Measurements:

While calculating vis-uv spectra, the light beam is split such that one half undergoes a solution of the sample (the sample beam) and the other half goes by the solvent alone (the reference beam). If energy is absorbed through the sample, the intensity of the sample beam (IS) after it has passed by the sample will be less than the intensity of the reference beam (IR) after it has passed via the solvent. This is computed like the absorbance (A) where:

A = log (I_R/I_S)

The absorbance will modify depending upon the wavelength of light employed and thus a spectrum is produced measuring wavelength (in nm) versus absorbance. There might be one or various broad absorption peaks in the spectrum. The wavelength at which the absorption is a maximum for any of these peaks is called λ_max and is reported for the compound. At this point the strength of the absorption is quantified by a word termed as the molar absorptivity (ε) that is related to absorbance (A), the concentration of the sample (C) and the length (l) in centimeters of the cell consisting of the sample. The units of ε are M^-1 cm^-1.

A =ε× C × l

Knowing λ_max and ε for a specific compound means that it is possible to compute the concentration of a sample if the absorbance of a solution is computed at λ_max. The capability to quantify concentrations by using vis-uv spectroscopy is significant in the analysis of mixtures using a method termed as high performance liquid chromatography (hplc). In this process, mixtures are passed down a column and exit the column at dissimilar rates. The compounds can be detected and quantified by passing them by a vis-uv spectrometer as they come off the column.