The principal components of a fluorescence spectrophotometer (fluorimeter)
are shown in Fig. 26.4. The instrument contains two monochromators, one
to select the excitation wavelength and the other to monitor the light emitted,
usually at 90° to the incident beam (though light is actually emitted in all
directions). As an example, the wavelengths used to measure the highly
fluorescent compound naphthalene are 270 nm (excitation) and 340 nm
(emission). Some examples of molecules with intrinsic fluorescence are given
in Table 26.1.
Compared with UV/visible spectrophotometry, fluorescence spectroscopy
has certain advantages, including:
- Enhanced sensitivity (up to IOOO-fold),since the emitted light is detected
against a background of zero, in contrast to spectrophotometry where small
changes in signal are measured against a large 'background' (see eqn [26.5]).
- Increased specificity, because not one, but two, specific wavelengths are
required for a particular compound.
However, there are also certain drawbacks:
- Not all compounds show intrinsic fluorescence, limiting its application.
However, some non-fluorescent compounds may be coupled to fluorescent
dyes, or fluorophores (e.g. alcohol ethoxylates may be coupled to
- The light emitted can be less than expected owing to quenching, i.e. when
substances in the sample (e.g. oxygen) either interfere with energy
transfer, or absorb the emitted light (in some instances, the sample
molecules may self-quench if they are present at high concentration).
|Fig. 26.4 Components of a fluorimeter
(fluorescence spectrophotometer). Note that
sample cells for fluorimetry must have clear
sides all round.
|Table 26.1 Examples of compounds with intrinsic fluorescence
The sensitivity of fluorescence has made it invaluable in techniques in which
specific chemicals, e.g. polycyclic aromatic hydrocarbons and alcohol ethoxylates,
are linked to a fluorescent dye for detection in high-performance liquid