U.V. -VISIBLE SPECTROSCOPY BY ANIQUA TAJASSUM SATTAR SHEIKH (M.PHARM SEM-1)

1. INSTRUMENTATION OF
UV-VISIBLE SPECTROSCOPY
Presented By
Aniqua Tajassum S. Sheikh
M. Pharm Sem - I
(P’ceutical Chemistry)
HOD : Dr. A. J. ASNANI
(M. Pharm, Ph. D)

2. CONTENT
■ Principle of UV-Visible Spectroscopy
■ Types of UV Spectrophotometer
■ Instrumentation
■ Components Involved in UV Spectrophotometer
1. Light Source
2. Monochromators
3. Sample Containers
4. Detectors
■ Advantages & Disadvantages
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INSTRUMENTATION OF UV SPECTROSCOPY
Priyadarshini J. L. College of Pharmacy, Hingna, Nagpur.

3. PRINCIPLE
■ It is based on the absorption of uv or visible light by a chemical
compound, which results in the production of spectra.
■ Spectroscopy is based on the interaction between light and matter.
■ It involves the measurement of absorption of light in visible
region :400-800 nm & UV region :200-400 nm.
■ The absorption of light by a substance at a particular wavelength is
directly proportional to the no. of molecules of the substance in the
path of light.”
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INSTRUMENTATION OF UV SPECTROSCOPY
-PRINCIPLE
Priyadarshini J. L. College of Pharmacy, Hingna, Nagpur.

4. TYPES OF UV SPECTROPHOTOMETER
1. Double Beam System :
 A double-beam spectrophotometer has two light paths. One path
passes through a “blank”, while the other passes through the
sample. This eliminates the need to manually re-zero the
spectrophotometer if the wavelength is changed. This allows
automation of wavelength scanning to produce an absorbance
spectrum.
 Double-beam uv spectrophotometer are more expensive than
single-beam.
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INSTRUMENTATION OF UV SPECTROSCOPY
-TYPES OF UV SPECTROPHOTOMETER
Priyadarshini J. L. College of Pharmacy, Hingna, Nagpur.

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INSTRUMENTATION OF UV SPECTROSCOPY
- INSTRUMENTATION OF DOUBLE BEAM SPECTROPHOTOMETR
Priyadarshini J. L. College of Pharmacy, Hingna, Nagpur.
Double Beam System :

6. 2. Single Beam System :
 A single-beam instrument has a single light path. The operator
must manually alternate between blank and sample whenever
the wavelength is changed.
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INSTRUMENTATION OF UV SPECTROSCOPY
-SINGLE BEAM UV SPECTROPHOTOMETER
Priyadarshini J. L. College of Pharmacy, Hingna, Nagpur.

7. COMPONENTS INVOLVED IN
UV SPECTROPHOTOMETER
1. Light Sources (UV and visible)
2. Wavelength selector (monochromator)
3. Sample containers
4. Detector
5. Signal processor and readout
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INSTRUMENTATION OF UV SPECTROSCOPY
-COMPONENTS INVOLVED IN UV
SPECTROPHOTOMETER
Priyadarshini J. L. College of Pharmacy, Hingna, Nagpur.

8. 1. LIGHT SOURCE
The most common radiation sources are;
a) Tungsten-Halogen filament Lamp (320nm – 1100nm)
b) Deuterium Arc Lamp (160nm – 375nm)
c) Hydrogen discharge lamp (160nm – 375nm)
d) Xenon discharge lamp (190nm - 1100nm)
e) Mercury Arc (250nm - 350nm)
 In all sources, excitation is done by passing electrons, through a gas
electronic, vibrational & rotational excitation in gas molecules.
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INSTRUMENTATION OF UV SPECTROSCOPY
- COMPONENTS INVOLVED IN UV SPECTROPHOTOMETER
Priyadarshini J. L. College of Pharmacy, Hingna, Nagpur.

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INSTRUMENTATION OF UV SPECTROSCOPY
- COMPONENTS INVOLVED IN UV SPECTROPHOTOMETER
Priyadarshini J. L. College of Pharmacy, Hingna, Nagpur.
Tungsten-Halogen Lamp Deuterium Lamp Hydrogen Discharge
Lamp
Xenon Discharge
Lamp
Mercury Arc
Lamp

10. 2. MONOCHROMATOR
■ All monochromators contains;
• An entrance slit
• A collimating lens
• A dispersing device (a prism or a grating)
• A focusing lens
• An exit slit
■ Polychromatic radiation (radiation of more than one wavelength)
enters the monochromator through the entrance slit. The beam is
collimated (made parallel), and then strikes the exit slit at an angle.
The beam is split into its component wavelengths by the grating or
prism. By moving the exit slit, radiation of only a particular
wavelength leaves the monochromator through the exit slit.
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INSTRUMENTATION OF UV SPECTROSCOPY
- COMPONENTS INVOLVED IN UV SPECTROPHOTOMETER
Priyadarshini J. L. College of Pharmacy, Hingna, Nagpur.

