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Agilent InfinityLab LC Series 1260 Infinity II FLD User Manual
5
Optimizing the Detector
Finding the Best Wavelengths
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 module 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 module 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 module to
a maximum of 250 nm. The excitation grating is blazed for highest efficiency at
300 nm.
