UV - Instrumention Details

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By – Dr. UMESH KUMAR SHARMA
& SHYMA M. S.
DEPARTMENT OF PHARMACEUTICS
MAR DIOSCORUS COLLEGE OF PHARMACY,
THIRUVANANTHAPURAM, KERALA, INDIA
UV – VISIBLE
SPECTROSCOPY
INSTRUMENTATION

2. CONTENTS
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• Instrumentation
• Types of instruments
• UV-Visible spectrophotometer
• Definition
• Internal components
• Instrumental Components
• UV -Visible radiation sources
• Sample cells
• Detectors
• Different types of spectrophotometer
• References

3. INSTRUMENTATION
Instruments are used for measuring molecular absorption at the
ultraviolet, visible and near infrared regions are produced by
dozens of companies.
Some instruments are simple & inexpensive, complex computer
controlled scanning instruments.
TYPES OF INSTRUMENTS
• Colorimeter
Small range of wavelength is 400 - 700nm.
• Spectrophotometer
High range of wavelength is 360 - 900nm.
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4. UV-VISIBLE
SPECTROPHOTOMETER
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The instrument used in UV - Visible Spectroscopy is called
UV -Visible Spectrophotometer.

5. DEFINITION
A spectrophotometer is an instrument that measure the amount of
light absorbed by a sample.
 It measure the intensity of light passing through a sample (I),
and compare it to the intensity of light before it passes through
the sample (Io).
 The ratio I/Io is called the transmittance, and is usually
expressed as a percentage (%T).
 The absorbance A is based on the transmittance.
A= log(%T/100%)
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6.  The UV-visible spectrophotometer can also be configured to
measure reflectance.
 In this case, the spectrophotometer measures the intensity of
light reflected from sample (I), and compares it to the intensity
of light reflected from a reference material (Io) (such as white
tile).
 The ratio I/Io is called the reflectance and usually expressed as
a percentage (%R).
INSTRUMENTAL COMPONENTS
1. Radiant Source 2. Wavelength selector
3. Sample containers 4. Radiation transducers
5. Signal processors 6. Readout devices 6

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INTERNAL COMPONENTS OF
UV-VISIBLE SPECTROPHOTOMETER

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SYSTEMATIC DIAGRAM OF
UV- VISIBLE SPECTROPHOTOMETER

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PARTS OF UV- VISIBLE
SPECTROPHOTOMETER
SOURCE OF LIGHT:
Basic requirements
• They must provide sufficient energy over the wavelength
region (400 – 800nm.) for easy detection & measurement.
• It must supply continuous radiation of adequate intensity,
stable and free from fluctuation over the entire wavelength
region in which it is used.
• The output power should be stable for reasonable period.

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Sources are of two types
 Continuum sources
Which emits radiation over a broad range of wavelength with a
relatively smooth variation in intensity.
 Line sources
Which emits a narrow band of radiation, which is important
because they are highly selective.
UV- VISIBLE LAMP
a. Hydrogen discharge lamp b. Tungsten lamp
c. Deuterium lamp d. Xenon discharge lamp

11. HYDROGEN & DEUTERIUM LAMP
• A continuum spectrum in the UV region is produced by electrical
excitation of hydrogen or deuterium.
• Initially the deuterium molecule absorb electrical energy. Which
results in the formation of an excited species followed by the
dissociation of excited molecule in to two atomic species plus a
UV photon.
D2+electrical energy D2 D1+D11+hv
Ee = electrical energy absorbed by the molecule
D2
* = excited deuterium molecule
D2 to get excited it should absorbs a fixed energy but the sum
of energies of D1 and D11 can vary from zero to D2
* energy.
• Thus a continuum spectrum is obtained from 160-375nm.
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• The energy of photon can vary continuously. Only quartz
cuvettes are used, because glass absorb radiation of
wavelength less than 350nm.
Advantages
• Radiation is stable .
• Intensity of radiation is emitted 3-5 times the intensity of
hydrogen lamp.
Disadvantages
• expensive
Hydrogen Lamp Deuterium Lamp

13. TUNGSTEN LAMP
 Most common light source used in spectrophotometer.
 The lamp consist of a tungsten filament in a vacuum bulb it
offers sufficient intensity.
 It has long life.
 Used wavelength region 350 - 2500nm.
 In the visible region the energy output varies with power of
operating voltage. The voltage control is essential for stable
radiation.
 Tungsten halogen lamps contain small quantity of iodine within
a quartz envelop that houses the filament.
 Quartz is required due to the high operating temperature. 13

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 The iodine react with tungsten gas molecules formed by
sublimation & the product is volatile.
 The molecule strike the filament, the decomposition occurs and
the tungsten lamp have long life time.
Advantages
• Have long half life
• Stable, cheap, easy to use.
Disadvantages
• Intensity at lower wavelength region in very feeble.
• Need voltage control for stable radiation.
Tungsten Lamp

