Turner Designs AquaFluor, Manual

Page: 23 / 23

Aqua Fluor
®
U
ser’s Manual
23
Appendix B: Linear Range, Quenching and Temperature Considerations
The linear range is the concentration range in which the readout of the Aqua Fluor
®
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.
Figure 1. Linearity and Calibration Response Curve
Temperature Considerations
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.