This document discusses the principles, instrumentation, and applications of UV spectroscopy. It begins with an introduction to UV spectroscopy and its uses in qualitative and quantitative analysis. It then covers the underlying principles of UV absorption, including Lambert's law and Beer's law. The key components of a UV spectrophotometer are described, including radiation sources, monochromators, sample containers, detectors, and recording systems. Finally, common applications of UV spectroscopy are outlined, such as determining functional groups, conjugation, and reaction monitoring.

1. Instrumentation and Pharmaceutical
Application of U.V Spectroscopy.
Shaikh Shakeel Shaikh Quader
M.Pharm (Pharmaceutics)
Ali Allana College of Pharmacy
Akkalkuwa Dist.Nandurbar
(2016-17)

2. Content:
Introduction
Principle
Theory
Instrumentation
Application

3. Introduction
 Ultraviolet and visible spectrometers have been in
general use for the last 35 years and over this period have
become the most important analytical instrument in the
modern day laboratory. In many applications other
techniques could be employed but UV-Visible
spectrometry for its simplicity, versatility, speed,
accuracy and cost-effectiveness.
 Absorption spectroscopy in the U.V and Visible region
is consider to be one of the oldest physical methods use
for the Qualitative, Quantitative analysis and structure
elucidation.

4. REGION WAVELENGTH
Ultraviolet 200-400
Visible 400-800
The wavelength of U.V radiation starts at the
blue end of visible light about 400 And end at 200
A. However, the wavelength of visible radiation of
starts at 800 A, and end at 400A.

5. Principle
 When U.V or visible radiation is passed through a
substance under examination, Absorption of energy
result in the promotion of electron from the ground
electronic state to the exited electronic state.
 During the process of absorption a large number of
photon-molecule collision is possible but only those
collision will cause absorption of energy in which the
energy of photon matches the energy difference
between the ground and exited electronic state of the
molecule.
 In ultra-violet and visible spectroscopy, electronic
transition take place.

6. Theory
Absorption Laws;
1) Lambert’s law
2) Beer’s Law

7. Lambert’s law
1) Lambert’s law : When a beam of monochromatic radiation is passes
through a homogeneous absorbing medium, the rate of decrease of
intensity of radiation with thickness of absorbing medium is proportional
to the intensity of incident radiation.
Mathematically, the law is expressed as:
- dl/dx = kl
Where; I= intensity of radiation after passing through a thickness x, of the
medium.
dl= infinitesimally small decrease in the intensity of radiation on passing
through infinitesimally small thickness, dx of the medium.
-dl/dx= rate of decrease of intensity of radiation with thickness of absorbing
medium.
K= proportionality constant.

8. Beer’s Law
Beer’s Law:
1) When a beam of monochromatic radiation is passes through a
homogeneous absorbing medium, the rate of decrease of intensity of
radiation with thickness of absorbing medium is proportional to the
intensity of incident radiation as well as the concentration of the
solution.
Mathematically, the law is expressed as:
- dI/dx = k’ Ic
Where; I= intensity of radiation after passing through a thickness x, of the
medium.
dl= infinitesimally small decrease in the intensity of radiation on passing
through infinitesimally small thickness, dx of the medium.
-dl/dx= rate of decrease of intensity of radiation with thickness of absorbing
medium.
K’= proportionality constant. C= concentration

9. Instrumentation
Fig: Diode array U.V Spectrophotometer

10. Fig: Schemic Diagram of and Double beam U.V spectrophotometer.

11. The instrument used in ultraviolet-visible spectroscopy is
called a UV/Vis spectrophotometer.
It measures the intensity of light passing through a sample
and compares it to the intensity of light before it passes
through the Reference sample. The ratio is called the
transmittance, and is usually expressed as a percentage
(%T). The absorbance, , is based on the transmittance.
Instruments for measuring the absorption of U.V. or
visible radiation are made up of the following
components;
•Radiation Sources (UV and visible)
•Wavelength selector (monochromator)
•Sample containers
•Detector
•Signal processor and readout

12. Radiation sources
In U.V spectrometer, the most commonly
used radiation source are:
 Tungstan lamp
 Hydroger or Deuterium lamp
 Xenon discharge lamp
 Mercury arc…

13. Tungstan lamp
1) Tungstan lamp:
Functioning same as an electric light bulb.
The tungsten filament lamp is commonly employed as a source of
visible light. This type of lamp is used in the wavelength range of
350 - 2500 nm. The energy emitted by a tungsten filament lamp is
proportional to the fourth power of the operating voltage. This
means that for the energy output to be
stable, the voltage to the lamp must be very stable indeed.
Electronic voltage regulators or constant voltage transformers are
used to ensure this stability.

