Chromatography Forum

Hi,

I was wondering if someone could help me please? I use a GCMS to identify fatty acid and hydroxy fatty acid (usually 14 to 18 carbon chain lengths) products produced in biological assays. I esterify the fatty acids using 3% sulphuric acid in methanol and also TMS derivatise the samples to produce the methyl ester and identify the hydroxy fatty acid. I have been doing this for the last 2 years with some success. However, recently I can't even identify my fatty acids on the GCMS. New columns have been used, but I can't even get a peak with my standard. Does anyone have any ideas why this is?

I use 7890A GC system (Agilent Technologies), 5975C mass spectrometer (Agilent Technologies) and a DB-5MS fused silica column with a 30m x 0.25mm x 0.25μm film thickness (J & W Scientific). Samples were injected in 1μl volumes in the splitless mode using helium as the carrier gas.

Any ideas and/or comments will be greatly appreciated.

Many thanks,
Claire

First - How does the tune look and are you able to get a chromatogram of some other compunds that do not need to be derivatized to be run? (Let's see if we can put the problem with either the instrument or with the samples the samples.)

Hi. Thanks.

Sterols are also run (these are TMS derivatised), but these run fine.

Get a few grams of plain old bar soap (not synthetic bar like Dove), stir/dissolve in your sulfuric acid/methanol, then heat a couple of minutes, cool, extract the fatty acid methyl esters and use as a readily available standard.

You'll get saturated FAME even numbered from C8 to C18, M/Z at 74 and 87 recognizable of GCMS. With a DB-5 type column or DB-1, the oleic acid methyl ester will elute before the C18 FAME, and the linoleic FAME before that. You'll also see a little palmitoleic, before the palmitic FAME (C16).

Thanks. I'll give that a try.

I have not dealt with fatty acids but have run other analysis using methylization and if you do not get rid of all the sulfuric acid it can cause problems, especially if it gets on the column. Can you do a base catalyzed methylization using tetrabutyl ammonium hydroxide?

Basic conditions do not fully derivatise the sample in my experience, but I have tried that to no available. I have got a second person to also try the acid derivatisation and they got the same result.

Try the bar of soap in the sulfuric methanol, no TMS. After heating a few minutes in a volumetric flask, cool to room temperature, add 10 ml hexane, swirl, add some saturated sodium chloride to force a separation, inject like 0.5 microliters of upper hexane layer.