Waters 2475, Operator'S Manual, Page: 16 / 228

1-2 Theory of Operation
Fluorescence theory
Fluorescence occurs when certain molecules absorb light at specific
wavelengths, promoting the molecules to a higher energy state. As they return
to their normal energy states, the “excited” molecules release their absorbed
energy as photons.
Many organic compounds absorb light, but few fluoresce. HPLC systems that
incorporate fluorescence detection effectively identify polyaromatic
hydrocarbons, aflatoxins, vitamins, amino acids, and so on. Chemical
derivatization methods extend fluorescence detection to some nonfluorescing
compounds such as carbamate pesticides.
Fluorescence detection requires both the excitation/emission wavelengths,
leading to a higher degree of sensitivity. As a result, this technique is useful
for analyses requiring low detection limits.
Certain conditions can interfere with a compound’s ability to fluoresce,
diminishing analytical performance:
• pH changes – The loss or gain of protons and their accompanying
increase or decrease of charge affects an analyte’s electronic structure
and can enhance or degrade fluorescence.
• Temperature changes – Fluorescence decreases as the sample
temperature increases.
• Changes in the amount of dissolved oxygen – For some molecules,
fluorescence is quenched (decreased) by the presence of dissolved
oxygen.
Fluorescence detectors can be adapted to measure chemiluminescence, where
a molecule without exposure to any excitation energy emits a low intensity
signal. This type of detection can be accommodated by disabling the light
source or (as in the case of the 2475 detector) enabling a shutter to stop any
excitation light from reaching the flow cell.
The process of fluorescence detection involves an excitation source and the
following processes:
• Filtering the source light
• Exciting the sample with filtered light
• Collecting and filtering the emitted fluorescence