The document discusses UV-visible spectroscopy, detailing its components (light source, monochromator, sample holder, detectors) and the functioning of spectrophotometers. It explains how light sources such as hydrogen, deuterium, tungsten, and xenon arc lamps are used, along with the importance of monochromators and various detectors. The document also compares single-beam and double-beam spectrophotometers, outlining their advantages and disadvantages.

1. INSTRUMENTATION OF
UV-VISIBLE SPECTROSCOPY
Prepared by,
Nilam Mistry
Roshni Panchal
1st Sem M.Pharm (Pharmaceutical chemistry)
Parul institute of Pharmacy
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2. CONTENTS:
o Introduction
o Components
o Light source
o Monochromator
o Sample holder
o Detectors
o Instrumentation design
o Reference videos
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3. INTRODUCTION:
 Spectroscopy is the branch of science that deals
with the study of interaction of electro-magnetic
radiation (EMR) with matter.
 Instrument used to measure the absorbance in
UV (200-400nm) or Visible (400-800nm) region is
called UV-Visible Spectrophotometer.
 A Spectrophotometer records the degree of
absorption by a sample at different wavelengths
and the resulting plot of absorbance (A) versus
wavelength (λ) is known as Spectrum.
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4. 4

5. COMPONENTS:
 Light sources
 Monochromator
 Sample holder
 Detector
 Recorder
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6. LIGHT SOURCE
Requirements:
 It should be stable.
 It should provide continuous radiation.
 It must be of the sufficient intensity for the
transmitted energy to be detected at the end
of the optical path.
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7. 1.HYDROGEN AND DEUTERIUM
DISCHARGE LAMPS
 A continuous spectrum in the UV region is
produced by electrical excitation of
deuterium or hydrogen at low pressure.
 Range: 3500-1200A (160-800nm)
 These are stable, robust and widely used.
 If deuterium lamp is used instead of hydrogen
lamp it increase the emission intensity.
 Deuterium lamp is more expensive then
hydrogen lamp but are used when higher
intensity is required.
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8. Hydrogen Lamp Deuterium Lamp
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9. 2.TUNGSTEN FILAMENT LAMP
 It is the most common light source used in a
spectrophotometer.
 Also called as “Quartz halogen lamp”
 These lamp consists of a tungsten filament
enclosed in a glass envelope, with a wavelength
range of about 320-2500nm, are used for the
visible region.
 Quartz allows the filament to be
operated at a temperature of about
3500K which leads to higher intensity in a
UV region.
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10. 3.XENONE ARC LAMP
 Xenone gas is stored in a lamps at 10-30 atm
pressure.
 It contain 2 tungsten electrodes that are
separated by a distance of about 8mm.
 When current passes through xenone cause
thermal excitation.
 It produces greater UV
radiation then the hydrogen
lamp.
 It is a continuous source.
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11. 4.MERCURY ARC LAMP
 In this mercury vapour is stored under high
pressure and the excitation of mercury atoms
is done by electric discharge.
 It is not suitable for continuous radiations.
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12. MONOCHROMATORS
 It is a device used to isolate the radiation of
the wavelength from wavelength of the
continuous spectra.
 These are types of monochromatic devices.
 Filters
 Prisms
 Grating
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13. 1.FILTERS
 Selection of filters is usually done on a
compromise between peak transmittance
and band pass width; the former should be as
high as possible and latter as narrow as
possible.
TYPES OF FILTERS:
1. Absorption filters
2. Interference filters
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14. Types Of
 Works by selective absorption
of unwanted radiation and
transmits the radiation which is
required.
 Examples – Glass and Gelatin
filters.
 These is less accurate
Merits:
 Simple in construction
 Cheaper
Filters
 Works on the interference
phenomenon, cause rejection of
unwanted wavelength by
selective reflection.
 It is constructed by using 2
parallel glass plates, which are
silvered internally and
separated by thin film of
dielectric material of different
(CaF2, MgF2) refractive index.
 Provide greater transmittance
and narrower band pass (10-
15nm)
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Absorption Filters Interference Filters