14. Hydroger or Deuterium lamp
• In this lamps, hydrogen gas is stored under relatively high
pressure. When a electric discharge is passed through the lamp,
excited hydrogen molecule will be produce which emit U.V
radiation.
• Its Cover the rang 3500-1200A.
• These are stable, robus and widely use.
•If deuterium lamp is used instead of hydrogen lamp it increase
the emission intensity.
• Deuterium lamp is more expensive then hydrogen lamp.
•But are use when higher intensity is required.

15. Xenon discharge lamp
•In this lamp xenon gas is store under pressure in the
rang of 10-30 atmosphere.
•Xenone lamp posses two tungsten electrodes separated
by about 8mm.
•When an intense arc is form between these two
electrodes by applying a low voltage, the U.V light is
produced.
•The intensity of U.V radiation produce by Xenone
lamp is mch greater then Hydrogen lamp.

16. Xenon discharge lamp

17. Mercury arc
•In this, the mercury vapour is under high pressure, and
the excitation of mercury atom is done by electric
discharge.
•Not suitable for continuous spectral studies because of
the presence of sharp lines and bands.
•Generally the low pressure mercury arc is very useful
for calibration work.

18. Monochromator

19. Most instruments use a monochromator to separate light from the
source into discrete wavelength segments
Components:
– Entrance slit
– Collimating/focusing device - mirror or lens,
– Dispersing device -filter, grating or prism
– Collimating/focusing device - mirror or lens
– Exit slit
The monochromator are use to dispersing the radiation
according to the wavelength.
The essential element of the monochromator are an entrance
slit, a dispersing element and exit slit.
The entrance slit sharply defined the beam of heterochromic
radiation.

20. The dispersing element disperses the the heterochromic
radiation into its component wavelength whereas exit slit allows
the nominal wavelength together with a band of wavelength on
either side of it.
The position of dispersing element is always adjusted by
rotating it to very the nominal wavelength passing through the exit
slit.
Dispersing element may be prism or grating.
Grating is generally made by Glass, Quartz or fused silica.
Glass has high resolving power but it is not transparent to
radiation having the wavelength between 2000 and 3000 A
because glass absorb in this region.
Quartz or fused silica are generally used due to their
transparency throughout the entire U.V range.

21. b) Prism monochromator

22. Gratings
Flat metallic grating
Concave grating – more accurate; eliminates mirrors and lenses
Holographic – Glass surface with photoresistor is grooved with
laser beam; where more accurate distance is achieved; higher
density of lines is possible – 6000/mm.
This master grating is coated with aluminum to replica of
holographic grating.

23. Sample containers (cuvettes)

24. Sample containers
The sample call that are to contain samples for analysis should
fulfill three main condition:
a) They must uniform in construction.
b) The material of construction should be inert to solvent.
c) They must transmit light of the wavelength used.
Most commonly use cell made up of quartz or fused silica.
When double-beam instrument are used they needed two cell
one as a reference and one for the sample, these two cell must
be same in construction to minimize the error called as
“Matched cells”.

25. CUVETTES
CUVETTES:
Virtually all UV spectra can be recorded in the solution phase
and the samples are placed in cells or cuvettes. Cells may be
made of glass, plastic or quartz. Quartz is transparent in all (200-
700nm) ranges and is normally used in UV region. Plastic and
glass are only suitable for visible spectra.

26. DETECTORS
Signal from detector have to be proportional to the amount
of
radiation
Response of detector have to be linear
 Detector should have wide linear response range
 DETECTORS:
The four common types of detectors are:
1.Barrier layer cell or photovoltaic cell
2.Phototube or photocell or photo-emissive tubes
3.Photomultiplier tubes(PMT)
4. The linear photodiode array
5. Charge-Coupled Devices (CCDs)

27. Barrier layer cell or photovoltaic
cell
Also known as photovoltaic.
Consist on the semiconductor such as selenium which is
deposited on a strong metal base, such as iron.
Very thin layer of silver and gold id sputtered over the
surface of the semiconductor as the second collector
electrode.
Radiation falling on the surface produce the electron on
the silver interface. Electron are accumulated on silver
surface and this accumulation produce the voltage different
between silver surface and the bas of cell.
Photocurrent is flow which is directly proportional to the
intensity of incident radiation beam.

