orbitals. Otherporbitals combine to form non-bindingnorbitals. The population
of binding orbitals strengthens a chemical bond, and, vice versa, the population of
anti-binding orbitals weakens a chemical bond.12.1.3 Lasers
Laser is an acronym forlight amplification by stimulatedemission ofradiation.
A detailed explanation of the theory of lasers is beyond the scope of this textbook.
A simplified description starts with the use of photons of a defined energy to excite an
absorbing material. This results in elevation of an electron to a higher energy level. If,
whilst the electron is in the excited state, another photon of precisely that energy arrives,
then, instead of the electron being promoted to an even higher level, it can return to the
original ground state. However, this transition is accompanied by the emission of two
photons with the same wavelength and exactly in phase (coherent photons). Multipli-
cation of this process will produce coherent light with extremely narrow spectral
bandwidth. In order to produce an ample supply of suitable photons, the absorbing
material is surrounded by a rapidly flashing light of high intensity (pumping).
Lasers are indispensable tools in many areas of science, including biochemistry
and biophysics. Several modern spectroscopic techniques utilise laser light sources,
due to their high intensity and accurately defined spectral properties. One of the
probably most revolutionising applications in the life sciences, the use of lasers in
DNA sequencing with fluorescence labels (see Sections 5.11.5, 5.11.6 and 12.3.3),
enabled the breakthrough in whole-genome sequencing.12.2 Ultraviolet and visible light spectroscopy
These regions of the electromagnetic spectrum and their associated techniques are
probably the most widely used for analytical work and research into biological problems.
The electronic transitions in molecules can be classified according to the partici-
pating molecular orbitals (See Fig. 12.4). From the four possible transitions (n!p*,
p!p*,n!s*,s!s*), only two can be elicited with light from the UV/Vis spectrum
for some biological molecules:n!p* andp!p*. Then!s* ands!s* transitions are
energetically not within the range of UV/Vis spectroscopy and require higher energies.
Molecular (sub-)structures responsible for interaction with electromagnetic radi-
ation are calledchromophores. In proteins, there are three types of chromophores
relevant for UV/Vis spectroscopy:- peptide bonds (amide bond);
- certain amino acid side chains (mainly tryptophan and tyrosine); and
- certain prosthetic groups and coenzymes (e.g. porphyrine groups such as in haem).
The presence of severalconjugated double bondsin organic molecules results in an
extendedp-system of electrons which lowers the energy of thep* orbital through
electron delocalisation. In many cases, such systems possessp!p* transitions in the
UV/Vis range of the electromagnetic spectrum. Such molecules are very useful tools in
colorimetric applications (see Table 12.1).482 Spectroscopic techniques: I Photometric techniques