Understanding spectrophotometer and its various applications

 

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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