15. XENON DISCHARGE LAMP
 This lamp produces intense radiation by passage of current
through an atmosphere xenon.
 The spectrum is continuous over the range between 200 -
600nm.
 Xenon gas is stored under pressure in the range of 10 - 30 atm.
 The excess xenon lamp possess two tungsten electrodes
separated by about 8nm.
 When an intense arc is formed between two tungsten electrodes
by applying a low voltage the UV light is produced.
 The intensity of UV radiation produced by xenon discharge
lamp is greater than that of hydrogen lamp.
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Advantages
• The intensity of UV radiation produced are greater than that
of hydrogen lamp.
• Emit both UV and visible wave length.
Xenon Discharge Lamp

17. WAVE LENGTH SELECTORS
• Most of the spectroscopic analysis radiation that consist of a
limited narrow continuous group of wave length is called as
band.
• Ideally the output from a wavelength selector would be a
radiation of single wavelength / frequency.
• A narrow band width represents better performance.
• A filter or monochromators is used which converts
polychromatic light to monochromatic light.
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18. A) MONO CHROMATOR
All monochromators contain the following component parts,
a. An entrance slit,
b. A collimating lens,
c. A dispersing device (a prism or a grating),
d. A focusing lens,
e. An exit slit,
• Polychromatic radiation (radiation of more than one
wave length) enters the monochromator through the
entrance slit.
• The beam is collimated, and then strikes the dispersing
element at the angle .
• The beam is split in to it component wavelengths by the
grating or prism.
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19. • By moving the dispersing element or the exit slit, the radiation of
only a particular wavelength leaves the monochromator through
the exit slit.
In spectrophotometer 2 types of wavelength selectors are used.
a. Monochromators b. Filters
a. TYPES OF MONOCHROMATORS
1) Prism type
1. Dispersive type 2. Littrow type
2) Grating type
1. Diffraction grating 2. Transmission grating 19

20. 1) PRISMS
 The prisms disperse the light radiation into individual colours or
wavelengths.
 These are found in inexpensive instruments.
 The band pass is lower than that of filters and hence it has better
resolution.
 The resolution depends upon the size and refractive index of the
prism.
 The material of prism is normally glass.
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21. Refractive type
(dispersive type) :
 The source of light through entrance
slit falls on a collimator.
 The parallel radiation from collimator
are dispersed into different colours or
wavelengths & by using another
collimator, the images of entrance slit
are reformed.
 The reformed ones will be either
violet, indigo, blue, green, yellow,
orange or red.
 The required radiation on exit slit are
be selected by rotating the prism or
by keeping the prism stationary
&moving the exit slit.
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Reflective type
( Littrow type):
• The principle of working is
similar to the refractive type
except that, a reflective surface
is present on one side of the
prism.
• Hence the dispersed radiations
gets reflected & can be collected
on the source of light.

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2) GRATINGS
• Grating are the most efficient ones in converting a
polychromatic to monochromatic light.
• As a resolution of ± 0.1 nm could be achieved by using
gratings, they are commonly used in spectrophotometer.
• Grating are of two type.
A. Diffraction Grating
B. Transmission Grating

24. A. Diffraction Grating
• These are rulings made on sample material like glass, quarts, or
alkyl halides ,depending upon the instrument.
• The number of rulings per mm also ranges from 3600 grooves or
more lines per mmm for UV - visible spectrophotometer.
• The mechanism is that diffraction produces reinforcement.
• The rays which are incident upon the grating gets reinforced
with the reflected rays.
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B. Transmission Grating
 It is similar to diffraction grating, but refraction takes place instead
of reflection, refraction, produces reinforcement.
 When radiation transmitted through grating reinforces with the
partially refracted radiation.

26. b. FILTERS
1) Absorption Filters :
o These filters are work by selective absorption of wave length.
o These filters are made up of glass, coated with pigments or
they are made up of dyed gelatine.
o The colour is produced by incorporating oxides of chromium,
manganese, iron etc.
o They absorb the unwanted radiation & transmit the rest of the
radiation which is required for colorimetry.
o The range of wave length transmitted is very wide and may
exceed 150nm.
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o Selection of filter is depend
upon sample.
o Draw wheel with 6 parts. Write
the colors of VIBGYOR in
clock wise or anti clock wise
manner omitting indigo.
o If the color of sample is red we
have to use green filter, and if
the color of sample is green we
have to use red filter ( the color
of filter is opposite to the color
of solution.
o That is the colour of filter is
opposite to the colour of the
solution.

