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UV/Vis.

INSTRUMENTATION

DR. BHARAT G. CHAUDHARI


M. Pharm., Ph. D.
VEERAYATAN INSTITUTE OF
PHARMACY

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Principle

 The principle involved in working of UV


spectrophotometers is Beer-Lambert’s law.
 Mathematical equation for Beer-Lambert’s law
is: A=εct
Where A=Absorbance or optical density or
extinction coefficient
ε=Molecular extinction coefficient
c=Concentration of drug (m mol/lit)
t=Path length (normally 10mm or 1cm)
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Instruments used:
 Colorimeters
• Inexpensive, less accurate. Measure either Absorbance or Transmittance or
both and has filters for use with different coloured solutions.
• Wavelength range: 400-700 nm.

 Spectrometers
• Spectrometer is a term that is applied to instruments that operate over
a very wide range of wavelengths, from gamma rays and X-rays into
the far infrared.
• If the region of interest is restricted to near the visible spectrum, the
study is called spectrophotometry.

 Spectrophotometers
• Little more expensive than colorimeters. Highly accurate since they employ
grating monochromators and photomultiplier tubes.
• WL range:360-900 nm.
• Supported by amplifier, recorders or plotters for hard copy. The recent one are
either microprocessor or computer based for easy data manipulation.
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PARTS OF UV
INSTRUMENTATION
 Radiation source
 Wavelength selector
 Sample cell
 Detector
 Readout device

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Block diagram of model
spectrophotometer

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Radiation sources:
Basic requirements:
 They must provide energy over the
wavelength region.
 They should maintain a constant
intensity over the time interval during
which the measurements are made.
 They should be stable and free from
fluctuations.

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Visible source:
 Incandescent filament lamps are a source of
electric light that works by incandescence (a general term for heat-driven
light emissions)
a. Tungsten lamp (Tungsten filament lamp):
WL range: 400-800 nm.
b. Carbon arc lamp: WL range: 400-800 nm, Very high intensity
UV Source:
 Hydrogen (or) Deuterium (or) Xenon
discharge lamps: Discharge lamps consisting of glass or
Quartz, It is an evacuated tube filled with gas. Contains anode and
cathode. On supplying electricity filled gas molecules will absorb energy
and goes to higher energy level. Excited molecules come back to ground
state gives emission of radiation.
a. Hydrogen discharge lamp: WL 120-350 nm, stable and robust
b. Deuterium lamp: WL 200-400 nm, 3-5 times more intense than others, Most
widely used but expensive.
c. Xenon Discharge lamp: Intensity greater than Hydrogen discharge lamp.
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Wave length selectors:

Filters
1. Absorption filters
2. Interference filters
Monochromators
1. Prisms (Refractive ,Reflective or Littrow
type mounting)
2. Gratings (Diffraction ,Transmission)

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Absorption Filters:
Made up of glass, coated with pigments or are made up of dyed gelatins.
They absorb the unwanted radiations and transmit the rest of the radiation
which is required for colorimetry.

Merits: Simple in construction, Cheaper, Selection of filter is easy.

Demerits:
1. Less accurate since band pass is more (±30nm). (Band pass is the
difference in wavelength between the points where the
transmittance is one-half the maximum.)
2. Intensity of radiation becomes more due to absorption by filters.
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Interference filter (Fabry-Perot)
Remove unwanted radiation by simple Interference or Reinforcement
(Physical effect of superimposing of two waves)
Mechanism: Radiation reflected by 2nd
Constructive Interference: When two film and the incoming radiation
waves are in same plane and have undergoes constructive interference
proper distance and frequency, the to give monochromatic radiation.
amplitude of resultant will increase.
Destructive Interference: When two
waves have movement opposite to
each other, on superimposing both
the wave will be destroyed.

Merits:
1. Inexpensive.
2. More accurate than
absorption filters.
Demerits:
1. Peak transmission is low.
2. Higher resolutions cannot be
obtained. Dielectric
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spacer film
Prism monochromator (Refractive ,Reflective)
Prisms disperse the light radiation in to individual colours or WLs.
Found in inexpensive instruments.
Better resolution than filters due to lower band pass.

The prism is made up of quartz for use in the UV light, since glass absorbs
wavelengths shorter than about 330 nm.
Glass prism are preferable for the visible region of the spectrum, as the
dispersion is much greater than that obtained with quartz.

The prism is made up of quartz for use in the UV light, since glass
absorbs wavelengths shorter than about 330 nm.

