Chromatographic Methods for Environmental Analysis - Fundamentals of Environmental Sampling and Analysis - page 273

Fluorescence Detectors

The second most popular HPLC detector, fluorescence detector, is similar in design to
that of the UV-VIS units. As described in Chapter 8 (Fig. 8.4), however, fluorescence
is very different from absorption. For fluorescence detectors, two wavelengths are
concerned rather than the single wavelength used in absorption-based UV detectors
(Fig. 10.10). The detector employs an excitation source (typically a UV lamp), which
emits UV radiation at a range of wavelengths. One or more filters or a grating
monochromator in more sophisticated instruments are used to acquire the needed
exciting beam (l excitation ). The emitted light (fluorescence) is most conveniently
measured at a 90 angle to the exciting light beam at a wavelength of l emission . This
right angle is important because at other angles, increase in light scattering for the
solution and the cell walls may result in large errors during fluorescence measurement.
Like UV detectors, fluorescence detectors are not universal. They are, in fact,
limited to certain chemicals, but, typically, they are at least an order of magnitude
more sensitive than UV detectors. To fully appreciate fluorescence detectors, one
should have some knowledge of fundamental chemistry regarding what chemicals
fluoresce. In Chapter 8, we have introduced various forms of electronic transition. For
the majority of fluorescent compounds, the radiation is produced by either an n ! p
or a p ! p transition. The most intense and the most useful fluorescence is found in
compounds containing aromatic functional groups with low-energy p ! p
transition levels. This explains why HPLC-fluorescent detector finds its wide
applications for the analysis of polycyclic aromatic hydrocarbons (PAHs) of
environmental concern. The intensity of fluorescence increases as the number of
fused benzene rings increases. Halogen substitution, generally, results in a substantial
decrease in fluorescence, and the substitution of a carboxylic acid (COOH) or
carbonyl group (C

O) on an aromatic structure may inhibit fluorescence.

Transducer

Filter
(λ emission )

Collecting lens

Sample cell

Lamp

Figure 10.10 Schematic diagram of a
fluorescence detector showing the light path
(Rubinson, Kenneth A.; Rubinson, Judith F.,
Contemporary Instrumental Analysis,
# 2000, p. 657. Reprinted by permission
of Pearson Education, Inc., Upper Saddle
River, NJ.)

Lens Filter

(λ excitation )

Lens