin sheet between a pair of
glass plates)
• Interferometer filter (consist of two parallel glass plates
silvered internally and separated by a thin film of cryolite or
some other dielectric material.)

8. Sample cell
• Gas or liquid samples for analysis for the UV and visible
region are examined in cuvette (absorption cell). Shapes
of the cell may be cylindrical or rectangular.
• For visible region, glass cells are used.
• For UV region, quartz cells are used.

9. Detectors
• Several types of the detectors are used but most
commonly phototubes and photomultiplier tubes are
used.
• Phototubes:
• It consists of semi cylindrical cathode and metal wire
anode sealed inside an evacuated glass envelop. A
potential difference is applied between the anode and
the cathode. When the photons are absorbed by the
detector, they transfer their energy to the photo-emissive
material on the cathode surface. The loosely bound
electrons leave the cathode surface and are collected at
the anode, causing current to flow in the phototube
circuit.

10. Detectors
• Photomultiplier tubes:
• A photomultiplier tube is a type of phototube that
contains a number of electrodes called dynodes in
addition to photosensitive cathode and anode. When an
electron leaves the photo-emissive cathode and strikes a
dynode, it transfer some of its energy and cause the
ejection of several no of electron. These electrons in turn
are accelerated toward the next dynode where the same
process takes place. After several stages of amplification
each photoelectron emitted by the cathode will have
been amplified to a great level.

11. Detectors
Other photodetectors include: Barrier layer cells (photovoltaic
cells) are also used.

12. Recorders
• The output from detector is amplified and is observed on a
recorder in term of %age transmittance or absorbance.
• Working:
• UV-visible spectrometer records the spectra of the compound
in the range of 200-800nm. This range consists of two parts,
one is uv range and other is visible range. For recording the
spectrum, the given sample is dissolved in a suitable solvent
which itself does not absorb light. The commonly used
solvents are 95% ethanol, methanol, hexane etc. A quartz cell
of path length 1cm is used as a container for the sample
solution. The solution is exposed to UV-visible light by the
prism selector. The prism selector is rotating continuously to
emit lights of varying wavelengths. This light pass through
the detector and amplified at recorder and graph is draw
between wavelength of radiation absorb and intensity of
absorption.

13. Double beam spectrophotometer
• In research purposes, double beam spectrophotometer is
mostly used.
• In this case, monochromatic radiations is split into two beams
of equal intensity. One beam is passed through the sample
cell. And the other beam is passed through the reference cell.
The two cells should be matched. Then its focused on the
detector that in turn is connected to an amplifier which
transmits the signal to the recorder. Double beam
spectrophotometer is more advantageous as compare to single
beam spectrophotometer because in single beam the blank
and the sample have to be replaced continuously according to
wavelength and the ratio of the absorption power. Because we
cant use the sample and the reference cell together.

14. Single beam Spectrophotometer:
Double-beam Spectrophotometer:
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15. Applications of UV-visible
spectroscopy
• There are two main application of UV-visible
spectroscopy.
• Detection of conjugation:
• It helps to establish the relationship with special
reference to conjugation in a compound i.e.
• Carbon-carbon double or triple bond
• And also the carbon-oxygen double bond and also in
aromatic ring.
• So , by finding the λmax , we can determine the location
of the substituents.

16. Applications (continue)
• Detection of functional group:
• It is possible to determine the certain functional group
with the help of UV-visible spectrum specially alpha, beta
unsaturated carbonyl system or any other functional
group.

17. Applications (continue)
• Detection of impurities:
• UV-visible spectroscopy is one of the best methods for
detection of impurities in organic compounds. The bands
due to impurities are very intense.
• Additional peaks can be observed due to impurities in
the sample and it can be compared with that of standard
raw material.

18. Applications (continue)
• Qualitative analysis:
• In case of qualitative analysis with the help of uv-visible
spectrometer, we can identify the given organic
molecule. Identification can be done by comparing the
absorption spectrum with the spectrum of known
compound.
Molecular weight determination:
o Molecular weights of compounds can be measured
spectrophotometrically by preparing the suitable
derivatives of these compounds.

19. Applications (continue)
. Quantitative analysis
o Prepare samples
o Make series of standard solutions of known concentrations
o Set spectrophotometer to the λ of maximum light absorption
o Measure the absorption of the unknown, and from the standard plot, read the
related concentration .
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20. Elucidation of the structure of Organic Compounds:
From the location of peaks and combination of peaks UV spectroscopy elucidate
structure of organic molecules:
o the presence or absence of unsaturation,
o the presence of hetero atoms
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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21. Reference Books
 Introduction to Spectroscopy
 Donald A. Pavia
 Elementary Organic Spectroscopy
 Y. R. Sharma
 Practical Pharmaceutical Chemistry
 A.H. Beckett, J.B. Stenlake
o Pharmaceutical Analysis by Anees A. Siddiqui