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The Dye Tracing Pages | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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The filter fluorometer is a device that exploits some of the principles of fluorescence
in order to identify and quantify fluorescent molecules (dyes). Fluorescent dyes absorb
light at one energy level (or wavelength) and emit light at a lower energy level ( or longer wavelength).
The difference between the absorbed wavelength and the emitted wavelength is referred
to as the Stoke's Shift or delta lambda. Depending on how much energy is lost in the
fluorescence process, the Stoke's Shift can be a very small or a very large number.
The wavelength range for which fluorescent molecules absorb light is relatively small
(usually less than 50 nanometers). What this means is that light outside a specific
avelength range will not cause the molecule to fluoresce. A filter fluorometer exploits
this fact to identify one fluorescent molecules within a sample that may contain many
several fluorescent molecules. Here is how it works: 1. A strong light source which produces light within a specific light range ( such as a xenon arc lamp) is focused down to a tight beam. 2. The tight beam of light is sent through a filter which removes most of the light outside of the target wavelength range for a particular fluorescent molecule. 3. The filtered light beam passes through the liquid target sample striking some of the fluorescent molecules in the sample. 4. Light emitted from the fluorescent molecules that is travelling orthogonal to the excitation light beam pass through a secondary filter that removes most of the light outside of the target wavelength range. 5. The filtered light then strikes a photodetector which allows the instrument to give a relative measurement of the intensity of the emitted light. This instrument was a breakthrough in fluorescence analysis when it was first introduced. For the first time, fluorescent molecules could be detected at concentrations below a level visible to the subjective human eye. The filter fluorometer provided one additional benefit as well--it allowed the user to determine the concentration of the fluorescent molecules in solution. Fluorescence intensity vs. concentration is a linear relationship except at very high concentrations where quenching becomes strong. Through the use of calibration curves, dye concentrations could be determined with a good degree of accuracy. At the time, it was a great breakthrough in fluorescence analysis, allowing quantification down to the 1 part per billion level. |
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