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Calibration Standards
Rhodamine WT dye solution must be used when completing a 2-point calibration. Purchase Rhodamine WT as a 2.5% solution to
follow the procedure below. Kingscote Chemicals (Miamisburg, OH, 1-800-394-0678) has historically had a 2.5% solution (item
#106023) that works well with this procedure. Note that there are many types of Rhodamine—make sure Rhodamine
WT
is selected.
If a 2.5% solution cannot be obtained commercially, prepare it from a solid or from another concentration of a liquid solution to a
2.5% final concentration, or adjust the dilutions below accordingly. It should be stored in the refrigerator when not in use.
For PC and chlorophyll channel calibrations, a 0.625 mg/L solution of Rhodamine WT should be prepared. For PE channel
calibration, a 0.025 mg/L solution of Rhodamine WT should be prepared. The steps below describe one procedure to prepare these
solutions.
1.
For any TAL sensor calibration, prepare a 125 mg/L solution of Rhodamine WT.
Transfer 5.0 mL of the 2.5% Rhodamine WT
solution into a 1000 mL volumetric flask. Fill the flask to the volumetric mark with deionized or distilled water and mix well to
produce a solution that is approximately 125 mg/L of Rhodamine WT. Transfer to a storage bottle and retain it for future use.
*This solution can be stored in the refrigerator (4°C). Its degradation will depend upon light exposure and
repeated warming cycles, but solutions used 1-2 times a year can be stored for up to two years. Users should
implement their own procedures to safeguard against degradation.
2.
For PC and chlorophyll channel calibrations, prepare a 0.625 mg/L solution of Rhodamine WT.
Transfer 5.0 mL of the 125
mg/L solution prepared in step one into a 1000 mL volumetric flask. Fill the flask to the volumetric mark with deionized
or distilled water. Mix well to obtain a solution that is 0.625 mg/L of Rhodamine WT. Use this solution within 24 hours of
preparation and discard it after use.
3.
For PE channel calibration, prepare a 0.025 mg/L solution of Rhodamine WT.
Transfer 0.2 mL of the 125 mg/L solution
prepared in step one into a 1000 mL volumetric flask. Fill the flask to the volumetric mark with deionized or distilled water.
Mix well to obtain a solution that is 0.025 mg/L of Rhodamine WT. Use this solution within 24 hours of preparation and
discard it after use.
In addition to preparing the Rhodamine solution(s), it is also necessary to determine temperature-compensated calibration values
for solutions. In general, fluorescence is inversely related with temperature. Measure the temperature of the Rhodamine solution(s)
and use the temperature of the solution at the time of calibration to select the compensated solution concentrations, in either RFU
(recommended) or µg/L pigment equivalents, from the table below.
As an example, assume that you will calibrate the chlorophyll channel in RFU, and that the temperature measured in the 0.625 mg/L
Rhodamine WT solution is 22°C. The first standard value entered will be 0, and the second standard value will be 16.4 (
). Likewise, if you intend to use the default µg/L unit when calibrating chlorophyll, the second standard value would
be 66 in this example. Using the same 0.625 mg/L Rhodamine WT solution to calibrate the PC channel will yield a second standard
value of 16.0 RFU or 16 µg/L. These values will be entered when performing a 2-point calibration.
Sensors, Calibration, and Maintenance