Calibrating the chlorophyll sensor

The Cyclops-7F™ chlorophyll sensor has two settings that can be optimized for pCO₂ measurements: the gain and calibration. The gain is a software setting that sets the sensitivity of the sensor. The user calibration may be needed periodically to ensure it is reporting accurate measurements.

Setting the gain

The sensor has three gain settings (X1, X10, and X100), which refer to the sensitivity configuration of the sensor. As the gain increases, the sensitivity increases and the concentration range decreases. The X10 gain is for samples representing typical conditions. It is desirable to obtain a signal from the sample that is significantly higher than a blank sample (deionized water or filtered aged offshore seawater), but not a signal that is close to the maximum of 5 volts. If the sample signal is high, use the X1 gain to avoid going over scale. If the sample signal is very low, the X100 gain setting may be used to achieve higher sensitivity but a smaller measurable range.

If the gain is set to auto-select, the program will determine the best gain and its corresponding equation. The approximate coefficients are displayed.

Note: At least one chlorophyll standard should be used to calibrate the chlorophyll sensor. Turner Designs also supplies a matching solid-state secondary standard, which can be used in place of a primary liquid standard once a correlation between a primary standard and the solid standard is established. This solid standard can be used to check stability and/or check for loss in sensitivity.

User calibration

The Cyclops-7F sensor has been calibrated using the standard solution (Turner Designs part number 2100-320) following the procedure recommended by the manufacturer. However, the standard solution based on rhodamine is only accurate to ±30%. It is best to create calibration curves using the expected algal groups and concentration with the probe installed in the pCO₂ system, which would eliminate a constant offset caused by the deviation in the distance between the container and the sensor from that shown in Figure A‑1.

1. Remove the Cyclops-7F sensor from the pCO₂ system but keep the cable connected.

2. Install the sensor over a non-florescent container, such as a 1-L glass beaker placed on a non-reflective black surface (Figure A‑1) so the sensor is suspended above the bottom.

   Do not use plastic containers, as they may interfere with the sample fluorescence. Leave the sensor submerged in DI water. Ensure that the sensor is more than 7.5 cm (3 inches) above the bottom of the container and that the sensor is in the center of the container and has more than 5 cm (2 inches) clearance between the circumference of the sensor and the beaker.

3. Connect the pCO₂ program to the pCO₂ system.

4. In the Manual DAQ tab, select the Omega modules and the chlorophyll sensor.

5. Choose the X1 gain setting.

6. Start continuous measurements and the readings should update in the table.

7. Read the fluorescence as a voltage in the pCO₂ program.

8. Repeat step 5 to 7 for gains X10 and X100.

9. Change the DI water to the chlorophyll standard solution (40 µg/L) and repeat steps 5 to 8.

   Note: The standard solution from Turner has a concentration of 40 µg/L. The gain of X100 only works for concentrations up to 5 µg/L. Use the X10 scale for this solution.

   After obtaining both voltage outputs for the blank and the standard for gain X1 and X10, a correlation between the chlorophyll concentration and the voltage output has been established. The chlorophyll concentration at gain X1 and X10 is calculated following:

   A‑1

   or

   A‑2

   where C stands for concentration, and Volt stands for the voltage output. We define:

   A‑3

   and

   A‑4

   then we have

   A‑5

   The gain X100 only works for concentration 0 to 5 μg/L and needs to scale from the readings at the gain X10. The chlorophyll concentration at gain X100 is calculated following:

   A‑6

   where the subscripts denote the gain setting and the material where the voltage is read. Similarly,

   A‑7

   and

   A‑8

   gives

   A‑9

10. For each gain of X1, X10, and X100, calculate a and b based on the voltage outputs in DI water and the standard solution and input them in the parameter window in the Test Config tab.

In practice, these caveats should be noted.

• The Cyclops-7F sensor is linear up to 500 μg/L at X1 gain, 50 μg/L at X10 gain, and 5 μg/L at X100 gain. Beyond these limits, a quenching effect is observed. As the concentration increases above the respective values, the light emitted by the sensor gets attenuated by the algae, which causes the measured concentration to decrease.
• When the Cyclops-7F sensor is installed in the pCO₂ system, it is less than 3 inches from the bottom of the plate. Therefore, the chlorophyll concentration reading is not 0 when DI water is measured. This is caused by the constant offset in the voltage. This offset should be subtracted from the voltage readings in the samples.

An optional secondary solid standard, not included in the package, is available from Turner Designs (part number 2100-900). It can be used in place of the liquid standard after the correlation between the two kinds of standard is established. It also can be used to check the stability and/or check for loss in sensitivity.