Waters 2475, Operator'S Manual, Page: 18 / 228

1-4 Theory of Operation
also be selected. Detectors with excitation and emission monochromators can
scan holding one monochromator at a constant setting while varying the
setting on the other. This type of operation is necessary when you are
evaluating mixtures or analyzing chemical structures.
Exciting the sample
The broad band of high-intensity light from the lamp passes through a filter or
monochromator, which selects a narrow band of wavelengths. This narrow
band of light is then directed onto the flow cell where it excites the analytes as
they pass through. Excitation wavelengths often correspond with the
absorbance wavelength of the analyte.
Flow cell
The quartz flow cell minimizes the amount of stray light that can affect the
measurement, and it maximizes the fluorescence signal. The sample
compartment is arranged so that the fluorescence energy is collected at an
angle perpendicular to the excitation (lamp) beam. This arrangement
minimizes the effect of Rayleigh scatter on background light levels.
Measuring fluorescence
To measure fluorescence in the flow cell, the detector must balance the need
for high selectivity (to distinguish specific fluorescence wavelengths) with the
need for high sensitivity (to measure low-fluorescence intensities).
Quantitation
Fluorescence is linear at low concentrations but can exhibit nonlinearity at
high concentrations.
Emission wavelength selection
A monochromator is used to select an emission wavelength.
Photomultiplier tube
The photomultiplier tube (PMT) produces a current proportional to the flux of
photons emitted by the molecules in the flow cell.