SPECTROPHOTOMETRY

SPECTROPHOTOMETRY

¡involves measurement of the light transmitted

by a solution to determine the concentration
of the light-absorbing substance in the
solution.
§ It is the measurement of light intensity in a narrower
wavelength
§ Photometric measurement – measurement of light
intensity without consideration of wavelength
¡Beer’s law states that the concentration of a
substance is directly proportional to the
amount of light absorbed or inversely
proportional to the logarithm of the
transmitted light.
¡Mathematically establishes the relationship
between concentration and absorbance
 PARTS OF SPECTROPHOTOMETER

1. Light source
1. Continuum source – emits radiation that changes in
intensity
2. Line source – emits limited radiation and wavelength
1. Tungsten light bulb – commonly used light source in the visible
and near IR region

Factors for choosing a light source: range,

spectral distribution w/in the range, the
source of radiant radiation, stability of the
radiant energy and temperature
¡Alternatives:
§ Mercury arc (visible and UV)
§ Deuterium lamp (UV)
§ Hydrogen lamp (UV)
§ Xenon lamp (UV)
§ Merst glower (IR)
§ Globar / silicon carbide (IR)
2. Entrance Slit
- Minimizes unwanted or stray light and prevents the entrance
of scattered light into the monochromator system
§Stray light – refers to any wavelengths
outside the band transmitted by the
monochromator; it causes absorbance error
(systematic error)
§Stray light limits the maximum absorbance
that a spectrophotometer can achive
3. Monochromator – isolates specific or
individual wavelength of light
§ Differentiates spectrophotometer and photometer
¡Kinds of monochromators:
§ Prisms (spectrophotometer)
§ Wedge-shaped pieces of glass, quartz or sodium chloride
§ A narrow of light focused on a prism is refracted as it enters
the more dense glass
§ Can be rotated, allowing only the desired wavelength to pass
through an exit slit
§Diffraction gratings (spectrophotometer)
§Most commonly used; better resolution than
prism
§Made by cutting grooves (parallel grooves) or
slits into an aluminized surface of a flat
piece of crown glass – wavelengths are bent
as they pass a sharp corner.
§Filters (photometer)
§ Simple, least expensive, not precise but useful
§ Made by placing semi-transparent silver films on both sides
of a dielectric such as magnesium fluoride.
§ Produce monochromatic light based on the principle of
constructive interference of waves – light waves enter one
side of the filter and are reflected at the second surface.
§ Usually pass a wide band of radiant energy and have a low
transmittance of the selected wavelength.
§ Sharp cut off filter – isolates stray light
4. Exit slit – controls the width of light beam
(bandpass)
§ Allows only a narrow fraction of the spectrum to
reach the sample cuvette.
§ Bandpass – the range of wavelengths between the
points at which transmittance is one half peak
transmittance.
¡ Cuvet – also called absorption cell/analytical
cell/sample cell
§ It holds the solution whose concentration is to be measured
¡ Kinds of cuvets:
§ Alumina silica glass – most commonly used
§ Quartz/plastic – used for measurement of solution requiring
ultraviolet spectrum
§ Borosilicate glass – strong alkaline solution
§ Soft glass – strong acidic solution
§ Cuvets with scratches on their optical surface scatter light and
should be discarded
5. Photodetector
§ Detects and converts transmitted light into
photoelectric energy
§ Detects the amount of light that passes through the
sample in the cuvet
¡Kinds of detectors:
¡Barrier layer cell / photocell / photovoltaic
cell
§ Simplest detector; least expensive; temperature-
sensitive
§ Composed of selenium on a plate of iron covered
with transparent layer of silver
§ It requires no external voltage source but utilized
internal electron transfer for current production – low
internal resistance
§ Used in filter photometer with a wide bandpass
¡ Phototube
§ It contains cathode and anode enclosed in a glass case
§ It has a photosensitive material that gives off electron when
light energy strikes it
§ It requires external voltage for operation
¡ Photomultiplier (PM) tube
§ Most common type – measures visible and UV regions
§ Excellent sensitivity and rapid response
§ Detects and amplifies radiant energy
§ Detects very low levels of light / low analyte concentration
¡ Photodiode
§ Not as sensitive as PM; excellent linearity
§ Measures light at a multitude of wavelengths
§ Detects less amount of light

6. Meter or read-out device

§ Galvanometer / ammeter
§ Displays output of the detection system