History
The First Fluorescent Dye Trace
Visual Tracing with Activated Charcoal
The Filter Fluorometer
The Synchronous Scanning Spectrofluorophotometer
The First Fluorescent Dye Trace
Fluorescein
Fluorescein
Fluorescent dyes are the most successful water tracers ever developed. Fluorescein (a green fluorescent dye) was first created in 1871. Six years later, in 1877, it was used to trace the sinking portions of the upper Danube river. A few years later a more water-soluble, disodium salt form of the dye was introduced under the trade name “Uranine” (Kass 1998). Today there many fluorescent dyes, but only about 10 of them are considered safe enough to use for dye tracing. Over one hundred and twenty years later, Uranine is still one of the best fluorescent dyes available for water tracing.
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Visual Tracing with Activated Charcoal
Initially, dye tracing was strictly a qualitative science. Dye was injected into a location while someone waited at the point of interest for the dye to emerge. Later, scientists revealed that some of the fluorescent dyes used could effectively be adsorbed onto activated charcoal grains. The dye could then be extracted from the charcoal grains with an alcohol-based solvent.
This vital discovery allowed for mesh packets containing activated charcoal to be placed in springs and streams and left unattended while the dye cloud passed by. No longer was it necessary to have people waiting at numerous locations for the dye to surface. The number of possible resurgence points was now inconsequential.
Activated Charcoal
The alleviation of the field issue did not solve the complications of the analysis process. Both techniques relied exclusively upon the human eye which was subjective for definitive dye results. Strong concentrations of dye were apparent, but as the amount diminished so did the consensus among the viewers. The issue was resolved with the inception of an instrument called a filter fluorometer.
Today, synchronously scanning spectrofluorophotometers are in use at every major fluorescence analysis laboratory in the United States, including the US EPA’s premier analysis laboratory in Las Vegas, Nevada. While filter fluorometers are still manufactured and still have their appropriate uses, the SSS has necessarily become the standard for fluorescence analysis.
The Filter Fluorometer
The filter fluorometer is an instrument that exploits some of the principles of fluorescence in order to identify and quantify fluorescent molecules (dyes).
Fluorescent dyes adsorb light at one energy level (or wavelength) and emit light at a lower energy level (or longer wavelength). The difference between the adsorbed 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 wavelength range will not cause the molecule to fluoresce. A filter fluorometer exploits this fact to identify one fluorescent molecule 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 photo detector 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 1 part per billion.
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The Synchronous Scanning Spectrofluorophotometer
In the mid 1980’s a new instrument became available that would eventually revolutionize fluorescence analysis along with dye tracing methodologies. The synchronously scanning spectrofluorophotometer (SSS), while still exploiting the same principles of fluorescence as the filter fluorometer, did not need special filters to remove unwanted wavelengths of light from the excitation beam. Instead it used a series of motorized mirrors, lenses, and thin slits to produce the precise wavelength range required for a particular molecule to fluoresce. It also used the same technique to measure the wavelength of the beam of light emitted from the sample. Another unique feature of this instrument was its ability measure the wavelength of both the excitation and emission spectra thanks to its dual-monocromator design. With this design, the following types of analyses could be performed:
Spectrofluorophotometer
1. Fixed excitation/fixed emission analysis–For this type of analysis, the excitation beam can be set to a particular wavelength and the monocromator can be set to read the intensity emitted light from the sample that has a particular wavelength. This type of analysis is the same type performed on a fluorometer, just without the filters.
2. Fixed excitation/variable emission analysis–For this type of analysis, the excitation beam is set to a particular wavelength and the emission monocromator is set to scan through a defined wavelength range measuring the intensity of any light emitted from the sample. This type of analysis produces a plot of fluorescence intensity vs. emission wavelength for that particular excitation wavelength. This plot allowed the user to determine what the maximum (peak) fluorescence intensity was for a particular excitation wavelength. Fluorometers cannot perform this type of analysis.
3. Variable excitation/fixed emission analysis–For this type of analysis, the excitation beam (being controlled by the second monocromator) is set to scan through a defined wavelength range and the emission monocromator is set to measure the intensity of any light emitted from the sample that is of a particular wavelength. This type of analysis produces a plot of fluorescence intensity vs. excitation wavelength for that particular emission wavelength. This plot allowed the user to determine what the maximum (peak) fluorescence intensity was for a particular emission wavelength. Fluorometers cannot perform this type of analysis.
4. Variable excitation/variable emission analysis (called synchronous scanning)–For this type of analysis, both the excitation and emission monocromators are programmed to scan through a defined wavelength range. In order to keep the emission monocromator from detecting scattered light from the excitation beam, the monocromators are typically programmed to scan slightly different wavelength ranges. Since fluorescent molecules typically absorb light at a lower wavelength range than they emit, the excitation monocromator is programmed to start its scan at a lower wavelength than the emission monocromator. This type of analysis produces a plot of fluorescence intensity vs. emission wavelength which is very different from the plot produced by a fixed excitation/variable emission analysis. This is the best type of analysis for identifying fluorescent molecules in an unknown sample. Fluorometers cannot perform this type of analysis.
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References:
Kass, Werner. 1998. Tracing Technique in Geohydrology. English Translation: A. A. Balkema, Rotterdam, Netherlands. 581 pages.