An analytical instrument called a UV-vis spectrophotometer measures how much ultraviolet (UV) and visible light a sample absorbs. In chemistry, biochemistry, and other sciences, it is a widely used method for locating and measuring compounds in a variety of materials.
The measurement and interpretation of electromagnetic radiation released or absorbed when molecules, atoms, or ions in a sample change energy levels is known as spectroscopy. In ultraviolet (UV) spectroscopy, molecules absorb light in the ultraviolet (200–400 nm) range, which excites electrons from their ground state to an excited state. This type of spectroscopy is similar to absorption spectroscopy.
UV-vis spectroscopy data can provide qualitative and quantitative information about a specific substance or molecule. Whether quantitative or qualitative information is desired, a reference cell should be used to zero the instrument for the solvent in which the compound is present. To obtain quantitative information on the compound, calibrate the instrument with known amounts of the compound in issue in a solution containing the same solvent as the unknown sample. If the information sought is just verification that a compound exists in the sample being tested, a calibration curve is not required; however, if a degradation study or reaction is being done and the concentration of the component in solution is required, a calibration curve is required.
To create a calibration curve, at least three concentrations of the substance are required, but five concentrations are preferred for a more precise curve. Concentrations should begin just above the predicted concentration of the unknown sample and gradually decrease to about an order of magnitude below the peak concentration. Calibration solutions should be spaced reasonably evenly apart and prepared as precisely as feasible using digital pipettes and volumetric flasks rather than graded cylinders and beakers. An example of an absorbance spectrum for Rose Bengal calibration solutions (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein).
To create a calibration curve, use the absorbance values from each of the spectral curves at the highest absorbing wavelength. An acceptable calibration has a correlation coefficient of at least 0.9. If the correlation coefficient is less than that, retry the solutions because the problem could be human mistake. However, if the calibration remains unsatisfactory after several solutions, something is amiss with the instrument; for example, the lights may be failing.