The interaction between radiation and matter is a fascinating area in its own right. Most drug molecules absorb radiation in the ultraviolet region of the spectrum, although some are coloured and thus absorb radiation in the visible region, e.g. a substance with a blue colour absorbs radiation in the red region of the spectrum. The absorption of UV/visible radiation occurs through the excitation of electrons within the molecular structure to a higher energy state; Figure 4.1 illustrates the nature of the transitions taking place. These transitions occur from the bottom vibrational state in the electronic ground state of the molecule to any one of a number of vibrational levels in the electronic excited state. The transition from a single ground state energy to one of a number of excited states gives width to UV spectra. Figure 4.1 shows a UV spectrum in which individual bands for different Vo to Vn transitions can be seen. Vibrational fine structure can be seen, although the bands overlap extensively; the vibrational bands themselves have width due to rotational transitions that are intermediate in energy between each vibrational transition. The relative energy of electronic:vibrational:rotational transitions is 100:1:0.01. In most molecules the vibrational behaviour is complex and the degree of overlap of the different energies of the vibrational transitions is too great for vibrational fine structure to be observed.

Fig. 4.1 Excitation of an electron from the ground to the excited electronic state.

Factors governing absorption of radiation in the UV/visible region

Radiation in the UV/visible region is absorbed through excitation of the electrons involved in the bonds between the atoms making up the molecule so that the electron cloud holding the atoms together redistributes itself and the orbitals occupied by the bonding electrons no longer overlap. Short wavelength UV radiation < 150 nm (> 8.3 eV) can cause the strongest bonds in organic molecules to break and thus is very damaging to living organisms. It is the weaker bonds in molecules that are of more interest to analysts because they can be excited by longer wavelength UV radiation > 200 nm (> 6.2 eV), which is at a longer wavelength than the region in which air and common solvents absorb. Examining a very simple organic molecule such as ethylene (Fig. 4.2) it can be seen that it contains two types of carbon–carbon bonds, a strong σ bond formed by extensive overlap of the sp² orbitals of the two carbons and a weaker π bond formed by partial overlap of the p orbitals of the carbon atoms. The σ bond would become excited and break when exposed to radiation at ca 150 nm. The weaker π bond requires less energetic radiation at ca 180 nm to produce the π* excited state shown in Figure 4.2. This excitation can occur without the molecule falling apart since the σ orbitals remain unexcited by the longer wavelength radiation at 180 nm. However, a single double bond is still not useful as a chromophore for determining analytes by UV spectrophotometry since it is still in the region where air and solvents absorb.

Fig. 4.2 Excitation of the carbon–carbon bonds in ethylene by radiation in the short wavelength UV region.

If more double bonds are present in a structure in conjugation (i.e. two or more double bonds in a series separated by a single bond), absorption takes place at longer wavelengths and with greater intensity, as detailed in Table 4.1 for a series of polyenes. The A (1%, 1 cm) value, which is described later, gives a measure of the intensity of absorption. The type of linear conjugated system which is present in polyenes is not very common in drug molecules.

Table 4.1 Longest wavelength maxima and absorption intensities of some polyenes

Polyene	λ max	A (1%, 1 cm)
CH₃(CH = CH)₃CH₃	275	2800
CH₃(CH = CH)₄CH₃	310	6300
CH₃(CH = CH)₅CH₃	342	9000
CH₃(CH = CH)₆CH₃	380	9800

Such extended systems of double bonds are known as ‘chromophores’. The most common chromophore found in drug molecules is a benzene ring (Table 4.2). Benzene itself has its λ max at a much shorter wavelength than a linear triene such as hexatriene (λ max 275 nm) and its strongest absorbance is at the wavelength of absorption of an isolated double bond at 180 nm. It also has a strong absorption band at 204 nm. This is due to the symmetry of benzene; it is not possible to have an excited state involving all three bonds in benzene because this would mean that the dipole (polarisation of the chromophore), a two-dimensional concept which is created in the excited state, would be symmetrical and thus would have to exist in three dimensions rather than two. There is a weak absorption in the benzene spectrum close to the λ max for hexatriene and this can occur because vibration of the benzene ring in a particular direction can distort its symmetry and thus allow all three double bonds to be involved in an excited state. If the symmetry of the benzene ring is lowered by substitution, the bands in the benzene spectrum undergo a bathochromic shift – a shift to longer wavelength. Substitution can involve either extension of the chromophore or attachment of an auxochrome (a group containing one or more lone pair of electrons) to the ring or both. Table 4.2 summarises the absorption bands found in some simple aromatic systems and these chromophore/auxochrome systems provide the basis for absorption of UV radiation by many drugs. The hydroxyl group and amino group auxochromes are affected by pH, undergoing bathochromic (moving to a longer wavelength) and hyperchromic (absorbing more strongly) shifts when a proton is removed under alkaline conditions, releasing an extra lone pair of electrons. The effect is most marked for aromatic amine groups. The absorption spectrum of a drug molecule is due to the particular combination of auxochromes and chromophores present in its structure.

Table 4.2 The UV absorption characteristics of some chromophores based on the benzene ring

Chromophore	Longest wavelength λ max	A (1%, 1 cm)
Benzene	255 nm	28
Benzoic acid	273	85
Cinnamic acid	273	1420
Protriptyline	292	530
Phenol	270 nm 287 nm → Bathochromic	72 271 → Hyperchromic
Aniline	255 nm 286 nm → Bathochromic	16 179 → Hyperchromic