By Sunil Bhardwaj

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Spectrophotometers are the instruments which are working at a wavelength where the absorbance is maximum. Such wavelength of the radiations whose absorbance is maximum is called $${ \lambda }_{ max }$$.

With the help of spectrophotometers we can have qualitative as well as quantitative analysis of any type of sample it may be solid, liquid or gas. It may be coloured or colourless.

The spectrophotometers are of two types;

(i) Single Beam Spectrophotometers,

(ii) Double Beam Spectrophotometers.

The following are the main components of a Double Beam Spectrophotometers:

1. Source of radiations: It may be U.V-light, visible light or I.R. light. U.V. Light can be obtained by heating the filament which is filled with either H2 gas or deuterium. Visible Radiations can be obtained by incandescent lamp with tungsten filament and I.R. Radiations can be obtained by heating Newtons glower at the temp. of 1500-2000 ℃. Newtons glower is metallic oxide of Yettrium, Erbium and Zirconium. I.R. radiations can also be obtained by heating glow bar which is Silicon carbide SiC (Carborandum) at the temp. of 1300-1700 ℃.

2. Collimating Convex Lens: The function is to collect all the rays coming from the source.

3. Diaphragm: To set the 100% transmittance.

4. Monochromators: To obtain the radiation of one wavelength. The Monochromators may be prism or diffraction gratings.

5. Cuvette or Sample Holder: For U.V. light it should be made up of quartz, for visible light it should be made up of glass and for I.R. light it must be that of rock salts for eg. NaCl.
a) If the sample is gas or volatile liquid having low B.P. must be taken in the closed container.
b) If it is liquid, one drop of the liquid must be placed between two parallel plates for U-V. the plates must be quartz and for visible it must be glass and for I.R. it is rock salt.
c) If it is solutions the solvent used for the preparation of solution must be Cyclohexane, methyl alcohol, ethyl alcohol or any other suitable solvent which is not capable to absorb the radiations incident on it.
d) If it is solids then we take about 1mg. of it and is mixed in 100-200 mg. of KBr - prepare slurry. It is cut into smaller circular discs (just like bindle) called pellets. They are dried and placed in the place of cuvette. The diameter of pellets is 10mm. and thickness is 1mm.

6. The Focusing Convex Lens: Its function is to collect all the transmitted light from cuvette and is focused at a point where photo cathode of the photo-cell is placed.

7. Photo-Cell: The transmitted light is incident on it photo cathode converts these radiations into current. It can be amplified using the amplifier if needed.

8. Read out meter or dial: The current is converted into OD or Absorbance in read out meter and by knowing OD we can find out the concentration of the solution i.e. quantitative analysis is possible by applying Beer Lamberts Law.

Working:

First of all the source of radiations from U.V. light to visible light to I.R. radiations is started. The light from the source is collected by Collimating convex lens and allowed to fall on monochromator. For each radiations incident on monochromator we are getting the radiations of one wave length only i.e. $$\lambda$$, the monochromatic light is then separated in two beams with the help of two mirrors. One beam is passed through one cuvette containing blank (solvent) and the other through the other cuvette containing sample solution and both are finally incident on detectors. These may be photo calls or thermocouples. Where the heat is produced which is then converted to current and finally O.D. or Absorbance (A).

The signal generated is platted O D Vs $$\lambda$$. From the graph we can find out $${ \lambda }_{ max }$$ and we can have the qualitative analysis. From O.D. or Absorbance we can have the quantitative analysis.

Value of the wavelength where the absorbance is maximum is known as $${ \lambda }_{ max }$$ which is characteristic property of the compound and gives the qualitative analysis of sample.While knowing the value of absorbance we can find out the concentration of solution using beer lambert’s law. Thus quantitative analysis is possible.

Double beam Spectrophotometer have following advantages:

1. As the two beams of radiations are passed simultaneously from sample solution and Blank Therefore any fluctuation in voltage can be compensated (cancelled).

2. As the two beams are passed from Blank and Sample solution any impurities present in the solvent will not affect the absorbance.

3. Accuracy is more than single beam spectrophotometer.

4. The main disadvantage is the instrument is costly.

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