Fluorescence spectrophotometry

Introduction

Figure 7.1 illustrates the behaviour of an excited electron in a fluorescent molecule. In a non-fluorescent molecule, when an electron is excited to the electronic excited state, it returns back to the ground state by losing the energy it has acquired through conversion of the excess electronic energy into vibrational energy. If a molecule has a rigid structure, the loss of electronic energy through its conversion into vibrational energy is relatively slow and there is a chance for the electronic energy to be emitted as ultraviolet or visible radiation. The energy emitted is of lower energy than the energy absorbed because, as indicated in Figure 7.1, the excited electron moves to the lowest energy vibrational state in the excited state before returning to the ground state. Thus fluorescence emission is typically shifted by 50–150 nm towards a longer wavelength in comparison with the wavelength of the radiation used to produce excitation. The fluorescence spectrum of a molecule is, ideally, a mirror image of the longest wavelength band in the absorption spectrum of the molecule, but often the spectrum is distorted due to partial overlap between the absorption and the emission spectra. Vibrational fine structure of the fluorescence band may be observed if the molecule does not interact with the solvent strongly (cf. UV spectra) and can be observed in the fluorescence spectra of polycyclic aromatic hydrocarbons such as anthracene. The shape of the fluorescence spectrum is independent of the wavelength used for excitation, since the transition producing the fluorescence spectrum is always from the first excited state to the ground state. In a molecule containing a number of UV absorption bands, the longest wavelength maximum is the one associated most strongly with the production of fluorescence. In addition, the wavelength usually used to produce excitation is close to the λ max of the longest wavelength absorption band in the spectrum of the analyte.

Fig. 7.1 Energy changes upon absorption of UV/visible radiation resulting in fluorescence.

Instrumentation

Figure 7.2 shows a schematic diagram of a fluorescence spectrophotometer. Since emission is being observed, the light being emitted is observed at right angles to the light being used to excite the sample.

Fig. 7.2 Schematic diagram of fluorescence spectrophotometer.

The instrument has two monochromators: one to select the wavelength to be used for excitation of the sample, the other to scan the wavelength range of the light emitted by the sample.

The lamp used, which is a quartz halogen lamp or xenon, produces radiation of high intensity to take advantage of the fact that the strength of the fluorescence is related to the number of photons absorbed multiplied by the fluorescence quantum yield (φ). For strongly fluorescent compounds, φ is close to 1; for non-fluorescent compounds, φ = 0. The wavelength which gives maximum excitation is not necessarily exactly the same as the longest wavelength absorbance maximum in the compound since the intensity of light emitted by the quartz halogen lamp varies markedly with wavelength, unlike the deuterium and tungsten lamps used in UV/visible spectrophotometers. The lamp gives radiation of maximum intensity between 300 and 400 nm.

Although the radiation emitted is observed at right angles to the exciting radiation, some of the exciting radiation can be detected by the emission detector because it is scattered by solvent molecules (Rayleigh scatter) or by colloidal particles in solution (Tyndall scatter). The presence of this scatter makes the use of the second monochromator necessary and also means that, for fluorescence measurements to be made without interference, the fluorescence band has to be shifted by at least 20 nm beyond the excitation band. Another, weaker, type of scatter which may be observed is Raman scatter. In Raman scatter, which is solvent dependent, the wavelength of the incident radiation is shifted to a longer wavelength by about 30 nm when methanol is used as a solvent and about 10 nm when chloroform is used as a solvent. Raman scatter is discussed in more detail later in this chapter.

Molecules which exhibit fluorescence

It is not entirely possible to predict how strongly fluorescent a molecule will be. For example, adrenaline and noradrenaline differ in their structures by only a single methyl group but noradrenaline exhibits fluorescence nearly 20 times more intensely than adrenaline. Generally, fluorescence is associated with an extended chromophore/auxochrome system and a rigid structure. Quinine (Fig. 7.3) is an example of a strongly fluorescent molecule, as might be expected from its extended chromophore and rigid structure. The chromophore in ethinylestradiol is just an aromatic ring but the presence of a phenolic hydroxyl group in combination with a rigid ring structure in the rest of the molecule renders it fluorescent (Fig. 7.3).

Fig. 7.3 Examples of fluorescent molecules.

Figure 7.4 shows the fluorescence spectrum of ethinylestradiol. When the fluorescence spectrum of the molecule is scanned with a wavelength of 285 nm being used for excitation, two maxima are seen. The maxima at 285 nm is due to scatter of the exciting radiation and the second, more intense, maximum at 310 nm is due to fluorescence. The separation of the exciting radiation and emitted radiation is not great in this example, but this is partly because excitation is taking place at a relatively short wavelength, where the displacement of wavelength with energy is lower. For example, the difference between 285 and 310 nm is 0.35 eV, whereas with an excitation wavelength at 385 nm, an energy displacement of 0.35 eV would give an emission wavelength at 443 nm.

Fig. 7.4 Fluorescence spectrum of a 20 μg/ml solution of ethinylestradiol.

Like ethinylestradiol, many other phenols exhibit fluorescence and, as is the case for ethinylestradiol, this fluorescence is pH dependent and does not occur under alkaline conditions, when the phenolic group becomes ionised. Table 7.1 shows some examples of fluorescent drug and vitamin molecules.

Table 7.1 Examples of drugs which yield fluorescence spectra

Factors interfering with fluorescence intensity

If the concentration of a solution prepared for fluorescence measurement is too high, some of the light emitted by the sample as fluorescence will be reabsorbed by other unexcited molecules in solution. For this reason, fluorescence measurements are best made on solutions with an absorbance of less than 0.02 at their maximum, i.e. solutions of a sample 10–100 weaker than those which would be used for measurement by UV spectrophotometry.

Heavy atoms in solution quench fluorescence by colliding with excited molecules so that their energy is dissipated, e.g. chloride or bromide ions in solution cause collisional quenching.

Formation of a chemical complex with other molecules in solution can change fluorescence behaviour, e.g. the presence of caffeine in solution reduces the fluorescence of riboflavin. This alteration of fluorescence upon binding is used to advantage when examining binding of fluorescent molecules to proteins or other constituents of cells.

Fluorescence is also temperature dependent since temperature can promote the loss of excitation by collision and bond vibration. A molecule that is not fluorescent at room temperature may become fluorescent at lower temperatures. In addition, more viscous solvents tend to promote fluorescence.