I have aqueous samples containing (amongst other things) a mixture of C16-18 fatty acid methyl esters (FAMEs) which I extract using NaCl (7.5 g per 25 mL sample) and ethyl acetate. Other solvents such as DCM and hexane have failed to give me sufficient recoveries, I have already tried. Only ethyl acetate works well in this particular case. Analysis is on an FFAP collum with GC-FID detection.
The problem is that the FAMEs degrade in my aqeous samples over the course of the test I'm performing. I therefore also need to analyse the free fatty acid. My GC-FID method of analysis only works for the FAMEs though, which means I need to derivatize the free fatty acid back to its methylated form.
Is there a simple derivatization method that works in ethyl acetate? The derivatization procedures that I have found so far say I have to use a non-polar solvent such as hexane or toluene, and I would like to avoid having to do extra preparative work by evaporating my ethyl acetate extracts first. Ideally I would like to add the derivatization agent directly to my ethyl acetate extracts (maybe with a heating step), deactivate the excess reagent, and analyse directly with my GC-FID method.
Many thanks for your help
Check though these methods http://lipidlibrary.aocs.org/Analysis/content.cfm?ItemNumber=40374
Check methods at Restek and the AOCS. Methanol is the most common solvent but I think yes (thus FAMEs). However in your case, you will find the most common fatty acid ester is an ethyl ester.
There is likely to be a problem with extraction of FFAs into ethyl acetate from water, unless you lower the pH, which will accelerate decomposition of the FAMEs. Are you interested in whether each acid is in its free or ester form, or are you going to sum them ?
Ethyl acetate is an ethyl ester. If you try to make methyl esters in this solvent you will get at least some if not total ester exchange and end up with fatty acid ethyl esters.
If the hydrolysis occurs before extraction with ethyl acetate (EtOAc), the free fatty acid may not be extracted well with EtOAc as pointed out by Peter. If this is correct, there is no point for delivertising the EtOAc extracts that may have only partially recovered free fatty acids.
Dear all
Thank you for your help.
I am interested in analysing just one of the FAMEs that are present in the mixture that I'm testing. I am looking at a C18 FAME. Because the carbon chain is so long even the free acid should be very insoluble in water and preferentially partition into my organic phase. Furthermore, because I have added a lot of salt to the aqeous phase I am going to assume the free acid has also been quantitatively extracted into the organic phase.
So let us assume that the free acid is also present in the ethyl acetate extract. My question is, does derivatization also work in this solvent, or does it have to be a non-polar solvent such as hexane (which is what the methods I have found called for). One of the standard methods I have found uses a BCl3-methanol or BF3-methanol derivatizazion reagent in hexane, heptane, or toluene. This mixture is then heated for a few minutes, after which water is added (I assume to quench the excess reagent? Can it also be quenched with something other than water?).
It seems like you might want to do some reading about esterification, ester exchange, and the physical organic chemistry involved. IMHO, Ethyl Acetate is not an optimum choice to begin with.
See my answer above. You will not get pure FAMES in this solvent there will be at least some ethyl esters. BF3 is acidic enough to cause ester exchange.
Dear Phil, listen to what John Peter Bartley is saying. As long as you have ethyl acetate, it will give you only problems. I think the best method is drying samples under vacuum to eliminate all solvent and then derivatize with BF3/MeOH
It all depends on how much you can experiment with this. If you have the time to tweak your method a little, there may be some options. I am not sure why EtOAc should be the only solvent... how about the common isopropyl acetate which is much less reactive, or even something like 2-methyl-THF which is outstanding for extractions of quite polar organic molecules into the org phase? Also, can your samples be manipulated/stored at low temperature to prevent the hydrolysis?
Unfortunately I have only limited resources to experiment with this project. Ethyl acetate was the first solvent that showed good recoveries for FAMEs during my extraction method development, so that's what we used when it was time to start testing the first analytical samples.
The samples (the unextracted and the extracted ones) can easily be stored in the fridge or freezer.
Thank you for the tip with 2-methyl-THF! I'll definitely keep that one in mind, also for future projects!
What is the pH of the aqueous solution you extracting your "FAME" from ? I doubt the methyl ester hydrolysis will occur in ethyl acetate after extraction (ethyl acetate will be hydrolyzed as well). Trans-esterification is more than likely, especially if you have traces of acid or base present in the extract.
Vladimir,
I understand where you are coming from, acidic or basic media should obviously be ecluded. However, methyl esters are beasts in that some of them are nonlinearly more prone to hydrolysis than you would otherwise expect, say from the series sec-butly->isopropyl->propyl>ethyl->methyl. Acidity of the vial glass wall :-) may be sufficient to induce hydrolysis. However, if you work relatively fast before the assay, and at lower temps, you may get good results. Again, it may depend on the case. Some solvents may be better here than others, too.
Jacek, thanks for your comments. I agree, methyl esters are much easier to undergo hydrolysis, and this might easily be one of the reasons for the problem we see.
At the same time, I think there could be many other factors involved; for example, why would CH2Cl2 extract C16 FAME with lesser efficiency then ethyl acetate ? Perhaps there are other questions to the whole isolation procedure, and we don't have the full picture.
Sometimes emulsions can cause a problem with a specific solvent. Testing various options may eventually give a good solution.
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