There is a linear relation between the concentration, C, of a fluorescent analyte and the intensity of
emission, IF, given by the equation
where ΦF and ε are the quantum yield and molar absorptivity respectively of the fluorescing species, Io
is the intensity of the incident (exciting) radiation and l is the path length of the sample cell. If Io and l
are constant and the absorbance (εCl) is small, i.e. the analyte concentration is low, then
Calibration curves are linear over several orders of magnitude but eventually show curvature and even
reversal due to quenching effects. These are caused by partial or total absorption of the emitted
radiation by unexcited analyte molecules, dissolved oxygen and other species, particularly if they are
paramagnetic. Unlike absorptiometry, sensitivity can be improved by increasing the intensity of the
exciting radiation, Io.
Fluorimeters have similar components to UV/visible spectrometers but differ in the geometry of the
radiation beams and in the need to be able to select or scan both excitation and emission wavelengths.
In most designs, the fluorescence, which is emitted from the sample equally in all directions, is
measured at 90° to the direction of the excitation beam. This prevents incident, scattered and reflected
radiation from reaching the detector. Filter fluorimeters are single-beam instruments capable of high
sensitivity for quantitative analysis but lacking in the versatility needed for investigative qualitative
scans. Spectrofluorimeters employ two monochromators which allows independent scanning of
excitation or emission spectra over the whole of the UV/visible region (200–800 nm) in addition to
making quantitative measurements at fixed wavelengths. Both filter fluorimeters and
spectrofluorimeters can be fitted with flow-through microcells for use as detectors for high-performance
liquid chromatography or capillary electrophoresis but purpose-designed detectors are preferable (p.
131).
Applications of UV/Visible Spectrometry and Fluorimetry
Quantitative analysis by UV/visible absorption spectrometry is practised by almost every analytical
laboratory at one time or another. Most inorganic, organic and biochemical substances can be
determined either directly or after the formation of an absorbing derivative or complex. A selection of
typical applications is given in Table 9.5. The technique is among the most sensitive and is
predominantly used for the determination of minor, trace and ultratrace level constituents. Its
applications to the determination of metals in trace amounts were particularly widespread because of
the many intensely coloured complexes known and the degree of selectivity introduced by proper
choice of organic reagent and masking reactions or by solvent extraction. In recent years, however,
atomic absorption and ICP spectrometry (pp. 297, 320) have largely superseded the use of UV/visible