Spectroscopic Techniques


A number of spectroscopic methods are available to the biochemical researcher. They give different information on a number of time and wavelength scales, and can be used to determine concentrations and molecular weights, as well as providing information on secondary and tertiary structure of proteins. Spectroscopic methods are utilised to detect rapid changes in the structures of biological macromolecules. Three‐dimensional structures can be calculated using information from nuclear magnetic resonance (NMR) and other spectra. Size limitations in NMR spectroscopy have been largely ameliorated through the application of ever‐higher magnetic fields and innovations in pulse sequences. Other recent innovations include applications of fluorescence to single molecules, as well as to the visualisation of particular molecules within cells.

Key Concepts:

  • Spectroscopic techniques form part of the basic toolset of research in the life sciences.

  • Spectroscopy utilises the various wavelengths of light in the electromagnetic spectrum to give information on various aspects of the structure, dynamics and function of molecules.

Keywords: ultraviolet; infrared; nuclear magnetic resonance; mass spectrometry

Figure 1.

Schematic illustration of the energy levels that are perturbed in a spectroscopic experiment. The frequency ranges for various forms of spectroscopy are summarised beneath the diagram.

Figure 2.

Examples of various forms of spectroscopy applied to the haem protein, myoglobin. (a) UV–visible spectrum of the carbon monoxide complex of myoglobin (MbCO) measured at pH 6 and 25°C. Inset: visible spectrum at higher gain. (b) far‐UV and near‐UVCD spectra of MbCO measured at pH 6 and 25°C. (c) One‐dimensional NMR spectrum of MbCO, pH 6, 25°C. (d) Two‐dimensional NMR spectrum (15N–1H heteronuclear single‐quantum coherence (HSQC) spectrum) of MbCO (reproduced from Thériault et al. (1994) Journal of Biomolecular NMR4: 491). (e) X‐band EPR spectrum of metaquomyoglobin at pH 8.4 (adapted from Maurus et al. (1994) Journal of Biological Chemistry269: 12606). (f) Fluorescence spectrum of myoglobin (pH 7.5) containing the porphyrin of the haem group but no Fe. The fluorescence of the Fe‐containing myoglobin is completely quenched at neutral pH. The excitation wavelength was 406 nm (reproduced from Postnikova and Yumakova (1991) European Journal of Biochemistry198: 241). (g) Resonance Raman spectrum of MbCO at pH 6 showing the Fe–CO stretching frequency at 500 cm−1 (reproduced from Wells et al. (1991) Journal of the American Chemical Society113: 9655). (h) Electrospray mass spectrum of myoglobin showing (top) numerous charge states, (bottom) reconstruction to show a single peak of the correct mass.


Further Reading

Croasmun WR and Carlson R (eds) (1994) Two‐Dimensional NMR Spectroscopy: Applications for Chemists and Biochemists. New York: VCH Publishers.

Fasman GD (ed.) (1996) Circular Dichroism and the Conformational Analysis of Biomolecules. New York: Plenum Press.

Howarth O (1973) Theory of Spectroscopy. London: Thomas Nelson and Sons Ltd.

Kapanidis AN and Strick T (2009) Biology, one molecule at a time. Trends in Biochemical Sciences 34: 234–243.

Siuzdak G (1996) Mass Spectrometry for Biotechnology. San Diego, CA: Academic Press.

Szabo AG (1990) Application of laser‐based fluorescence to study protein structure and dynamics. Biotechnology 14: 159–186.

Wertz JE and Bolton JR (1972) Electron Spin Resonance. New York: McGraw‐Hill.

Wüthrich K (1986) NMR of Proteins and Nucleic Acids. New York: Wiley.

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Dyson, H Jane(Jun 2010) Spectroscopic Techniques. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002703.pub2]