I´m working with an alpha proteobacteira. In specific growth condition I detected, using the CAS assay, production of a chelating agent. I performed the classical chemical test to identify the chemical features of the chelating agent and I obtained positive results with the Rioux test which is specific for cathecol siderophores. Subsequently I extracted from my spent medium the siderophore using ethyl acetate. I evaporate the solvend and I resuspend the compound in Acetonitrile 70%. Again the extract has an immediate positive CAS reaction. Afterwards I tried to isolate my compound via RP-HPLC. I detect several peak which have assorbance in the UV, but when I collect them none of them is CAS positive. Moreover none of the fraction I collect during the RP-HPLC run is CAS positive. How can this be possible? Do you have any explaination/adviase to manage to isolate the compound?
Have tried looking at your extract using NMR? It's possible your siderophore is unstable by nature and breaks down quickly. NMR might be able to capture that process.
Thank you very much for your answer. NMR is definetly something I would like to do, but I think I need a real clean compound for that. In my extract I´m sure I have several compounds and for this reason I would like to separate them, colelct the siderophore containing fraction and go then for NMR:
I had the same problem a few years ago. I was using RP-HPLC in the last step of the purification of the siderophore and lost activity. The problem is the stability of the compound with that column. I have solved the problem using special RP columns for highly polar compounds, such as Atlantis from Waters or Discovery from Aldrich-Sigma.
Hopefully this will solve the problem
You're either not collecting the siderophore following RPLC, chemically changing the siderophore during RPLC, or losing it due to irreversible adsorption.
If you have a visible detector, and assuming you're looking for an iron-binding siderophore, try adding some Fe (iii) to your sample prior to RPLC and monitoring the absorbance at about 505 nm, looking for the bound iron. I've found that the more selective detection is useful in figuring out which peak I'm interested in.
Be careful to collect the void peak too. If you're injecting a 70% ACN solution, it is possible that the desired material isn't retained, but is moving in the injection solvent.
Tell us more information about the RPLC conditions you're using - is it the typical acidic aqueous/ACN gradient on a C18 column? You can test the stability of your putative siderophore by putting the crude material in that solvent and testing for CAS activity as a function of time.
Good luck.
Thank all of you for the answers!
So I extract my siderophore with ethyl acetate. After removing the solvent I dissolve the extract in 70% ACN. I test the extract in ACN woth CAS and I have a really fast reaction, in about 5 min. After that I run the same sample in RP-HPLC; using a C18 Nucleodur-Isis, eluent A is 10%ACN ph 6, and eluent B is 70% ACN pH 6.
I assume you're running a gradient of some tens of minutes from A to B. Do you get a large void peak?
While the pKas of the catecholic phenols are higher than the pH 6.0 of your mobile phase, the acidity is different for the complexes than the neutral molecule.
And don't forget that the chromatographic process dilutes the eluting component.
Hello, you may use a gradient system up to 100 % acetonitrile, did you? Otherwise, if the concentration of the siderophore is only small you need to run your solvent before chromatography over a chelex 100 column in order to bind any ferric ions from the solvent.
Good luck Wolfram
May be you have an protein like compound, perhaps you better should use LH-20 for isolation, but I believe you have already isolated the compound?
Bye Wolfram
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