An understanding of the UV – VIS absorption spectrum is important for full exploitation of the potential of your spectrophotometer. UV- VIS spectroscopy is not only the oldest spectroscopic technique but is also a forefather of instrumental methods of analysis. A sound understanding and clarity of concepts of absorption of light will also help you gain a sound understanding of other spectroscopic techniques.
The absorption of light is governed by Beer- Lambert law which states that the absorbance of light is in direct relation to the path length of the absorbing medium and the number or concentration of absorbing molecules in the light path. A graphical representation of absorption over a range of wavelengths is called a spectrum and is characteristic of the absorbing species present in the sample.
The sample solution is taken in the absorption cell or cuvette and is exposed to the light beam from the light source. The broad band light beam is resolved into individual wavelengths by the monochromator and the isolated wavelengths are led to the sample in a sequence called scanning. The absorbing species absorb selectively and the absorbance is plotted as ordinate against the wavelength as abscissa. The resulting plot is called a spectrum.The wavelength of maximum absorbance is important and referred to as λ max which is selected for quantitative studies.
The spectrum of gases comprises of several closely spaced peaks whereas liquid phase spectrum appears as broad peaks. The reason for this is that in the liquid phase the absorbing species are surrounded by solvent molecules and are in constant collisions with them. Such collisions result in dissipation of energies and a spread of absorptions over a range of wavelengths. This results in smooth continuous peaks in the solution phase. Data are scanned at all wavelengths over the selected range to find the peak of maximum absorption for a particular species and then the sample is irradiated with this selected wavelength for quantification measurements.