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46
Calibration
Rhodamine WT Dye Solution Preparation
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. 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 liquid solution to a 2.5% final
concentration, or adjust the dilutions below accordingly. Kingscote Chemicals (Miamisburg, OH, 1-800-394-0678)
has historically had a 2.5% solution (item #106023) that works well with this procedure. 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 how 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 (
see table on page 47 ). 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.