Agilent Technologies 1100 Series, Reference Manual, Page: 62 / 346

64 1100 Series FD Reference Manual
3
Optimizing the Fluorescence Detector
Finding the Best Wavelengths
The most important parameters to be optimized in fluorescence detection are
the excitation and emission wavelengths. Generally, it is assumed that the best
excitation wavelength can be taken from the excitation spectrum acquired on
a spectrofluorimeter. It is also assumed that once the optimal excitation
wavelength has been found for one particular instrument type this wavelength
can also be applied to other instruments.
Both assumptions are wrong.
The optimum wavelength for the excitation depends on the absorption of the
compounds but also on the instrument characteristics, for example the lamp
type and the gratings. As most organic molecules absorb best in the
ultra-violet range the Agilent 1100 fluorescence detector was designed to give
an optimum signal-to-noise ratio in the 210 nm to 360 nm range of the
spectrum. To achieve greatest sensitivity, the absorbance wavelength of your
sample molecule should match the wavelength range for your instrument. In
other words, an excitation wavelength in the ultra-violet range. Your
Agilent 1100 fluorescence detector has a broad excitation wavelength range,
but for higher sensitivity you should choose a wavelength in the ultra-violet
range (near 250 nm).
The design elements that contribute to lower efficiency in the lower
ultra-violet range are the xenon flash lamp and the gratings. Flash-type lamps
shift the optimum wavelength to lower wavelength ranges with the
Agilent 1100 fluorescence detector to a maximum of 250 nm. The excitation
grating is blazed for highest efficiency at 300 nm.