28. Phototubes (PT)
Photon Transducers: Covert photon energy to electrical
signal (current, voltage, etc.)
• Detectors based on photoelectric effect:
Phototubes, Photomultiplier tubes
• Phototube:
– Incident photon causes release of an electron
– Photocurrent
– Not best for low-light scenarios

29. Phototubes (PT)
Its consist on the high sensitive cathode in the form of a half
cylinder of metal which is contained in an evacuated tube.
The anode is also present in the tube.
Inside surface on the photo cell is coated with the high
sensitive layer.
When the light incident upon the photo cell the surface
coating emits the electrons.
These are attracted and collected by an anode.
The current which is created between the cathode and anode
is regarded a measure of radiation falling on the detector.

30. Phototubes(PT)

31. Photomultiplier tubes(PMT)
The photomultiplier tube is a commonly used detector
in UV-Vis spectroscopy.
It consists of a photo-emissive cathode (a cathode
which emits electrons when struck by photons of
radiation), several dynodes (which emit several
electrons for each electron striking them) and an anode.
A photon of radiation entering the tube strikes the
cathode, causing the emission of several electrons.
These electrons are accelerated towards the first
dynode (which is 90V more positive than the cathode).

32. Photomultiplier tubes(PMT)
 The electrons strike the first dynode, causing the emission of
several electrons for each incident electron.
 These electrons are then accelerated towards the second
dynode, to produce more electrons which are accelerated towards
dynode three and so on.
 Eventually, the electrons are collected at the anode. By this
time, each original photon has produced 106 - 107 electrons.
 The resulting current is amplified and measured.
 Photomultipliers are very sensitive to UV and visible radiation.
They have fast response times.
 Intense light damages photomultipliers; they are limited to
measuring low power radiation.

33. The linear photodiode array
The linear photodiode array is an example of a multichannel
photon detector.
These detectors are capable of measuring all elements of a beam
of dispersed radiation simultaneously.
A linear photodiode array comprises many small silicon
photodiodes formed on a single silicon chip.
For each diode, there is also a storage capacitor and a switch.
The individual diode capacitor circuits can be sequentially
scanned.
They are useful for recording UV absorption spectra of samples
that are rapidly passing through a sample flow cell, such as in an
HPLC detector.

34. The linear photodiode array

35. Charge-Coupled Devices (CCDs)
 Charge-Coupled Devices (CCDs) are similar to diode
array detectors, but instead of diodes, they
 consist of an array of photo-capacitors.

36. Recording system
 The signals from the photomultiplier tube is finally
received by the recording system.
 The recording is done by the recorder pen.

37. Application
The Ultra-violet spectroscopy has been mainly applied
for the detection of functional group, the extent of
conjugation, detection of polynuclear compound by
comparison etc.
The wavelengths of absorption peaks can be correlated
with the types of bonds in a given molecule and are
valuable in determining the functional groups within a
molecule.

38. Some important application is as follow:
1. Extent of conjugation:
Conjugation may be between two or more C=C or triple
bond, between C-C or C-O bond, between double
bond in aromatic rings.
2. Identification of unknown compound:
Unknown compound can be identified by their
characteristic absorption peaks.
3. Detection of functional group.
4. Distinction of conjugation and non conjugation
compound.
5. Examination of poly nuclear carbon atom.
6. Elucidation of the structure of Vit A and Vit K.
7. Preference over two Tautomeric form.
8. Detection of impurity.

39. 9. Identification of compound in different solvent.
eg: Chloral hydrate shows absorption maximum at 290 mu in
Hexane while the absorption is disappear in aqueous
solution.
10. Determination of configuration of Geometrical isomers:
Cis compound is absorb at different wavelength as compare to
trans compound.
eg: Cis- stilben and Trans-stilben
11. Determination of strength of hydrogen bonding.

40. 12. Chemical Reaction
A common method of analysis is to change the required
component by adding a chemical reagent which reacts with it
specifically to form a highly absorbing compound. An example
of this is shown in Figure 16. A quantity of the reagent added to
the mixture reacts only with one component and both increases
its absorption and changes the wavelength of the absorption
maximum so that there is no longer interference between the
components. The analysis is then reduced to a simple case and
its sensitivity is improved. Many hundreds of such specific
reagents are now available for all sorts of analyses and sample
matrices and are thoroughly detailed in the literature.

41. Improving sensitivity by adding a chemical reagent

42. REFERENCES
 Chatwal Gurdeep R & Anand Sham K “Instrumenral Method of Chemical
Analysis” Fifth edition, 2007, Published by Himalaya Publication House,
page No: 2.107-2.148
 Sharma Y.R & Chand.K “Elementary Organic Chemistry Organic
Spectroscopy, Principle and Chemical Applications”, Forth Edition 2009,
Published by S.Chand & Company Ltd. Page No: 09-64
 http://learnspectroscopy.blogspot.com
 wikipedia.org
 www.molecularinfo.com
 www. shu.ac.uk.com