Biochemical Techniques and Instrumentation

1.1.3. UV-Vis Spectroscopy

UV-Vis spectroscopy is based on the selective absorption of electromagnetic radiation in the wavelength range of 180-800 nm. Electronic transitions in molecules are classified according to the participating molecular orbitals. There are four possible transitions: n → π*, π → π*, n → σ*, σ → σ*. Transitions among these orbitals are by the radiation having the same energy as that of the energy difference between the specific orbitals. The UV-Vis radiation has sufficient energy to cause only two transitions between n → π* and π → π*. The other transitions, n → σ* and σ → σ* requires higher energies. The molecular structures such as bonds and functional groups responsible for interaction with electromagnetic radiation are called chromophores.

Transitions to a π* orbital requires the presence of an unsaturated functional group. The outer nonbonding n electrons form hydrogen bonds with water and alcohols, whereas inner π electrons are unaffected by solvents. Organic molecules with conjugated double bonds, carbonyl groups, carboxyl groups, and nitro groups have good absorbance in the UV-Vis region. The absorption maxima are strongly affected by substituent groups.

In proteins, there are three types of chromophores: peptide bond, certain aminoacid side chains and certain prosthetic groups/ coenzymes. Electronic transition of the peptide bond occur in the far UV with an intense peak at 190 nm and weak one at 210-220 nm due to π → π* and n → π* transitions. The aromatic aminoacids, phenylalanine, tyrosine and tryptophan haveh their absorption maxima at 257, 274 and 280 nm, respectively. The proteins that contain prosthetic groups such as haem, falvin, caroteniod have strong absorption bands in the UV-Vis region. The absorption of UV light by nucleic acids is due to n → π* and π → π* transitions of the purine and pyrimidine bases that occur between 260 nm and 275 nm.