A
spectrophotometer is a device that measures the absorption of light at
different wavelengths. It is similar to a calorimeter, except that it
produces monochromatic light using a prism or diffraction grating. It
may operate in the ultraviolet (UV) spectrum, visible spectrum, and
infrared (IR) spectrum of the electromagnetic spectrum.
The
photometric approach asserts that when a beam of incident light of
intensity I0 passes through a solution, a portion of the light is
reflected (Ir), a portion is absorbed (Ia), and the remaining light is
transmitted (It).
I0 = Ir + Ia + It
(Ir)
is not measured in photometers (colorimeters and spectrophotometers)
because the measurements of (I0) and It are adequate to determine the
wavelength (Ia). The amount of light reflected (Ir) is kept consistent
for this purpose by using cells with identical characteristics. The
values of (I0) and (It) are then calculated.
The two fundamental equations of photometry, on which the Double Beam UV spectrophotometer is based, show the mathematical link between the amount of light absorbed and the concentration of the substance.
The
Spectrophotometer's operating concept is based on Beer-law, Lambert's
which states that the amount of light absorbed by a colour solution is
exactly proportional to the solution's concentration and the length of a
light path through it.
A ∝ cl
Where,
A = Absorbance / Optical density of solution
c = Concentration of solution
l = Path length
or, A = ∈cl
∈ = Absorption coefficient
SPECTROPHOTOMETER TYPES
There are two types of spectropotometers:
- Single
beam spectrophotometer - A single beam spectrophotometer uses a single
beam of light to operate between the wavelengths of 325 nm and 1000 nm.
The test solution and blank are both read in the same direction as the
light.
- Double beam spectrophotometer - The wavelength range of a
double beam spectrophotometer is 185 nm to 1000 nm. It is equipped with
two photocells. The light from the Monochromator is separated into two
beams by this device. One beam serves as a reference, while the other is
used to read samples. It eliminates the inaccuracy caused by
fluctuations in the light output and the detector's sensitivity.
A spectrophotometer has seven key components -
Light
source - Three separate light sources are routinely employed in
spectrophotometers to create light of different wavelengths. A tungsten
lamp is the most frequent light source used in spectrophotometers for
the visible spectrum. The hydrogen lamp and the deuterium lamp are
popular sources of ultraviolet radiation.
Monochromator - A prism or diffraction grating splits the light from the light source to select a certain wavelength.
Sample holder - Colored solutions are held in test tubes or Cuvettes. At a visible wavelength, they are made of glass.
The
beam splitter is only found in double beam spectrophotometers. It is
used to divide a single light beam from a light source into two beams.
Both
in single beam spectrophotometer and a double beam spectrophotometer
have a mirror. It is utilised to steer the splitted light from the beam
splitter in the proper direction.
When light strikes the photodetector system, an electric current is generated, which reflects the galvanometer reading.
The
current from the detector is supplied into the measuring device, which
is a galvanometer. The metre reading is related to the amount of light
present.
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