Linearity Fluorescence Detector

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

4 posts Page 1 of 1
Hi,

We are currently setting up qualification tests for Agilent HPLC equipment.
We also have a fluorescent detector in the system for which we would like to do a linearity test.
How should you perform the linearity test:
- What would be a suitable concentration range assuming we don't use a column?
- What is the response range (upper limit) for the fluorescence detector? (for example for UV detection this is ± 2 AU)
- What should be the acceptance criteria?

Is it correct that the linear range for a fluorescence detector is much smaller than for UV detection?

Regards,
Leon
Dear Leon, if you want to fill the detector’s cell with a series of solutions by e.g. syringe, you will only roughly estimate the linearity range.
If you need this for HPLC, keep in mind that detector may perform differently as it is a part of a complex system. Column bleeds or removes impurities from the flow that may quench the signal. However you should be able to estimate. We normally use range from ~½ LOD-LOD to upper limit. You keep going up and evaluate residuals as soon as non linearity starts you will catch this by statistical methods (available many). But after you established the upper point, I would recommend to go back for about 20% to be inside the range all the time. And again, from the practical point of view if your detector is linear up to 100 ppm but your aflatoxin samples are around 10 ppb most of the time, do not calibrate that high, use working range 0.1-1000 ppb.
"If your experiment needs statistics, you ought to have done a better experiment." Rutherford
Alexandre wrote:
Dear Leon, if you want to fill the detector’s cell with a series of solutions by e.g. syringe, you will only roughly estimate the linearity range.
If you need this for HPLC, keep in mind that detector may perform differently as it is a part of a complex system. Column bleeds or removes impurities from the flow that may quench the signal. However you should be able to estimate. We normally use range from ~½ LOD-LOD to upper limit. You keep going up and evaluate residuals as soon as non linearity starts you will catch this by statistical methods (available many). But after you established the upper point, I would recommend to go back for about 20% to be inside the range all the time. And again, from the practical point of view if your detector is linear up to 100 ppm but your aflatoxin samples are around 10 ppb most of the time, do not calibrate that high, use working range 0.1-1000 ppb.


Dear Alexandre,

thank you for you reply! We want to test the linearity for HPLC. Maybe we want to use a column, but it can also be that we use a restriction capillary.
I think it's a good idea to just test it as you suggest. Which component would you choose for the experiment? Vitamin B12?

Regards,
Leon
One tests the linearity of a fluorescence detector like any other type.
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