|Compared with UV/visible spectrophotometry, fluorescence spectroscopy
has certain advantages, including:
However, there are also certain drawbacks:
- 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.
- 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.