Hi Dear Friends,
I want to ask a question about metabolite extraction for HPLC. My sample is a plant cell suspension culture. I freeze dried whole culture (22,5 ml/Tube). But now I have a huge content of salt within other ingredients. Filtration of media from cells is not an option because my metabolite also exists in media. So what should I do? I have to get rid of salt (only salt) but protocols which I know for extraction does not consist about desalting.
Thanks,
Dr. Deniz KOM
Dear colleague,
Have you tried solidphase extraction (e.g. with Seppak C18) before the freeze drying.
If your metabolites of interest are partly hydrophylic this could work. If they are weak acids you should adjust the pH of your sample to a value at least 1.5 pH unit below the components pKa to be able to extract them. The same way vice versa if your metabolites are weakly basic components.
The salt will be in the effluent and by eluting the SepPak with a proper solvent (buffered water/methanol for partly hydophobic constituents, neutral low ionic strenght buffer for weak acids and bases) you will have isolated your metabolites of interest.
Good luck
Hi Friend,
I have the same problem, and it was easily solved. Generally plant metabolites has good solubility in methanol. Additionally, salt cannot be solved in methanol. By adding methanol or acetonitrile you can easily separates your metabolites.
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Dear Wielders, I don't have any SPE instruments or equipments right now and aim of the project is lowering the budget for metabolite production and exraction. So I think project manager will not supply it. Thanks for precious tip. If I can persuade pm, I will try that way.
Dear Yahyazadeh, after dissolving methanol how should i proceed? in each protocol that I read they offer filtering with 0,45 um filter. But I think cells in suspension can cause blockage on filter.
Generally, all metabolites are soluble in methanol and acetonitrile except salt. By adding the solvent to the residue of freeze drier, all of metabolites are solved and then with centrifugation you can separate upper phase from lower phase that is salt by pipetting.
Try using a reverse phase column. It doesn't need to be SPE, and it could be loose C18. I had a similar problem, removing DMSO from a plant extract. A small column did the trick.
this link shows how I did it: http://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Application%20Notes/Removal%20of%20Non-volatile%20Solvents%20with%20RediSep%20Rf%20Gold%20C18Aq%20Columns%20App%20Note.pdf#search=c18Aq
This also works for fairly water soluble compounds- here, I removed phosphate salt from elution form ion exchange using reverse phase: http://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Application%20Notes/Desalting%20Samples%20with%20RediSep%20Rf%20Gold%20C18Aq%20Columns%20App%20Note.pdf#search=c18Aq
There is no reason why, after washing with water, a gradient couldn't be run instead of a step to start purifying the extract.
Jack's suggestion is based on the same principleas using SPE C18 cartridges, I guess cartridges are much cheaper and easier to handle.
Yes, it uses the same principles of SPE, but as people may not have dedicated SPE cartridges in their lab, I suggested the use of columns or packing that they may already possess. If they have loose C18 or CHP-20 around, that can be packed into a small column and used for desalting as well.
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