UV – Visible Spectrophotometers Instrumentation
At the end of the
session students will be able to
the essential components of UV Visible spectrophotometers
the construction and working of radiation sources and dispersive devises used
in UV- Visible spectrophotometers
UV Visible Spectrophotometer
- Wavelength selectors (filters,
- Sample containers
- Readout devices
A single beam
The above essential features of a spectrophotometer shows
that polychromatic light from a source
separated into narrow band of wavelength (nearly monochromatic light) by a wavelength selector, passed through
the sample compartment and the
transmitted intensity, P, after the sample is measured by a detector
In a single beam
instrument, the light beam follows a single path from
the source, to the monochromator, to the sample cell and finally to the
1- Sources of light
Sources used in UV-Vis Spectrophotometers are continuous
sources emit radiation of all wavelengths within the spectral region
for which they are to be used.
of radiation should also be stable and of high intensity.
2. Wavelength Selectors
Ideally the output of a wavelength selector would be a
radiation of a single wavelength.
No real wavelength selector is ideal, usually a band
of radiation is obtained.
The narrower this bandwidth is , the better
performance of the instrument.
Wavelength selectors for
100 ~ 40,000
3000 lines/mm for vacuum UV,
50 lines/mm for far IR
120 ~ 30,000
200 ~ 14,000
380 ~ 750
Dispersion of radiation along the focal plane AB of a
typical prism(a) and echellette grating (b).
Schematic diagram of diffraction from a grating.
nl = (a
d sin q
– d sin f = b
nl = d
(sin q + sin f )
permit certain bands of wavelength (bandwidth of ~ 50 nm) to pass
simplest kind of filter is absorption filters , the most common of this
type of filters is colored glass filters.
used in the visible region.
colored glass absorbs a broad portion of the spectrum (complementary color) and
transmits other portions (its color).
• They are not very good wavelength selectors
and can’t be used in instruments utilized in research.
• This is because they allow the passage of a
broad bandwidth which gives a chance for deviations from Beer’s law.
• They absorb a significant fraction of
the desired radiation.
(a) Schematic cross section of an interference filter.
(b) Schematic to show the conditions for constructive
of interference filters.
band pass filter has ~90% transmission in the 3- 50 5- mm wavelength range but <0.01%
transmittance outside this range.
band-pass filter has a transmission width of 0.1 mm
centered around 4 mm.
– selection of filters
are used for spectral scanning (varying the wavelength of
radiation over a considerable range ).
can be used for UV/Vis region.
monochromators are similar in mechanical construction.
monochromators employ slits, mirrors, lenses, gratings or prisms.
radiation from the entrance slit is collimated (made into beam of parallel rays)
by a concave mirrors
M These rays fall on a reflection grating,
whereupon different wavelengths are reflected at different angles.
M The orientation of the reflection grating
directs only one narrow band wavelengths, l2,
to the exit slit of the mono-chromator
M Rotation of the grating allows different
to pass through the exit slit
The reflection grating monochromator Device consists of
entrance and exit slits, mirrors, and a grating to disperse the light
Echellette Reflection Grating
reflection grating is ruled with a series of closely spaced, parallel grooves
with repeated distance d.
grating is covered with Al to make it reflective.
polychromatic light is reflected from the grating, each groove behaves as a new
point source of radiation.
adjacent light rays are in phase, they reinforce one another (constructive
adjacent light rays are not in phase, they partially or completely canceled one
another (destructive interference).
Reflection followed by either constructive or destructive interferences
For more detail see Skoog text book p. 159-160
Echellette Grating equation
= d (sin qi
+ sin qr)
where n = 1, 2, 3,….
Since incident angle qi = constant;
therefore l µ qr
For each reflection angle qr , a certain wavelength is observed
2- Prism monochromators
G Dispersion by prism depends on refraction of
light which is wavelength dependent
A Violet color with higher energy (shorter
wavelength) are diffracted or bent most
B While red light with lower energy (longer
wavelength are diffracted or bent least
F As a result, the poly-chromatic white
light is dispersed to its individual colors
The advantages and disadvantages of decreasing monochromator
The size of the monochromator exit slit determines the width
of radiation (bandwidth) emitted from the monochromator.
A wider slit width gives higher
sensitivity because higher radiation intensity passes to the
sample but on the other hand, narrow slit width gives better
resolution for the spectrum.
In general, the choice of slit width to use in an experiment
must be made by compromising these factors. Still, we can
overcome the problem of low sensitivity of the small slit by increasing
the sensitivity of the detector.
3- Sample compartment (cells)
For Visible and UV spectroscopy, a liquid
sample is usually contained in a cell called a cuvette.
Glass is suitable for visible
but not for UV spectroscopy because it absorbs UV radiation. Quartz
can be used in UV as well as in visible spectroscopy
The detectors are devices that convert
radiant energy into electrical signal.
A Detector should be sensitive, and has a
fast response over a considerable range of wavelengths.
In addition, the electrical signal produced
by the detector must be directly proportional to the transmitted intensity
Phototube emits electrons from a photosensitive,
negatively charged cathode when struck by visible or UV radiation
The electrons flow through vacuum to an
anode to produce current which is proportional to radiation intensity.
a) Barrier-layer photocell:
Barrier layer cell
one of the simplest detectors, which has the advantage that it requires no
power supply but gives a current, which is directly proportional to the light
intensity. It is consists of a metallic plate, usually copper or iron, upon
which is deposited a layer of selenium.
An extremely thin transparent layer of a good conducting metal, e.g. silver,
platinum or copper, is formed over the selenium to act as one electrode, the
metallic plate acting as the other. Light passes through the semitransparent
silver layer causes release of an electron, which migrates, to the collector.
The electron accumulating on the collector resulting in a potential difference
between the base and collector, which can be measured by a low resistance
The useful working range of selenium photocell is 380-780 nm. Their lack of
sensitivity compared to phototube and photo multiplier tube, restricts their
use to the cheapest colorimeters and flame photometers.
b) Photo emissive tube:
It consists of an anode and a cathode sealed in an evacuated glass tube,
which may have a quartz or silica window for UV measurement.
Photo emissive tube:
The cathode is coated with a layer of light sensitive material that emits
electrons upon absorption of photons.
A power supply maintains the anode positive with respect to the cathode so
that the photoelectrons are collected at the anode.
This current is directly proportional to the light intensity. Phototubes are
available for use over the entire UV/visible region of the spectrum, but no
single tube covers the entire range satisfactorily.
Therefore many instruments with phototube detectors employ interchangeable blue
and red sensitive phototube in order to provide sufficient sensitivity over the
c) Photo multiplier tube:
It is very sensitive detector with very
short response times. It contains a photo cathode and a series of dynodes,
which are also photosensitive.
of photomultiplier with nine dynodes.
ii. Photomultiplier tube
It is a
very sensitive device in which electrons emitted from the photosensitive
cathode strike a second surface called dynode which is positive
with respect to the original cathode.
are thus accelerated and can knock out more than one electrons from the dynode.
above process is repeated several times, so more than 106 electrons
are finally collected for each photon striking the first cathode.
Schematic diagram of Photo Diode Array
The components of a single beam spectrophotometer
typical spectrophotometer consists of
radiation sources, dispersive devise, sample compartment, detector and
read out system
radiation sources are a tungsten lamp or
halogenated tungsten lamp
radiation sources include a Hydrogen or deuterium discharge lamp
prisms and gratings constitute dispersive devises
are sample holders. They can be cylindrical or rectangular.
can be a barrier layer cell, a photo cell, a photo multiplier tube, a diode or
diode array detector
beam, double beam and diode array spectrophotometers are available in the
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