28. Merits
• Simple in construction, Cheaper, Selection of filter is easy.
Demerits
• Less accurate, Intensity of radiation become less due to
absorption by filters.
2) Interference Filters
 These filters are also known as Fabry - Perot filter.
 It has a dielectric spacer film made up of CaF2, MgF2 and SiO
between two parallel reflecting silver films.
 Maximum transmission is 40%.
 The mechanism the radiation reflected by the second film & the
incoming radiation undergoes constructive interference to give a
monochromatic radiation. Band pass is 10-15nm. 28

29. Merits
• Inexpensive.
• Lower band pass when compared to absorption filters &
hence more accurate.
Demerit
• Peak transmission is low & becomes 50 when additional
filters are used to cuts off undesired wave length.
• The band pass is only 10-15 cm.
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30. SAMPLE CELLS
A variety of sample cells available for UV region. The
choice of sample cell is based on –
• The path length, shape, size.
• The transmission characteristics at the desired
wavelength.
• 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 2000 nm.
• The thickness of the cell is generally 1 cm. cells
may be rectangular in shape or cylindrical with
flat ends.
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DETECTORS
• They are two types one is respond to heat and other is respond to
photon.
• The detectors respond to light or photon are called photoelectric
detectors.
• They are actually transducers that convert radiant energy to an
electrical signals.
• An ideal transducer should have high light sensitivity, high signal
noise ratio, response over a considerable range of wavelength.
In order to detect radiation, three types of
photosensitive devices are
a. Photo Voltaic cells or barrier- layer cell
b. Photo tubes or photo emissive Cell
c. Photo multiplier tubes

32. PHOTO VOLTAIC CELL
• Photovoltaic cell are also known as barrier layer or photronic cell.
• The photo voltaic cell is simple and used for measuring radiation
in the visible region.
• The barrier layer cell consists of a metallic plate usually copper or
iron, which act as the anode.
• Upon this layer a semi conducting material like selenium or
copper is deposited.
• Then the surface of selenium is covered by a very thin layer of
silver or gold, which act as a second collector tube.
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• When the radiation is incident upon the surface of selenium,
electrons are generated at the selenium- silver surface and the
electrons are collected by the silver.
• This accumulation at the silver surface creates an electric
voltage difference between the silver surface and the basis of
the cell.
• The radiation of sufficient energy falls on the semi conductors
covalent bonds are broken so that electrons & holes flow to the
wire anode generating a photo current about on length greater
than produced by photo voltaic cell.
• On increasing the potential a saturation potential is reached
when all the emitted electrons will reach the anode The current
then to radiant power .
• The operation potential is usually 90v.the exact metal coated on
the photo emissive cathode determine the wave length response

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• Advantages
• Simple in design do not need external power supply.
• Cheapest and inexpensive.
• Disadvantages
• Amplification of detector is not possible.
• Lesser response of detector with light other than blue or red light.

35. PHOTOTUBES
• Phototubes are also known as photo emissive cells.
• A phototube consists of an evacuated glass bulb.
• There is light sensitive cathode inside it.
• The inner surface of cathode is coated with light sensitive layer
such as potassium oxide and silver oxide.
• When radiation is incident upon a cathode, photoelectrons are
emitted.
• These are collected by an anode. Then these are returned via
external circuit. And by this process current is amplified and
recorded.
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36. THE PHOTOMULTIPLIER TUBE
• The photomultiplier tube is a commonly used detector in UV
. spectroscopy.
• It consist of a cathode covered with a photo emissive material,
which emits electrons when exposed to radiation.
• The tube contain additional electrodes called dynodes.
• Dynodes is maintained at a potential 90v more positive than the
cathode and so the electrons emitted by the cathode are attracted
towards it.
• Each photo electron striking the dynodes causes emission of
several additional electrons.
• These electrons are then accelerated towards the second dynode,
which is at 90v more than dynode 1. 36

37. • Again several electrons are emitted for each electron.
• Repetition of the process leads to formation of 106 to 107
electrons for each photon striking the cathode.
Advantages
• Ideal for measuring weak light intensities.
• Fast in response.
Disadvantages
• Very intense light cause irreversible damage of photo emissive
surface .hence should be protected from day light other stringer
radiations.
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Cross section of a photomultiplier tube

39. TYPES OF SPECTROPHOTOMETER
I. Single beam
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40. • A spectrophotometer can be either single beam or double beam.
• In a single beam instrument (such as the Spectronic 20), all of
the light passes through the sample cell.
• Io must be measured by removing the sample.
• This was the earliest design and is still in common use in both
teaching and industrial labs.
Advantages
• This type is cheaper.
• The system is less complicated.
• Low cost.
• High sensitivity.
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41. 2. Double-beam instrument
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42. • In a double-beam instrument, the light is split into two beams
before it reaches the sample.
• One beam is used as the reference, and the second beam
passes through the sample.
• The reference beam intensity is taken as 100% Transmission
(or 0 Absorbance), and the measurement displayed is the
ratio of the two beam intensities.
• Measurements from double beam instruments are easier and
more stable.
Advantages
• High stability because reference and sample are measured
virtually at the same moment in time.
Disadvantages
• High cost, Lower sensitivity.
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43. REFERENCES
1. Text book of pharmaceutical analysis 4th
edition Dr. Ravi Sankar, page no. 1-25.
2. Principles of instrumental analysis Douglas,
Skoog, F.James Holler, page no. 345-358.
3. Instrumental methods of chemical analysis B.K
Sharma, page no. 1.12-1.25.
4. Instrumental methods of analysis 7th edition
Willard, Merritt, Dean, Settle, page no118-129. 43

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