UV Spectroscopy: Principle, Instrumentation

 

Coloured compounds absorbs visible light while colourless substances don’t. UV radiation is required for the detection of colourless substances in UV visible spectrophotometer.

Must read the Basics of UV spectroscopy first.

Principle

The sample to be analysed contains either sigma, pi or non-bonding (n) electrons. On irradiation, these electrons absorbs characteristic amount of radiation and undergoes transition from ground state to the excited state.

The greater is the number of molecules capable of absorbing light of particular wavelength, the more will be light absorption.The intensity of light absorption depends upon the path length and concentration as according to the BeerLambert’s law (covered earlier).

By knowing the characteristic absorption peaks, the various types of electrons present in the compound can be analyzed and hence would help in structure ellucidation. 

The UV-Visible spectrum is often plotted as Absorbance versus Wavelength. However, most of the spectra are described by characterizing the wavelength maxima and absorbtivities of principle absorption peaks of the compound.

 

 

Instrumentation

The typical Uv-Visible spectrophotometers composes of the following:

1.   Light Source: For UV radiation – Deuterium lamp is used while Tungsten lamp is used as a source of visible light. The light should be stable and of proper intensity.

2. Monochromators: Diffraction gratings are used in UV-Visible spectrophotometers. These are used to convert the polychromatic light to the monochromatic light. As the name indicates, polychromatic light composes light of different wavelengths while monochromatic light has single wavelength light. 

3.  Sample cells: Glass or plastic cells can be used to the visible light but in case of UV light; sample cells made up of quartz are used as glass can absorb uv radiations while quartz couldn’t.

4.   Solvents: Hexane, water and 95% Ethanol are commonly use solvents. Following are the factors to be considered while choosing the solvent:

a.    Solvent should not absorb the radiation in the region in which the sample to be analyzed absorbs.

b.    As the polar solvents could interact with the sample species by forming H-bonds with them, this will lead to the loss of fine spectrum.  Non polar solvents don’t interact.

c.     The solvent has the ability to influence the property of sample to absorb the light of particular wavelength by providing the stability to sample species in either ground state or excited. One should consider this while choosing the solvent.

5.     Detectors: Among various detectors available, Photomultiplier tube detectors are used. In modern instruments, photodiodes are used.

6.     Recorders: It can be done on plotters or digital displays.

 

The UV – visible spectrophotometers can be single beam or double beam. As the name indicates, single beam spectrophotometer has single beam of light to get passed through the sample only for analysis. While the double beam spectrophotometer has a rotating disc to split the single beam into two. From the resulting two beams, one is passed through sample compound and other through the reference compound. The results are then plotted. A typical diagram of double beam spectrophotometer is given below.

Must read : Red and blue shifts and Auxochrome