Spectroscopy and Mass Spectrometry

Aromatic amino acid UV absorbance

In proteins and peptides, absorbance in the near-UV region above about 250 nm comes almost entirely from aromatic side chains. Tryptophan, with its indole ring, has its main band near 280 nm. Tyrosine, with its phenol ring, has a strong band near 275 nm. Phenylalanine has only a weak band near 257 nm, and its molar absorptivity at 280 nm is negligible. The peptide bond absorbs strongly only below about 230 nm, so a peptide containing none of Trp, Tyr or Phe is effectively transparent above 250 nm.

Nucleotides and nucleic acids are different. Their purine and pyrimidine rings absorb maximally near 260 nm and keep a measurable tail at 280 nm, so they can contribute to readings at 280 nm. Guanine-containing polymers such as poly dG are an example. Before concluding that a molecule is silent at 280 nm, check which chromophore it contains.

The quantitative link is the Beer-Lambert law, A = \varepsilon\, l\, c, where A is absorbance, \varepsilon is the molar absorption coefficient in M^{-1}cm^{-1}, l is the path length in cm and c is the molar concentration in M. Absorbance is defined as A = -\log_{10} T, where T is transmittance, so a solution that transmits 1% of the light has A = 2. Rearranging gives \varepsilon = A/(l\,c). For example, a 2 \times 10^{-5} M solution with A = 0.36 in a 1 cm cuvette has \varepsilon = 0.36/(1 \times 2 \times 10^{-5}) = 18000 M^{-1}cm^{-1}.

The same relation converts transmittance data into \varepsilon. A solution that transmits 1% of the light at 0.5 mM in a 1 cm path gives \varepsilon = 2/(0.5 \times 10^{-3}) = 4000 M^{-1}cm^{-1}. Always convert the path length to cm and the concentration to M before dividing.

Fluorescence spectroscopy principles

Fluorescence is the emission of a photon when an electron drops from the lowest vibrational level of the first excited singlet state (S1) back to the singlet ground state (S0). Absorption is the step that lifts the electron from S0 to a higher vibrational level of S1. The excited molecule then relaxes rapidly to the lowest vibrational level of S1 by internal conversion and vibrational relaxation, a principle known as Kasha's rule. The emitted photon therefore has less energy than the absorbed photon, which means a longer wavelength. This wavelength difference is the Stokes shift.

Because emission always starts from the relaxed S1 level, the emission maximum (\lambda_{max}) does not depend on the excitation wavelength. Changing the excitation wavelength changes which molecules are excited and how intense the emission is, but it does not move the emission band.

The emission maximum is sensitive to the environment. Polar solvents stabilize a more polar excited state, which lowers its energy and red-shifts emission. This is called solvatochromism. Temperature changes solvent relaxation around the excited fluorophore and the thermal population of vibrational levels, which can also shift \lambda_{max}. Quenching is different: it lowers fluorescence intensity and lifetime, but it does not shift the emission band.