The linear range is the concentration range in which the fluorometer’s output is directly proportional to the concentration of the signal. The linear range begins with the smallest detectable concentration and spans to an upper limit (concentration) that is dependent upon the properties of the material, filters used, and path length. A non-linear relationship is seen at very high concentrations where the signal does not increase at a constant rate in comparison to the change in concentration (see figure below). At even higher concentrations, the signal will decrease even though the sample concentrations are continuing to increase. This effect is known as “signal quenching.” Linearity can be checked by diluting a sample 1:1 or some other convenient ratio. If the sample is still in the linear range, the reading will decrease in direct proportion to the dilution. If the reading does not decrease in direct proportion to the dilution, or if the reading increases, the sample is beyond the linear range.

Fluorescence is temperature sensitive. As the temperature of the sample increases, the fluorescence decreases. For greater accuracy, record the sample temperature and correct the sensor output for changes in temperature. For further information on how temperature, light, water quality and the physiological state of the algal cells can all affect the measurement of chlorophyll, please refer to the application section of Turner Designs’ website.

The linear range is the concentration range in which the readout of the AquaFluor® is directly proportional to the concentration of the fluorophore in the sample. The linear range begins with the lowest detectable concentration and spans to an upper limit concentration that is dependent on the properties of the fluorescent material and the cuvette optical path length. For Rhodamine or Fluorescein dye in the 10 mm square cuvette, the upper limit of linearity is approximately 400 ppb (µg/L). At concentrations above this upper limit, the fluorescence reading will not increase at a linear rate in comparison to the change in concentration. At concentrations 10 times higher than the upper limit, the readings will start to decrease even though the sample concentration is increasing.

This effect is known as "sample quenching" and is due to light absorption losses in the sample. See the Figure 1 graph below. When you start to see visual color in the sample, this is an indicator that the sample may be above the upper limit of linearity. The linearity can be verified by diluting a sample 1:1 or another convenient dilution ratio. If the sample is within the linear range, the reading will decrease in direct proportion to the dilution. If the reading does not decrease in direct proportion to the dilution or if the reading increases, the original sample concentration was above the linear range.

Fluorescence is temperature sensitive. As the temperature of the sample increases, the fluorescence decreases. For best accuracy make sure your samples are at the same temperature as the calibration standard. If you have the ability to measure sample temperature, a temperature coefficient may be applied to your measurements, see Appendix A - Application Specifications table in the user manual: Aquafluor Handheld Fluorometer (turnerdesigns.com)