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I am trying out a new standard addition method and having some very basic problems. I am hoping someone could point me in the right direction.

I am using a fluorometric method to quantify ammonium in solution. The calibration curve is produced using the standard addition method, in which known amounts of ammonium chloride is spiked into collection tubes containing the unknown test solution and a fluorometric reagent. I am generating an evenly spaced 6-point calibration curve using this method, with an R2 of 0.99.

When using purified water, values are close to zero, as they should be. I am also getting appropriate values in the natural/environmental samples I am testing. However I would like to assess accuracy. This is where the difficulty has been.

To test accuracy, I prepared a concentrated ammonium chloride stock solution in pure water, and used this to 1) create a diluted "dummy" solution to test the method with, and 2) a solution to be used for standard spikes. For some reason, I am having difficulty reproducing the original dummy concentration, with values that are 10-30% higher than expected (i.e., 3.3-3.7 uM instead of 3 uM).

I am using volumetric flasks, pipettes, and serological pipettes for precision, and acid rinsing all materials before use. I don't believe contamination is a problem since the blanks are very low. Because the measured concentrations are consistently too high, I think I could be losing ammonium to bottle adsorption, and fluorescence values are therefore inaccurately reflecting the standard concentrations (not accounting for the wall loss). However, this happens even after testing freshly made solutions, using Teflon bottles (instead of glass or polycarbonate which supposedly minimize adsorption), and switching to a salt water base in case ammonium behaves unpredictably in deionized water. I don't believe the fluorometer is the problem, since the curve is linearly increasing and only 5 years old.

I am concerned I am missing something basic. Please let me know if you have an ideas, and I am grateful for your thoughts.

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  • $\begingroup$ It seem you are doing everything carefully. The key question is are the standards and this dummy solution are prepared from the same ammonium chloride standard? Where do you get ammonium ion standards? $\endgroup$
    – ACR
    Commented Jul 1, 2021 at 12:44
  • $\begingroup$ If you were losing ammonium ion due to ion absorptio you would not be getting higher values but rather lower values. $\endgroup$
    – ACR
    Commented Jul 1, 2021 at 12:47
  • $\begingroup$ Yes, both the standards and dummy solution are made from the same concentrated stock, which was prepared by weighing out solid ammonium chloride powder. $\endgroup$
    – user112717
    Commented Jul 1, 2021 at 14:19
  • $\begingroup$ 1. Even if there was slight error in this molarity calculation, it should have affected the standards and dummy solution evenly, since they were prepped from the same solution. 2. Loss of ammonium in the standard solution (adsorption) would decrease fluorescence, but since original standard concentrations are used in the calibration curve, it would make the measured concentrations appear artificially high $\endgroup$
    – user112717
    Commented Jul 1, 2021 at 14:24
  • $\begingroup$ Could you provide a reference to your method. I mean the original protocol. Where was it published. $\endgroup$
    – ACR
    Commented Jul 1, 2021 at 17:50

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