Absorption Spectroscopy: Practical Aspects

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 (Norden et al. 2010; Rodger and Norden 1997). Nucleic acids (DNA and RNA), proteins, and peptides absorb very little light above 300 nm in the absence of ligands or prosthetic groups with chromophores (absorbing units). However, it is usually wise to collect absorbance data from about 350 nm. If the spectrum is not flat between 350 and 310 nm, 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. The extreme of this is when an absorbance spectrometer is used to measure cell density in a culture using say 450 nm light – this is only light scattering despite...