Spectrophotometry is a non-destructive and rapid analysis method which is widely used in the laboratory. This technique makes it possible to determine the absorbance of a chemical substance in solution, that is to say its capacity to absorb the light passing through it. UV-Visible spectrophotometry makes it possible to provide qualitative information on the nature of the bonds present within the sample (via the order of magnitude of λmax and εmax) but also to quantitatively determine the concentration of species absorbing in this spectral range (via Beer-Lambert law).
The absorbance of a chemical depends on the nature and concentration of this analyte as well as on the wavelength chosen.
A polychromatic source, emitting in the UV or visible, is placed in front of a prism. This dispersive system will decompose this light into a monochromatic radiation I0 (at a selected wavelength) and will irradiate the cuvette containing the sample (or the blank). The choice of the cuvette is important to avoid interference; either plastic for the visible, or quartz for the UV. A blank is made to minimize the influence of the solvent and the cuvette. This value is subtracted from that of the sample.
The intensity of the It radiation, transmitted after crossing the analyte is measured by the photodetector and translated into absorbance A via the formula: A = log(I0/It). This is possible since the intensity of the monochromatic light emitted I0 is known. The transmittance T can also be defined using the formula: T = It/I0 and will be expressed as a percentage. For information, there is also a relation between T and A: A = -logT.
We know that the absorbance is proportional to the concentration of the chemical according to the Beer-Lambert law: A = e*L*C where (C) is the concentration of the analyte, (A) its absorbance, (e) its molar absorbance coefficient and (L) the length of the path of the light through the cuvette.
This technique will also make it possible to follow the kinetics of a chemical reaction.