Exciting The Sample; Flow Cell; Measuring Fluorescence; Quantitation - Waters 2475 Operator's Manual

Multi fluorescence detector

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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.

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.

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).

Fluorescence is linear at low concentrations but can exhibit nonlinearity at

high concentrations.

A monochromator is used to select an emission wavelength.

The photomultiplier tube (PMT) produces a current proportional to the flux of

photons emitted by the molecules in the flow cell.

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Theory of Operation

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