Calculation of concentration (C = A/(L x Ɛ))
In order to derive the concentration of a sample from its absorbance, additional information is required. The Lambert-Beer law, which forms the physical basis for photometric applications, describes that the absorption of light by a sample is directly proportional to its concentration and its path length. Altogether, three parameters contribute to the absorbance value of a sample: first, the concentration (C) of the molecule; second, the path length (L) of the sample, which generally equals the path length of the cuvette. Then there is the extinction coefficient (Ɛ). The extinction coefficient is a physical constant unique to the molecule; it describes its property of absorbing light at a specific wavelength. This material-specific constant is known for a number of substances, including nucleic acids and various proteins, and the values have been published in the pertinent literature. In these cases, concentration can be determined instantly. If the value is not known, however, it is possible to enlist the help of a calibration curve. In order to generate a calibration curve, standards are required, i.e. solutions which contain known concentrations of the substances to be analyzed. These are measured in the photometer prior to the actual sample. The concentration of the analyte is then calculated using the standard curve.
Further to quantification, absorbance measurements may also reveal qualitative information about the sample: for example, the purity of nucleic acids and proteins may be determined by subjecting the sample to measurements at additional wavelengths, whereas information on enzyme activity is generally obtained through repeated measurements over time.