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INSTRUMENTATION OF UV SPECTROSCOPY
- COMPONENTS INVOLVED IN UV SPECTROPHOTOMETER
Priyadarshini J. L. College of Pharmacy, Hingna, Nagpur.

12. 3. SAMPLE CELLS
 Sample containers or cuvettes may be made up of
– Quartz
– Fused Silica (350 and 2000 nm)
– Borosilicate/Glass
– Plastic
■ The sample cell is based on the path length, shape, size &
transmission characteristics at the desired wavelength.
■ It should be transparent to the wavelength region to be recorded
(Quartz or Fused Silica).
■ The most common cuvette size is 1 cm, it can vary from 0.1-10 cm,
which may be rectangular in shape or cylindrical with flat ends.
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INSTRUMENTATION OF UV SPECTROSCOPY
- COMPONENTS INVOLVED IN UV SPECTROPHOTOMETER
Priyadarshini J. L. College of Pharmacy, Hingna, Nagpur.

13. 4. DETECTORS
■ To detect radiation, three types of photosensitive devices are;
a. Photovoltaic cells or barrier- layer cell
b. Phototubes or photo-emissive tubes
c. Photomultiplier tubes
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INSTRUMENTATION OF UV SPECTROSCOPY
- COMPONENTS INVOLVED IN UV SPECTROPHOTOMETER
Priyadarshini J. L. College of Pharmacy, Hingna, Nagpur.

14. a. Photovoltaic cells or barrier- layer cell
■ Photovoltaic cell detector works on the principle of the generation of
potential difference when the transmitted UV radiation falls on the
semiconductor layer.
■ It consists of a semiconductor metal like selenium deposited over a
metallic base plate like iron or aluminium. Then the surface of
selenium is covered by a very thin layer of silver or gold which acts as
a second collector tube.
■ Radiation → Barrier layer cell (silver) → electrons generated at the
selenium- silver interface → barrier layer prevents electrons flow into
iron → electrons collected on silver surface → electric voltage
difference between the silver surface & the base of the cell.
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INSTRUMENTATION OF UV SPECTROSCOPY
= COMPONENTS INVOLVED IN UV SPECTROPHOTOMETER
Priyadarshini J. L. College of Pharmacy, Hingna, Nagpur.

15. 2. Phototubes or photo-emissive tubes
■ Photo-emissive tube detector works on the principle of emission of
electrons from the photocathode and subsequent capturing of
electrons by the anode resulting in the flow of current.
■ They are concave surfaces of cathode and anode inside a glass bulb.
The cathode is coated with light sensitive layer e.g. potassium oxide,
silver oxide, etc.
■ Radiation → upon cathode → photoelectrons are emitted →
collected by anode → current flow → current is amplified and
recorded.
■ Photo-emissive cell detector is more sensitive as compared to
photovoltaic cell detectors because higher degree of amplification
can be used.
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INSTRUMENTATION OF UV SPECTROSCOPY
- COMPONENTS INVOLVED IN UV SPECTROPHOTOMETER
Priyadarshini J. L. College of Pharmacy, Hingna, Nagpur.

16. 3. Photomultiplier tubes
■ Photomultiplier tube detector works on the principle of
multiplication of photoelectrons by the secondary emission of
electrons.
• The multiplication of electrons is achieved by using a
photocathode and a series of anodes called dynodes. The
photomultiplier tube can use up to 10 dynodes. Hence the
detector can detect even very weak signals.
• In the vacuum tube, the photocathode tube is fixed which receives
the transmitted radiation from the sample.
• Radiation → strike on the first dynode → electrons emitted →
strike on second dynode → produce more electrons & so on →
each photon produce 106-107 electrons → collected at the anode.
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INSTRUMENTATION OF UV SPECTROSCOPY
- COMPONENTS INVOLVED IN UV SPECTROPHOTOMETER
Priyadarshini J. L. College of Pharmacy, Hingna, Nagpur.

17. ■ ADVANTAGES :
1. The core advantage is the accuracy of the UV-VIS
spectrophotometer.
2. It can be utilized in the qualitative and quantitative analysis.
3. The Derivative graph can be obtained by UV-VIS
spectrophotometer.
■ DISADVANTAGES:
1. Only those molecules are analyzed which have
chromophores.
2. The results of the absorption can be affected by pH,
temperature, contaminants, and impurities.
3. Only liquid samples are possible to analyze.
4. Cuvette handling can affect the reading of the sample.
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INSTRUMENTATION OF UV SPECTROSCOPY
-ADVANTAGES & DISADVANTAGES
Priyadarshini J. L. College of Pharmacy, Hingna, Nagpur.

18. THANK YOU!
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Priyadarshini J. L. College of Pharmacy, Hingna, Nagpur.