Fluorescence Detection; Overview; Excitation Sources; Types Of Light Sources - Waters 2475 Operator's Manual

• Measuring the emitted fluorescence
• Amplifying the emitted signal

Fluorescence detection

Overview

The scanning fluorescence detector illuminates a sample with a narrow band
of high-intensity light. The detector then measures the low levels of
fluorescence emitted by the sample. The emitted light is filtered, amplified,
and converted to electrical signals that can be recorded and analyzed.

Excitation sources

The typical energy source used for fluorescence detection is a lamp that
provides an intense, stable spectrum of light in the UV and visible ranges. The
resulting fluorescence intensity is directly related to the intensity of the
excitation spectrum. Thus high-sensitivity detectors use the most intense
excitation source available.

Types of light sources

Xenon lamps are the preferred source for general-purpose fluorescence
detectors.

Excitation wavelength selection

The excitation wavelength of choice requires some source-light filtering. In
modern detectors, a monochromator is typically used for the same purpose.
A monochromator is an adjustable device that you use to select wavelengths
over a wide range of the spectrum. A grating monochromator uses a
diffraction grating that passes only a small range, or bandwidth, of
wavelengths. By moving the grating, you can select wavelengths within a
particular range of wavelengths. A grating monochromator also passes
fractions, or orders, of a selected wavelength. For example, if the
monochromator is set to pass light energy at 600 nm, it also passes energy at
the second-order wavelength of 300 nm. A long-pass filter can be used to
absorb the higher-order energy produced by a monochromator. As the
excitation is selected by a monochromator, the emission (radiated energy) can
Fluorescence detection 1-3