15. 2.PRISM
 Prism is made from glass, Quartz or fused silica.
 Quartz or fused silica is the choice of material of
UV spectrum.
 When white light is passed through glass prism,
dispersion of polychromatic light in rainbow
occurs.
 Now by rotation of the prism different
wavelength of the spectrum can be made to pass
through in exit slit on the sample.
 The effective wavelength depends on the
dispersive power of prism material and the
optical angle of the prism.
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17. 3.GRATINGS
 Gratings are most effective one in converting a
polychromatic light to monochromatic light.
 As a resolution of +/- 0.1nm could be achieved by
using gratings.
MERITS:-
o Grating gives higher and liner dispersions compared to
prism monochromator.
o Can be used over wide wavelength ranges.
o Gratings can be construed with like materials
aluminium which is resistant to atmospheric moisture.
o Provide light of narrow wavelength.
o No loss of energy due to absorption
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18. TYPES OF GRATINGS:
1. DEFRACTION GRATING
 More refined dispersion of light is obtained by means of diffraction
grating.
 These consist of large number of parallel lines (grooves) about 15000-
30000/ inch is ruled on highly polished surface of aluminum.
 These gratings are replica made from master gratings by coating the
original master grating with a epoxy resin and are removed after setting.
 To make the surface reflective, a deposit of aluminum is made on the
surface. In order to minimize to greater amounts of scattered radiation
and appearance of unwanted radiation of other spectral orders, the
gratings are blazed to concentrate the radiation into a single order.

2. TRANSMISSION GRATING
 It is similar to diffraction grating but refraction takes place instead of
reflection. Refraction produces reinforcement.
 This occurs when radiation transmitted through grating reinforcement
with the partially refracted radiation
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19. SLITS AND MIRRORS
 Slits are an important device in a resolving
polychromatic radiation into monochromatic
radiation.
 To archive these, entrance slit and exit slit are
used.
 The width of slit plays a important role in
resolution of polychromatic radiation.
 Mirrors are used to reflect, focus light beams in
a spectrophotometers.
 To minimize the light loss, mirrors are
aluminized on there front surfaces.
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20. SAMPLE HOLDER
 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 colour
corrected fused glass is used for visible region.
 The surfaces of absorption cells must be kept
clean. No fingerprints or blotches should be
present on cells.
 Cleaning is carried out washing with distilled
water or with dilute alcohol, acetone.
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21. 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.
Requirements of an ideal detector:-
 It should give quantitative response.
 It should have high sensitivity and low noise level.
 It should have a short response time.
TYPES OF DETECTORS
1. Barrier layer cell/Photovoltaic cell
2. Phototubes/ Photo emissive cell
3. Photomultiplier tube
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22. 1.BARRIER LAYER 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.
 This creates a potential difference between two electrodes
and 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.
 Also called as photovoltaic cell
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23. 23

24. 2.PHOTO-EMMISSIVE 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 caesium, 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.
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25. 25

26. 3.PHOTO MULTIPLIER TUBE
 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 where weaker or low radiation is received
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27.  https://www.youtube.com/watch?v=s-
xy8YOItwQ
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28. AMPLIFIER AND RECORDER
 Amplifier amplifies signal coming from
detector
 Recorder records them which is displayed on
readout device.
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29. INSTRUMENTATION DESIGN
 Depending upon the monochromators (filters or
dispersing device ) used to isolate and transmit a
narrow beam of radiant energy from the incident
light determines whether the instrument is
classified as Photometer or a
Spectrophotometer.
 Spectrophotometer used here detects the
percentage transmittance of light radiation,
when light of certain intensity and frequency
range is passed through the sample.
 Both can be a single beam or double beam
optical system.
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30. SINGLE BEAM SPECTROPHOTOMETER
 Light from the source is carried through lens or 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.
 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 reading of solution of both standard and unknown are
recorded in optical density unit a after adjusting instrument to
a reagent blank.
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31. ADVANTAGES:
 Simple in construction, Easy to use and economical .
DISADVANTAGES:
 Any fluctuation in the intensity of radiation sources affects the
absorbance.
 Continuous spectrum is not obtained. 31

32. DOUBLE BEAM SPECTROPHOTOMETER
 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.
 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.
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33. `
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D
ADVANTAGES:
 It facilitates rapid scanning over wide (λ) region.
 Fluctuations due to radiation source are minimised.
 It gives ratio of intensities of sample and reference beams
simultaneously.
DISADVANTAGES:
 Construction is complicated.
 Instrument is expensive.

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35. REFERENCE VIDEOS:
 https://www.youtube.com/watch?v=pxC6F7b
K8CU
 https://www.youtube.com/watch?v=sVc-
XIysw0c
 https://youtu.be/lQTsN5pHmKc
 https://youtu.be/XAp-5r3LxQo
 https://youtu.be/jyDXjQDxb-8
 https://www.directindustry.com/prod/nanodr
op/product-36205-1688787.html
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37. THANK YOU
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