Chromatography Forum

Hi, javascript:emoticon(':D')

I am new in this forum, so I apologize if I this is a repetitive question.

I have been trying to elute free long chain fatty acids (C10, C12, C14, C15 (internal standard), C16, C18, C18:1, C18:2) in my HP 5890 II with some moderate success. My standards are these FFA in a DCM solution (plus a 1:1 solution of HCl:1-propanol) in concentrations that go from 25 to 1000 mg/L.

My problem is that I only can get peaks the first 2 or 3 samples/standards, but after that the peaks do not elute, and I get a flat line for the next standards. It seems that does not depend on the concentration because it happens regardless of the order of the samples.

I have tried everything, and I am pretty sure I don't have any leaking in the system. I also tried both of my injectors, i.e. split/splitless capillar and packed, but I saw the same response.

If anybody have any idea of what's going on here, or if you have a recommendation, please let me know!

Thanks so much!

Rodrigo

Acids + alcohol in the presence of hydrochloric acid = esters ?

Peter

I've worked with fatty acids for 30 years, and we make into fatty acid methyl esters, the industry standard. I've never had much "luck" with assaying these length fatty acids as the acids.

Like Peter stated, fatty acids and your 1-propanol will esterify, even at room temperature - are you intentionally "trying" to esterify them? If so, I'd choose methyl esters (but I've also done ethyl, n-propyl, isopropyl, and n-butyl a few times). If not, ditch the alcohol. You need your fatty acids to be one way or the other, not a combination.

What exactly is your sample (matrix), and what are you trying to do? Most likely someone else has done exactly that. By the way, we also use a 5890 Series II for this assay (fatty acid methyl esters), use dilute sulfuric acid in methanol to esterify, the extract in petroleum ether. Injecting HCl into the GC like you're doing is not a good idea....

Thanks for the replies... my sample comes from an anaerobic digester - my goal is to quantify hydrolysis of neutral lipids in to FFAs.

I found a couple of papers with a similar approach for FFA GC analysis - one using sulfuric acid:solvent, and the other with HCl:solvent. I was trying to avoid inorganic acids, of course, but at the same time, since I am injecting only the organic phase, I thought that that would be ok (for the column).

In any case, I would be open to hear about any other methods that could work out for quantifying FFAs. I considered the FAME option at the beginning, but I was not sure if that would have worked for my objective. Hope you can enlighten me on this. Thanks!

Rodrigo

If you have a mix of Fatty Acids where some are free acids and some are connected to glycerol(mono-, di, or tri-) or alcohols you can measure the two groups by selectively esterifying the non-free acids by first removing water from the sample, then reacting the sample with a Tetramethylammonium hydroxide solution with ethyl ether. After extracting the FAMEs with hexane and NaCl(aq) you can then remove the FA salts from the aqueous layer and esterify using a normal esterification procedure.

Sigma technical service can assist you with the proper reagents.

Rodney George
consultant USA

Thanks Rodney and all for your replies!

I am not an expert at all on this, and I really appreciate everybody's time to reply to my questions. In fact a have a couple more

Rodney: do you think I could apply the method you recommend to quantify both FFA and bound-FA in my sample - that is, could I distinguish between natural occurring FFA (from microbiological hydrolysis) and FA in neutral lipids? Also, would this work for my samples, which consist of anaerobic digester sludge?

To all: since I don't know pretty much anything about FAMEs, is there any particular document/paper I could read to learn and conduct FAMEs for liquid and solid samples?

Thanks, and please let me know if anybody else has any other suggestions to analyse my samples. Thanks again!

Rodrigo

Rodrigo,

Here are a couple of very useful web sites that cover all aspects of fatty acids.

http://lipidlibrary.aocs.org/

http://www.cyberlipid.org/index.htm

I hope these are helpful.