Nucleic acids: Nucleic acids absorb very strongly in the far and near UV region of the electromagnetic spectrum. The absorption is largely due to the nitrogenous bases. The transitions in the nucleic acid bases are quite complex and many π → π* and n → π* transitions are expected to contribute to their absorption spectra. A 260 nm wavelength radiation is routinely used to estimate the concentration of nucleic acids. Though the molar absorption coefficients vary for the nucleotides at 260 nm, the average ε_max can be taken as ~10⁴ M^-1 cm^-1. It is important to mention that nucleotides show hyperchromicity when exposed to aqueous environment. The absorbance of the free nucleotides is higher than that of single stranded nucleic acid which is higher than that of the double stranded nucleic acid (assuming equal amount of the nucleotides present in all three).

Other chromophores: Nucleotides like NADH, NADPH, FMN, and FAD; porphyrins such as heme, chlorophylls and other plant pigments; retinal (light sensing molecule); vitamins; and a variety of unsaturated compounds constitute chromophores in the UV and visible region.

Having studied the principles of the UV/visible absorption spectroscopy and various factors that influence the electronic transitions, we can now have a look at its applications, especially the applications for analyzing the biological samples.

Applications:

i. Determination of molar absorption coefficient: From Beer-Lambert law, A = εcl. It is therefore straightforward to calculate the molar absorption coefficient of a compound if the concentration of compound is accurately determined.

ii. Quantification of compounds: This is perhaps the most common application of a UV/visible spectrophotometer in a bioanalytical laboratory. If the molar absorption coefficient at a wavelength is known for the compound, the concentration can easily be estimated using Beer-Lambert law. The compounds can still be quantified if their molar absorption coefficients are not known. Estimation of total protein concentration in a given solution is an important example of this. As the given solution is a mixture of many different proteins, the ε is not available. There are, however, dyes that specifically bind to the proteins producing colored complex. The color produced will be proportional to the amount of the protein present in the solution. Performing the experiment under identical conditions using known concentrations of a protein gives a standard graph between absorbance of the dye and the amount of protein. This standard graph is then used to estimate the concentration of the given protein sample.

iii. Quality control: A given organic compound such as a drug can be studied for its purity. Comparison of spectrum with the standard drug will detect the impurities, if any. UV/Visible absorption is often used to detect the nucleic acid contamination in the protein preparations. Aromatic amino acids as well as the nucleotides show absorption band in the near UV region and there is a considerable overlap in the absorption spectra of aromatic amino acids and the
nucleotides. A nucleic acid contamination in a protein, however, can be determined by measuring absorbances at 260 and 280 nm. A typical nucleic acid containing all four bases shows an absorption band centered ~260 nm while a protein having aromatic amino acids shows absorption band centered ~280 nm. It is possible to
determine the purity of protein preparations by recording absorbances at both 260 and 280 nm. A ratio of the absorbance at 260 nm to that at 280 nm i.e. is a measure of the purity.