Absorption Spectroscopy: Practical Aspects

Definition

UV-visible absorbance is most commonly used to determine the concentration of a sample and also to give an indication of its purity. It is very easy to collect absorbance spectra of biomolecules; however, they are not always useful because of some of the issues outlined below (Nordén et al. 2010; Rodger and Nordén 1997). Nucleic acids (DNA and RNA), proteins, and peptides absorb very little light at wavelengths greater than 300 nm in the absence of ligands or prosthetic groups with chromophores (absorbing units). However, it is usually wise to collect absorbance data from 200 nm to about 350 nm. For a biomolecule that does not absorb between 350 and 310 nm, if the spectrum is not flat in this region, then the sample includes particles whose size is of the order of the wavelength of light; therefore, what is being measured includes scattering of the incident light rather than simply absorption. An extreme example of this is when an absorbance spectrometer is used to measure...