Many molecules absorb in the ultraviolet (UV) and visible (Vis) range of the electromagnetic spectrum. UV radiation covers the range from 190-350 nm, while visible radiation covers the 350-800 nm range. The absorption of radiation corresponds to the excitation of outer electrons in the molecule. In a spectrophotometer, radiation with a specific intensity is passed through a liquid sample that is contained in a cuvette (usually made of quartz). If the sample contains species that absorb(s) in that specific radiation wavelength, the radiation intensity emerging on the other side of the cuvette is reduced. This phenomenon is used to identify and/or quantify a molecule in a sample. Compounds have unique UV-Vis spectra, including a maximum absorption wavelength (λmax) and molar extinction coefficient (ε), so UV-Vis can be used to identify the presence of chemicals in samples. Because of this, UV-Vis is a popular technique in chemistry, foods, pigments, pharmaceuticals, polymers, and the life sciences, for basic or applied research as well as quality control.
Quantitation in UV-Vis spectrophotometry is expressed by Beer-Lambert’s Law:
Aλ = ελ c l
where Aλ = absorbance at wavelength λ, ελ = extinction coefficient at wavelength λ, c = concentration, and l = pathlength. In most experiments, ε and l are constant, therefore absorbance is proportional to the concentration of the compound.
UV-Vis spectrophotometers can be either single beam or double beam in design. In single-beam instruments, all the light passes through the sample cell, therefore a correction for the loss of light intensity as the beam passes through the solvent must be made. This is done by replacing the sample by a blank or reference sample to account for any matrix effects. Dual-beam spectrophotometers split the light into two beams before it reaches the sample. One beam is used as a reference beam, and the other passes through the sample. Correction is performed automatically.
Feel free to express your comments
Your opinion is important to us. Let us know if this information was useful, or if there was something missing that you'd like to see. Contact us.