Glass prism are preferable for the visible region of the spectrum, as the
dispersion is much greater than that obtained with quartz. 11
Diffraction grating
 Most modern UV
spectrophotometer uses
diffraction grating as a
Monochromator.
 It consisting of a very
large number of
equispaced lines (200-
2000 per mm) ruled on a
glass plate.
 They can be used either
as transmission grating or
when aluminized, as
reflection grating.

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Sample cells

The sample holders in UV instruments


should be made of quartz as it does not absorb UV
radiation. For Visible spectroscopy glass is used.
 Sample volume: Small volume cells(0.5 ml or
less)
 Large volume cells(5-10ml)
 Shape of cell: Mostly rectangular.
 Path length (internal distance): Normally 1 cm.
 Material: Quartz
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Radiation detectors and
indicators

1. Photovoltaic cells or Barrier layer


cells

2. Photo emissive (vacuum) Tubes

3. Photomultiplier tubes

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Photovoltaic or Barrier layer cells
MECHANISM:
 Electrons are transferred at
the interface from the
semiconductor to metal when
the semiconductor is
irradiated.

 The electric current produced


is proportional to the
radiance power of the
incident beam and to the
area of photosensitive being
irradiated.

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Merits:
1. At low level of illumination it produces photo current proportional to
the radiant power received on it.

2. It is relatively cheap and widely used in filter photometers,


nepheloterbidimeters and cheap spectrophotometers.

Demerits:
1. Slow response.

2. Fatigue effect.

3. While using it there should be no fluctuations in power and low


external electric resistance.

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Photo tubes or Photo emissive cells
 It consists of a photosensitive
cathode containing an alkali
metal oxide, such as cesium
oxide and an anode mounted
in an evacuated glass tube.

The spectral response of
phototube depends upon the
nature of material coated on
photocathode.
 Ex; Coatings of Cs-Sb (200-
650nm)
Cso2-Ag(250-1200nm)

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Mechanism:
 Radiant energy striking the photocathode causes photoelectrons to be
emitted and the number of electrons is proportional to the radiant power of
the incident beam .The velocity of electrons is directly proportional to
frequency of incident radiation energy .These electrons are collected by the
anode and a photocurrent is generated.

Merits :
1. More sensitive when compared to photovoltaic cell.

Demerits:
1. High voltage(90-100v) is required.

2. Current produced is small and hence amplification is required.

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Photomultiplier tubes
.Mechanism:
 It has a number of photo emissive
electrodes each charged at a
successively higher potential and
so arranged that the electrons
ejected from the photocathode travel
successively from one electrode to
the next.
 In each step the photocurrent is
increased by the secondary emission
of electrons.
 Each Dyanode is maintained at 75-
100V higher than proceeding one.
 At each stage, the electron emission
is multiplied by a factor 4 or 5 due to
secondary emission of electrons(106) 19
Merits:
1. It is highly sensitive to light.

2. Best suited where weaker or low radiation is received (even 200


times weaker than Photovoltaic cell). Hence useful in fluorescence
measurements.

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UV INSTRUMENTS-TYPES

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Colorimeters:
 Tungsten lamp,
 absorption filters,
 photovoltaic cell.
 Read either %Transmission or Absorbance.
 Normally, single beam,
 non-recording type.
 WL accuracy are normally ±30nm.

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Spectrocolorimeters:
 Tungsten lamp,
 Prism (monochromator),
 either photovoltaic cell or phototube.
 Single beam,
 non-recording type,
 Read either %Transmission or Absorbance.
 WL accuracy ±5nm.

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Spectrophotometers:
 Expensive,
 More sophisticated,
 read %Transmission or Absorbance,
 record absorption spectra, Double beam.
 Storage of spectrum, comparison of spectra, quantitative
techniques,
 rapid WL scanning, data manipulation, derivative spectral
mode are the options in more automated instruments.
 Either microprocessor based or software driven using
computer.
 WL accuracy is ±0.1nm.
 More accurate, sensitive, higher range, reliable and
repeatable than others.

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Double beam colorimeter

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SPECTROPHOTOMETERS TYPES:

 Single beam spectrophotometers

 Double beam spectrophotometers

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Single beam spectrophotometer

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Double beam spectrophotometer

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APPLICATIONS
Qualitative analysis :
 Detection of impurities.

 Structure elucidation of organic compounds.

 Prediction of functional groups of organic compounds.

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Quantitative analysis:
 Determination of molecular weight.

 Quantitative analysis of pharmaceutical substances.

 Assay of medicinal substances.

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References
1. Instrumental methods of analysis
Willard , Merritt

2. Textbook of pharmaceutical analysis


Ravishankar

3. Pharmaceutical analysis-Industrial methods


Nirali Prakashan

4. Spectroscopy B.K.Sharma

5. Industrial methods of instrumental analysis


Chatwal

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Thank you

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