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Principles of Operation Section 5
YSI Incorporated Environmental Monitoring Systems Manual 5-30
RHODAMINE WT STANDARD SOLUTION – PREPARATION AND USE
CAUTION
: Before using concentrated Rhodamine WT solution to prepare standards, be certain to
read the safety instructions provided by the supplier with this chemical. Remember that only trained
personnel should handle chemicals.
PREPARATION
Use the following procedure to prepare a 100 ug/L solution that can be used to calibrate your Rhodamine WT
sensor for field use:
1. We recommend that Rhodamine WT concentrate be purchased from Keystone Aniline Corporation, 2501
W. Fulton Street, Chicago, IL 60612 (Telephone: 312-666-2015) under the name KEYACID
RHODAMINE WT LIQUID (Part # 70301027). As purchased, the solution is approximately 20 %
Rhodamine WT by weight, i.e., 200 g/L.
2. Accurately weigh 0.500 g of the 20 % Rhodamine concentrate, quantitatively transfer the viscous liquid
to a 1000-mL volumetric flask and fill the flask to the top graduation. This solution contains 100 mg of
Rhodamine WT per 1000 mL of water.
3. Accurately transfer 1.0 mL of the solution prepared in the above step to a 1000 mL volumetric and then
fill the flask to the top graduation with purified water. Mix well to obtain a solution that is 100 ug/L
(0.10 mg/L) in water (a 1000:1 dilution of the concentrated solution).
4. Store the concentrated standard solution in a darkened glass bottle in a refrigerator to retard
decomposition. The dilute standard prepared in the previous step should be used within 5 days of its
preparation.
When Rhodamine WT standards are required in the future, perform another dilution of the concentrated dye
solution after warming it to ambient temperature. Our experience has indicated that the concentrated solution
that has been kept at cold temperatures is much more stable than the dilute solution stored at room
temperature.
EFFECT OF TURBIDITY ON RHODAMINE WT READINGS
As described above, the filters in front of the photodiode in the YSI 6130 Rhodamine probe prevent most of
the green light which is used to excite the Rhodamine molecules from reaching the detector after being
backscattered off of non-fluorescent particles (turbidity) in environmental water. However, the filter system
is not perfect and a minor interference on Rhodamine WT readings from suspended solids may result.
Laboratory experiments indicate that a suspension of typical soil measured with a turbidity sensor will have
turbidity interference characterized by a factor of about 0.03 µg/L per NTU. For example, the turbidity of
the water must be above 100 NTU to produce an apparent Rhodamine WT reading equal to 3 µg/L. In
very cloudy water, the user may wish to use the independently-determined turbidity value and the above
compensation factor to correct measured chlorophyll values using, for example, a spreadsheet.
EFFECT OF CHLOROPHYLL ON RHODAMINE WT READINGS
While the green LED used in the Rhodamine WT sensor is not ideal for excitation of the chlorophyll in
phytoplankton, some fluorescence of environmental chlorophyll will always be induced by the Rhodamine
sensor. Because the filter system for the Rhodamine photodiode is not perfect in excluding chlorophyll
fluorescence, a minor interference on Rhodamine WT readings from phytoplankton may result. Laboratory