Encyclopedia of Biophysics

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

Absorption Spectroscopy: Relationship of Transition Type to Molecular Structure

  • Paul WormellEmail author
Living reference work entry
DOI: https://doi.org/10.1007/978-3-642-35943-9_781-1
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Synonyms

Definition

A very wide range of organic and inorganic substances can be analyzed using UV and visible absorption spectroscopy. The wavelengths, intensities, and fine structure of the absorption bands are determined by the structures of the neutral or ionic species concerned. For almost all substances, UV and visible absorption spectra are caused by electronic transitions. A spectrum may contain more than one region of absorption, known as a band or band system, as molecules may undergo a range of different electronic transitions.

Basic Characteristics

To a first approximation, electronic transitions may be described as involving different combinations of orbitals: bonding (e.g., σ and π for organic molecules), so-called nonbonding (n), and antibonding (e.g., σ* and π*) orbitals. A simple bonding-to-antibonding promotion model of electronic transitions is an oversimplification that does not adequately explain the spectra of all compounds – for example, benzene, porphyrins (e.g., in heme groups), and proteins – but transitions are commonly and usefully characterized as being, for example, ππ* or nπ* in character, and detailed assignments are possible using combinations of experimental spectroscopy and computational chemistry (Atkins and Friedman 2011). However, detailed analysis and computational studies may show that excited electronic states have mixed orbital character, as in some ruthenium and platinum complexes of biological interest (Campagna et al. 2007; Mackay et al. 2009).

Given the number of possible transitions that may occur in the UV/visible absorption spectrum of an organic compound or metal complex, many bands are composite, with overlapping band systems, often including vibrational (also referred to as vibronic) fine structure. Figure 1 shows vibrational structure in the absorption spectrum of phenylalanine and other amino acids with aromatic side chains. The vibronic bands of vapor-phase spectra may also show rotational fine structure (Hollas 2004).
Fig. 1

Aromatic absorption spectra of tryptophan, tyrosine, and phenylalanine scaled to give similar long-wavelength maximum absorbances. The concentrations to achieve these spectra are indicated on the figure

At the wavelengths, λ, that are commonly used for absorption spectrophotometry (λ = 170–800 nm), electronic transitions are commonly associated with conjugated regions of organic molecules, or with metal complexes. The structural components that cause absorption are often referred to as chromophores. Conjugated chromophores can be linear, as in β-carotene, or cyclic, as in purine and pyrimidine bases, heme groups, and the amino acids that have aromatic side chains. The chromophores may consist entirely of CH groups or may include heteroatoms such as nitrogen, sulfur, or oxygen. Typically, the longest absorption wavelength (lowest energy) increases with the length or, for cyclic systems, the size of the chromophore (Atkins and Friedman 2011; Atkins and de Paula 2006).

The color and hence electronic spectrum of a molecule will change when its structure is changed, for example, by lowering the pH to protonate a species. This is well illustrated by the color changes that occur when compounds such as phenolphthalein and Coomassie Blue are protonated, causing changes in the extent of π-delocalization within the molecules (Bell 2006). The neutral form of phenolphthalein, which predominates at neutral and low pH, is colorless as there is no conjugation between the aromatic chromophores in the molecule, which absorb at UV wavelengths. However, in basic solutions, the molecule is deprotonated, leading to extended conjugation in the resulting dianion, producing a distinctive pink color (Fig. 2).
Fig. 2

Phenolphthalein at low and high pH

At wavelengths greater than 200 nm, most absorption spectra of organic molecules are dominated by transitions in which electrons are redistributed between π and π* orbitals; these are known as ππ* transitions. However, heteroatoms introduce so-called nonbonding or n-orbitals or lone pairs to the chromophore. These are σ-type orbitals that can give rise to nπ* transitions in an absorption spectrum (Hollas 2004). The transitions are typically weaker than ππ* transitions and are often suppressed in hydrogen-bonding solvents such as water, but they may be seen when the accompanying ππ* transitions are weak or when they occur in a different wavelength range. n → π* transitions make an appreciable contribution to the absorption spectra of some proteins, and give rise to the color of some compounds such as trans-azobenzene, and may become more prominent in circular dichroism (CD) spectra (Nordén et al. 2010).

As discussed above, absorption wavelengths are sensitive to the extent of conjugation in a chromophore. They are also sensitive to interactions between neighboring chromophores in the same molecule (intramolecular), or in neighboring molecules (intermolecular). Examples of intramolecular coupling between chromophores include π stacking of aromatic residues in proteins and nucleic acids, and strong interactions between peptide nπ* transitions in protein α-helices. Examples of intermolecular coupling include π stacking of ligands bound externally to DNA and the effects of intercalation between DNA base pairs on the spectroscopy of the ligand.

Metal complexes often absorb in the range of 170–800 nm, giving rise to the familiar colors of many transition-metal compounds, and contributing to the colors of some metalloproteins. The band systems in these spectra may be assigned to electronic transitions that are predominantly metal-centered (MC), ligand-centered (LC), ligand-to-metal charge-transfer (LMCT) or metal-to-ligand charge-transfer (MLCT) in character. For example, [Ru(en)3]2+ is not strongly colored, and its absorption spectrum comprises metal-centered d-d transitions. However, the replacement of the ethylenediamine ligands by a π-conjugated polypyridine ligand such as 2,2′-bipyridine (bpy) or 1,10-phenanthroline (phen) causes profound changes to the spectrum, by introducing ligand-centered π- π* transitions, and strong MLCT transitions associated with the transfer of metal d-electrons to empty ligand π*-orbitals. The MLCT bands are predominantly responsible for the bright colors of complexes such as [Ru(bpy)3]2+ and [Ru(phen)3]2+ (Campagna et al. 2007) (Fig. 3). It should be noted that, for example, the orange color of [Ru(phen)3]2+ arises because it absorbs the shorter wavelength colors of light and does not absorb the red, orange, yellow, and some green wavelengths. The spectra of these complexes are sensitive to the chemical environment, and this is exploited in DNA binding studies. As stated above, some transitions may have mixed orbital character.
Fig. 3

Absorbance spectrum of the orange colored [Ru(phen)3]2+

Cross-References

References

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  2. Atkins PW, Friedman RS (2011) Molecular quantum mechanics, 5th edn. Oxford University Press, OxfordGoogle Scholar
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Copyright information

© European Biophysical Societies' Association (EBSA) 2018

Authors and Affiliations

  1. 1.School of Science and HealthWestern Sydney UniversityPenrithAustralia

Section editors and affiliations

  • Alison Rodger
    • 1
  1. 1.Department of ChemistryUniversity of WarwickCoventryUK