Spectroscopy Methods

By BSc (Hons)

Ultraviolent and Visible Light Spectroscopy

The techniques associated with these regions of the electromagnetic spectrum are probably the most widely used for analytic work.

The molecular substructures that are responsible for interacting with the electromagnetic radiation are called chromophores. In proteins, the relevant types in UV/Vis spectroscopy are peptide bonds, certain amino acid side chains (primarily tryptophan and tyrosine) and certain prosthetic groups and coenzymes (e.g. porphyrin groups present in haem).

Colorimetric assays require a calibration curve to be plotted (concentration versus absorbance) which should be linear as long as the Beer–Lambert law applies. Using this, absorbance of unknowns are then measured and their concentration can be interpolated from the linear region of the plot.

Qualitative analysis can be used to identify certain classes of compounds both as pure samples and in biological mixtures. This type of spectroscopy is most commonly used for quantification of biological samples either directly or via colorimetric assays.

In many cases, proteins can be directly quantified using their intrinsic chromophores, tyrosine and tryptophan. Protein spectra are acquired by scanning from 500 to 210 nm. The characteristic features in a protein spectrum are a band at 278/280 nm and another at 190 nm. The region from 500 to 300 nm provides valuable information about the presence of any prosthetic groups or coenzymes.

Fluorescence Spectroscopy

Fluorescence occurs where an energy transition from a higher to a lower state is accompanied by radiation. There are many and highly varied applications for fluorescence despite the fact that relatively few compounds exhibit this characteristic. The effects of pH, solvent composition and the polarization of fluorescence may all contribute to structural studies.

Intrinsic protein fluorescence

Proteins possess three intrinsic fluorophores: tyrosine, tryptophan and phenylalanine, although the latter contributes little to protein fluorescence emission. Intrinsic protein fluorescence is usually determined by tryptophan fluorescence which can be selectively excited at 295–305 nm. Excitation at 280 nm leads to tyrosine and tryptophan fluorescence; the resulting spectra might then contain contributions from both types of residues.

Extrinsic fluorescence

If a molecule of interest is non-fluorescent, an external fluorophore can be introduced by chemical coupling or non-covalent binding. 1-anilino-8- naphthalene sulphonate (ANS) is a commonly used extrinsic chromophore which emits only weak fluorescence in polar environment, e.g. in aqueous solution.

However, in non-polar environments, e.g. when bound to hydrophobic regions of proteins, its fluorescence emission significantly increases. These characteristics make ANS valuable for assessing the degree of non-polarity and to monitor binding of ligands and prosthetic groups.

Scattering Spectroscopy

The scattering of light can yield valuable insights into the properties of macromolecules, including the molecular mass, association/dissociation properties and internal dynamics. When incident light strikes a macromolecule, it is scattered into all directions and the intensity of the scattered light is only a fraction of the original intensity.

Most of the scattered light possesses the same wavelength as the incident light; this phenomenon is called elastic light scattering. When the scattered light has a wavelength higher or lower than the incident light, the phenomenon is called inelastic light scattering (Raman spectroscopy). The special properties of lasers with high monochromaticity, narrow focus and strong intensity, make them ideally suited for light scattering applications.

Atomic Spectroscopy

With regards to the general theory of electronic transitions, molecules give rise to band spectra while atoms yield clearly defined line spectra. In atomic emission spectroscopy (AES), when the atoms are excited, the wavelengths emitted of particular wavelength (color) may be identified using a spectrophotometer.

In a spectrum of an element, the absorption or emission wavelengths are associated with electron transitions due to an energy change. Electron transitions in an atom are limited by the availability of empty orbitals and the rules governing how these orbitals are filled together mean that emission and absorption lines are characteristic for an individual element.

In order for atoms to emit or absorb monochromatic radiation, they need to be volatilized using high thermal energy.

Atomic emission spectroscopy (AES) and atomic absorption spectroscopy (AAS) are generally used to identify specific elements and their concentrations within a sample. The energy absorbed or emitted is proportional to the number of atoms in the optical path. Concentration determination with AES or AAS is carried out by comparison with calibration standards.



Further Reading

Last Updated: Nov 23, 2015

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