I am trying to prepare Hutner's trace elements solution, but I keep having issues with formation of insoluble complexes (of EDTA with one or more of the metals in the solution, probably iron, I think).
I first completely dissolve EDTA (disodium salt dihydrate) in MQ water by stirring + heating and keeping the pH at 8 by adding KOH pellets. Then I prepare the individual trace element solutions.
I mix all of the trace element solutions together and heat the mixture to 40-50°C, after which I slowly add the EDTA solution. When I add the EDTA solution, however, the mixture turns dark green (almost black) instead of the light green that it should be. At this point, the pH is very acidic (around 2.3). When I try to adjust the pH to 6.5 with KOH, a brownish precipitate starts to form.
Could the issue be that the iron sulphate is perhaps not fully dissolved enough, therefore forming insoluble complexes with EDTA? Or could the temperature be the issue and should I re-heat the EDTA to 50°C before adding? Or should I perhaps only adjust the pH later on?
Many thanks for any input!
I thought the problem might be the pH is actually precipitating Fe:
From the Michigan University Extension website: https://www.canr.msu.edu/news/selecting_which_iron_chelate_to_use
I think the pH8 might be too high to chelate the Fe.
On re-reading your protocol, are you adding all of the trace elements to the EDTA? That might be the problem, you might have to prepare [Fe + EDTA] separately from [all other Trace elements].
Also, check that you have the right molar mass to chelate the iron. I think you were effectively chelating everything and there was too much of something, leading to a black precipitate.
Elsewhere I found the following protocols:
Cold Spring Harbor protocol:https://cshprotocols.cshlp.org/content/2006/1/pdb.rec8030.full?sid=7e38ebd1-ed55-4287-8aba-8bda25966e5d
Merck website:
https://www.sigmaaldrich.com/AU/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/plant-tissue-culture/iron-chelate-solution
THis undergrad experiment may not be what you need, but it might help with dissolving things in the right order: https://cuhk.edu.hk/chem/doc/s6_resourcebk/en-s_expt_06.pdf
Best of luck.
Hi Matthew, this information helps a lot, thank you so much for your answer!
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