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Bulletin 103
Fluorescein
Overview
Fluorescein was the first fluorescent dye
used for water tracing work (1) and is still used for qualitative (visual)
studies of underground contamination of wells. In recent years, Rhodamine
WT has almost completely replaced fluorescein for flow measurements (2)
and circulation, dispersion, and plume studies (3). Nonetheless, Fluorescein
has a role in such studies, and can be used for masking, hydraulic model
studies, and underground studies.
Advantages
Fluorescein has the following advantages
over other tracer dyes:
- Its low sorption rate is far better
than Rhodamine B, and comparable to Rhodamine WT.
- It has a temperature coefficient of
only -0.36%C, about one-eighth of the temperature coefficient of Rhodamine
dyes (2,4).
- It emits a brilliant green fluorescence,
which gives an excellent visual or photographic contrast against the
backgrounds normally encountered in water transport studies. Therefore
it is easy to visualize the progress of an experiment.
- It is more aesthetic than the red dyes.
This is psychologically important, especially in ocean areas subject
to the explosive growth of certain dinoflagellates, called “red
tide.” Less public resistance will be encountered using a dye that
does not resemble red tide (5).
Disadvantages
Fluorescein has been replaced by other
dyes, principally Rhodamine WT, for the following reasons:
- It is rapidly destroyed by sunlight.
Reference 4 reports that a 50% loss occurred in three hours of sunlight
exposure, with dye being held in an Erlenmeyer flask. Other tests in
an flat, uncovered Pyrex dish showed an almost complete destruction
in two hours (6).
- Many naturally occurring fluorescent
materials have similar characteristics and thus interfere with measurement.
When carefully chosen optical filters are used, the situation is better
than that reported in Reference 4, but higher concentrations are required
to overcome the effect of higher and more variable "blank"
fluorescence.
- Fluorescein is more pH-sensitive than
Rhodamine dyes. Fluorescence drops very sharply at pH values below 5.5.
For optimum results, pH should be between 6 and 10.
Masking Techniques
In river, harbor, and ocean tests, Fluorescein
can be used to mask the objectionable color of the Rhodamine dyes. Tests
show that Fluorescein is an effective mask, subject to the following conditions
(6):
- The concentration of Fluorescein should
be at least five times that of the active ingredients in the Rhodamine
B or Rhodamine WT concentrate.
- Where the receiving water is shallow,
clear, and in full sunlight, the dyes must be dispersed quite rapidly.
With slow dispersion, the photosensitive Fluorescein will be destroyed
before the masking effect is complete.
- Masking can be very subjective. Lower
(hence less costly) amounts of fluorescein may be effective, depending
on water clarity, bottom color, wave action, etc. Small scale addition
of the mixed dyes to the receiving water should be made in advance of
a large scale test. This test should be made on a bright sunny day,
if possible.
- Note that the Fluorescein is not the
ingredient measured, but acts only as a mask. The optical filter and
light source in the fluorometer are selected for the Rhodamine dye (7).
Model Studies
Fluorescein may be used in hydraulic model
studies in exactly the same way that Rhodamine WT is used (See Refs. 2
and 3 for details).
The major advantage of using Fluorescein
is its visibility, allowing visualization as the test proceeds. The major
disadvantage is Fluorescein’s light sensitivity. It can be destroyed
by light entering the test area, both from windows and from indoor lights,
especially fluorescent ones.
Containers used for dye destruction tests
must be transparent to light at shorter wavelengths. Clear borosilicate
glass baking pans are handy, since they transmit light at shorter wavelengths
than window glass or the glass envelopes of fluorescent lamps.
Test samples must be at low concentrations
(around 0.2 PPM) so that the Fluorescein in the bottom of the pan is not
protected from the incident light by absorption of the Fluorescein in
the top of the pan.
In certain cases, deliberate destruction
of the Fluorescein by sunlight may be a convenience instead of a problem.
Hydraulic models often recycle water. With the very stable Rhodamine WT,
the concentration of dye in the entire system will build up over a sequence
of several tests, requiring replacement of the water. If a shallow holding
tank can be placed outdoors, the degradation of Fluorescein by sunlight
may eliminate the need to replace the water.
Underground Studies
Fluorescein can be used quantitatively
for underground tests, subject to limitations imposed by the higher background
of naturally occurring fluorescent materials.
An advantage of fluorescein in underground
studies is its light sensitivity. Should it reach an open receiving body
of water, the color will be less of a problem because it will disappear
rapidly in the sunlight.
Filter and Light Source Selection
Using Fluorescein, the following light
sources and filters are recommended (referenced part numbers are specific
to Turner Designs products):
We have found that background fluorescence
can be very high in natural systems. If you cannot suppress background
fluorescence and the sensitivity knob is turned full counterclockwise,
then a mask may be added to the excitation filter holder to reduce its
effective diameter. Attenuation by a factor of about five can be attained
with our 10-318 Attenuator Plate.
Fluorescein, known as “Acid Yellow
73”, “Acid Yellow T”, “DNC Yellow 7”, etc., can
be obtained from the following sources (addresses checked and confirmed
June 1996):
Pylam Products Company, Inc.
1001 Stewart Avenue
Garden City, NY 11530
516/222-1750
Tricon Colors, Inc.
16 Leliarts Lane
Elmwood Park, NJ 07407
201/794-3800
Lissamine FF
The properties of uses of Lissamine FF
are reported in Reference 9. Its spectral characteristics are similar
to those of Fluorescein, but it does not decompose as rapidly in sunlight.
Use the Fluorescein filters detailed above with Lissamine FF. Pylam Products
(address shown above) offers Lissamine FF as “Brilliant Acid Yellow
8G” or “Brilliant Sulphoflavine FFA”.
References
- Dole, R. B., Use of Fluorescein in the
Study of Underground Waters, USGS Water Supply Paper 160, 73-85 (1906).
- A Practical Guide to Flow Measurement,
monograph by Turner Designs, 845 W. Maude Avenue, Sunnyvale, CA 94085.
- Circulation, Dispersion, and Plume Studies,
monograph by Turner Designs, 845 W. Maude Avenue, Sunnyvale, CA 94085.
- Feuerstein, D.L., Sellick, R.E., Fluorescent
Tracers for Dispersion Measurements, Journal of Sanitary Engineering,
ASCE 89 (SA4), 1-21 (1963).
- Murakami, Ken, Water Quality Section,
Water Quality Control Division, Public Works Research Institute, 5-41-7,
Shimo, Kita-Ku, Tokyo, 115, personal communication.
- Turner Designs Laboratory Tests conducted
July 23, 1975.
- “Filter Selection Guide” for
Turner Designs Fluorometers, by Turner Designs, 845 W. Maude Avenue,
Sunnyvale, CA 94085.
- Smart, P.L., Laidlaw, I.M.S., An Evaluation
of Some Fluorescent Dyes for Water Tracing, Water Resources Research,
13 (1), 15-33 (1977).
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