Chapter 3 – Making Light Work in Biology  61

Technical experts of IR spectrometers generally do not cite absorption wavelengths but

refer instead to wavenumbers in units of cm⁻¹, with the range ~700–​4000 cm⁻¹ being rele­

vant to most of the different covalent bonds found in biomolecules, which corresponds to a

wavelength range of ~2.5–​15 μm. Although broadband IR sources are still sometimes used in

older machines, it is more common now to use IR laser sources.

The pattern of IR absorption peaks in the spectrum, their relative position in terms of

wavelength and amplitude, can generate a unique signature for a given biochemical compo­

nent and so can be invaluable for sample characterization and purity analysis. The principal

drawback of IR spectroscopy is that water exhibits an intense IR absorption peak and samples

need to be in a dehydrated state. IR absorption ultimately excites a transition between

different bond vibrational states in a molecule (Figure 3.1a), which implies a change in elec­

trical dipole moment, due to either electrical polar asymmetry of the atoms that form the

bond or the presence of delocalized electronic molecular orbitals (Table 3.1).

FIGURE 3.1  Simple spectroscopy and fluorescence excitation. (a) Some typical vibrational modes of a carbon atom–​

centered molecular motif that are excited by infrared absorption. (b) Jablonski energy level transition diagram for single-​

photon excitation resulting in fluorescence photon emission, characteristic time scales indicated. (c) Schematic of a typical

fluorescence-​assisted cell sorting device. (d) Typical output response from an SPR device showing injection events of a

particular ligand, followed by washes and subsequent ligand injections into the SPR chamber of increasing concentration.