Agilent Technologies 1100 Series Reference Manual page 42

2 First Steps with the Fluorescence Detector
Procedure II - Take two LC runs with the FLD
The conditions for the separation of organic compounds such as polyaromatic nuclear
hydrocarbons (PNAs) are well described in various standard methods, including
commonly used EPA and DIN methods. Achieving the best detection levels requires
checking for the optimum excitation and emission wavelengths for all compounds. Yet
taking fluorescence scans individually makes this a tedious process. A better approach
is to acquire spectra online for all compounds during a run. This speeds up method
development tremendously. Two runs are sufficient for optimization.
During the first run, one wavelength is chosen in the low UV range for the excitation
wavelength and one emission wavelength in the spectral range for the emission
wavelength. Most fluorophores show strong absorption at these wavelengths and the
quantum yield is high. Excitation is sufficient for collecting emission spectra.
Figure 13
of 15 PNAs. This set of spectra is used to set up a timetable for optimum emission
wavelengths for all compounds.
The individual compound spectra in the isofluorescence plot show that at least three
emission wavelengths are needed to detect all 15 PNAs properly:
Table 6
0 min:
8.2 min:
19.0 min:
In the second run, three setpoints for emission wavelengths are entered into the time-
program and excitation spectra are recorded, as shown in figure 8. The area of high
intensity (red) is caused by stray light when emission spectra overlap with the
excitation wavelength. This can be avoided by fitting the spectral range automatically.
Excitation at 260 nm is most appropriate for all PNAs.
Table 7
Column
Mobile phase
42 1100 Series FD Reference Manual
on page 46 contains all emission spectra obtained in a single run from a mix
Timetable for PNA analysis
350 nm
420 nm
500 nm
Conditions for Figure 13
for naphthalene to phenanthrene
for anthracene to benzo(g,h,I)perylene
for indeno(1,2,3-cd)pyrene
and Figure 14 on page 47
Vydac, 2.1 x 200 mm, PNA, 5
A = water; B = acetonitrile (50:50)
µ
m