UV – Visible Spectrophotometers Instrumentation – Instrumental Methods of Analysis B. Pharma 7th Semester

Type	Wavelength range (nm)	Note
Continuously variable
Grating	100 ~ 40,000	3000 lines/mm for vacuum UV, 50 lines/mm for far IR
Prism	120 ~ 30,000
Discontinuous
Interference filter	200 ~ 14,000
Absorption filter	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
– b)

d sin q
= a

– d sin f  = b

nl = d
(sin q + sin f )

i- Filters

•         
Filters
permit certain bands of wavelength (bandwidth of ~ 50 nm) to pass
through.

•         
The
simplest kind of filter is absorption filters , the most common of this
type of filters is colored glass filters.

•         
They are
used in the visible region.

•         
The
colored glass absorbs a broad portion of the spectrum (complementary color) and
transmits other portions (its color).

Disadvantage

•       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
interference

Transmission spectra
of interference filters.

(a)    Wide
band pass filter has ~90% transmission in the 3- 50 5- mm wavelength range but <0.01%
transmittance outside this range.

(b)   Narrow
band-pass filter has a transmission width of 0.1 mm
centered around 4 mm.

Complimentary colours
– selection of filters

ii- Monochromators

Ø  They
are used for spectral scanning (varying the wavelength of
radiation over a considerable range ).

Ø  They
can be used for UV/Vis region.

Ø  All
monochromators are similar in mechanical construction.

Ø  All
monochromators employ slits, mirrors, lenses, gratings or prisms.

1-Grating monochromators

Reflection grating

M Polychromatic
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
wavelengths, l₁,
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

1.       The
reflection grating is ruled with a series of closely spaced, parallel grooves
with repeated distance d.

2.       The
grating is covered with Al to make it reflective.

3.       When
polychromatic light is reflected from the grating, each groove behaves as a new
point source of radiation.

4.       When
adjacent light rays are in phase, they reinforce one another (constructive
interference).

5.       When
adjacent light rays are not in phase, they partially or completely canceled one
another (destructive interference).

Reflection followed by either constructive or destructive interferences

Note:
For more detail see Skoog text book p. 159-160

Echellette Grating equation

•         
n l
= d (sin q_i
+ sin q_r)          
where n = 1, 2, 3,….

•         
Since incident angle q_i = constant;
therefore l µ q_r

•         
For each reflection angle q_r , 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
slit width

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.

Bandwidth Choice

3- Sample compartment (cells)

Ø 
  For Visible and UV spectroscopy, a liquid
sample is usually contained in a cell called a cuvette.

Ø 
  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

4- Detectors

$
  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
(linear response).

i- Phototube

      
  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
galvanometer circuit.
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
entire spectrum.

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.

Phototube

Schematic diagram
of photomultiplier with nine dynodes.

ii. Photomultiplier tube

Ø 
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.

Ø 
Electrons
are thus accelerated and can knock out more than one electrons from the dynode.

Ø 
If the
above process is repeated several times, so more than 10⁶ electrons
are finally collected for each photon striking the first cathode. 

Photo Diode

Schematic diagram of Photo Diode Array

The components of a single beam spectrophotometer

Summary

•      
A
typical spectrophotometer consists of 
radiation sources, dispersive devise, sample compartment, detector and
read out system

•      
Visible
radiation sources are  a tungsten lamp or
halogenated tungsten lamp

•      
UV
radiation sources include a Hydrogen or deuterium discharge lamp

•      
Filters,
prisms and gratings constitute dispersive devises

•      
Cuvettes
are sample holders. They can be cylindrical or rectangular.

•      
Detectors
can be a barrier layer cell, a photo cell, a photo multiplier tube, a diode or
diode array detector

•      
Single
beam, double beam and diode array spectrophotometers are available in the
market

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