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

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

of  spectrophotometers

  1.  Sources

  2.  Wavelength selectors (filters,

  3.  Sample containers

  4.  Detectors

  5.  Readout devices

  • Instrumentation

  • 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
    , 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

    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


    Wavelength range




    100 ~ 40,000

    3000 lines/mm for vacuum UV,

    50 lines/mm for far IR


    120 ~ 30,000


            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

    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.

    They are
    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

    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

    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

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

    Echellette Grating equation

    n l
    = 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
    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
    because higher radiation intensity passes to the
    sample but on the other hand, narrow slit width gives better
    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)

      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

    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
    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.


    Schematic diagram
    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.

    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


    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

     For PDF Notes Click on Download Button

    Leave a comment