= Fluorometer
A <fluorometer> measures <fluorescence> emitted by a sample under specified excitation and collection conditions. A spectral instrument can vary the excitation or observation <wavelength>; a simpler instrument can select fixed bands with filters. The reading depends on both the sample and the instrument, so converting an intensity into <concentration> requires an appropriate <fluorescence calibration curve>.
A typical spectrofluorometer sends source light through an excitation <monochromator>, illuminates the sample, then selects an emission band before a <photodetector>. Collection at $90^\circ$ to the incident beam reduces direct transmitted excitation reaching the detector. JASCO's illustrated configuration also sends a reference beam to a separate detector; dividing the fluorescence reading by the reference reading helps compensate for source fluctuations. These optical choices separate useful emission from excitation and stabilize its measurement.<Principles of fluorescence spectroscopy (3) - Instrument configuration by JASCO>[Optical system and Fig. 7, excitation monochromator, right-angle fluorescence detection and reference-beam ratio]{ref}
For an illustrative wavelength-selection example, hold the excitation band near $470\,\mathrm{nm}$ and scan the observation band across longer <wavelengths> to record an <emission spectrum>. Holding the observation band near a selected emission peak while varying excitation instead records an <excitation spectrum>. The numbers specify a hypothetical setting, not a measurement of an identified dye. Slit widths select finite bands, so a nominal setting does not mean a single exact <wavelength>.
For quantitative work, measure a <fluorescence blank> containing the solvent and other background-contributing materials without the target fluorescent substance, then measure standards and unknowns with matching settings. Check detector saturation, source drift and repeatability before interpreting differences as changes of <concentration>. Changes in optical absorption, <fluorescence quenching> or <photobleaching> can change the response even when the intended <concentration> comparison is unchanged. A <fluorometer> measures an optical signal; a calibrated model supplies the inference about the sample.
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