# Encyclopedia of Biophysics

Living Edition
| Editors: Gordon Roberts, Anthony Watts, European Biophysical Societies

# Absorbance Spectroscopy: Overview

Living reference work entry
DOI: https://doi.org/10.1007/978-3-642-35943-9_784-1

## Definition

Most spectroscopic phenomena arise from the interaction of a molecule or other chemical species with one photon at a time. Multiphoton processes are less commonly encountered. The molecule may either absorb or scatter the photon; it may also emit a photon. The simplest form of spectroscopy is absorption, which measures how much electromagnetic radiation (e.g., light) of a given frequency is absorbed by a collection of molecules. If a molecule absorbs a photon of frequency ν, it increases its energy by
$$\Delta E= h\nu = h c/\lambda$$
(1)
where h is Planck’s constant, λ is the wavelength of the light, and c is the speed of light. Only light of the correct frequency to cause a molecule to jump from one energy level to another may be absorbed. When molecules absorb photons of visible or ultraviolet (UV) light, the magnitude of the energy of these photons is (2–12) × 10−19 J/molecule (λ = 170–800 nm) which is the amount required to rearrange the valence electron distribution of a molecule. Thus, UV-visible absorption spectroscopy gives information about the bonding in a molecule or collection of molecules.
In practice, in a collection of molecules, the photons absorbed by different molecules will be of slightly different energies, so what we measure is a curve such as the one in Fig. 1, where the signal that is plotted is a measure of the probability that a transition will occur at that energy (or wavelength). Such a plot of the absorption of light versus λ or ν is known as an absorption spectrum. The spectrum of Fig. 1 is the spectrum that would be measured for a 1 M solution in a 1 cm path length cuvette if any instrument could do it. In practice, either the path length or concentration (or a combination) needs to be reduced by about two orders of magnitude. Fig. 1Absorption spectrum of the metal complex [Co(en)3]3+, en ethylenediamine
Absorbance is a common measure of absorption and is defined in terms of the intensity of incident, Io, and transmitted, I, light (Hollas 2004; Atkins and Friedman 2011; Atkins and de Paula 2010) when a light beam passes through a sample:
$$A={\log}_{10}\left({I}_o/I\right)$$
(2)
The Beer-Lambert law for the absorbance, A, of a sample of concentration C is
$$A=\varepsilon C\mathrm{\ell}$$
(3)
where is the length of the sample through which the light passes and ε is known as the extinction coefficient. If C is measured in mol dm−3 and is measured in cm, then ε has units of mol−1 dm3 cm−1. ε depends on the identity of the molecules in the sample, their environment (e.g., solvent or buffer), and λ. The situation is more complex if methods of sample presentation such as required for attenuated total reflectance (ATR) measurements are used. A is a function of wavelength, and the Beer-Lambert law is valid as long as the spectrometer can measure I accurately and there are no concentration-dependent interactions between molecules. The most common application of UV/visible absorption spectroscopy is to determine the concentration of a species in solution using the Beer-Lambert law, as solution absorption spectra are usually too broad for conclusive identification of a chemical species. However, gas-phase atomic absorption bands are much narrower and can be used for both qualitative and quantitative analysis, as in atomic absorption spectrophotometry (AAS).

## References

1. Atkins PW, de Paula J (2010) Atkins’ physical chemistry, 9th edn. Oxford University Press, OxfordGoogle Scholar
2. Atkins PW, Friedman RS (2011) Molecular quantum mechanics, 5th edn. Oxford University Press, OxfordGoogle Scholar
3. Hollas JM (2004) Modern spectroscopy, 4th edn. Wiley, ChichesterGoogle Scholar