Chromatography Forum

I am going to start doing FAMES on my 5890. It seems everyone here has an old 5890 that they are using for FAMES. Anyways I found Consumer Product Guy's method on an old post. It looks like a very simple method I could use.

The procedure states you can acidify with either BF3 or H2SO4. Can I use HCl. I have a huge tank full of 11.4N HCl I inherited and BF3 is expensive. From what I understand I just need an excess acid?

Finally instead of a steam bath can I use a near boiling water bath?

viewtopic.php?f=3&t=18963&p=91319&hilit=BSTFA#p91319

I currently have an rtx-wax 30m .32mm .5um film. That should be good enough for a basic profile though when it dies I will replace it with one of the nice high cyanopropyl columns. Right now I am trying to get my boss to buy me a 1701 column for my capsaicinoids and amino acid methods and I doubt they'll spring for two columns right now.

There are many procedure for FAME analysis and HCl is commonly used. check out the following information.

http://lipidlibrary.aocs.org/GC_lipid/0 ... /index.htm

http://lipidlibrary.aocs.org/lit_surv/g ... t_meth.htm

http://www.cyberlipid.org/cyberlip/home0001.htm

Another complete reference:
Christie, W. W., "Preparation of ester derivatives of fatty acids for chromatographic analysis" in Advances in Lipid Methodology - Two, pp. 69-111 (1993) [Ed. W. W. Christie, Oily Press, Dundee, Scotland

All the information you'll need and more.

The presence of water is the big issue here.

If your acid is DRY, then HCl or H2SO4 make little difference, although the BF3 allows much FASTER esterification. 8-10 minutes at 100C in BF3/methanol will esterify 99.9% of the free acids with little decomposition of polyunsaturated fatty acids. A very little water can cause big losses in the esterification reaction.

Since analysts often do not purge the headspace in the reaction vessel with nitrogen when using HCl and H2SO4 reactions, this decomposition of PUFA does occur. These losses may affect your results as well as contaminate your injection liner and your column with partially oxidized junk.

Due to time is money issues, I have always used BF3/methanol (use only 0.5mL and add the extraction solvent (1mL of DCM or Toluene or 50/50 hexane-DCM) BEFORE heating the reaction mixture @ 110C for 6-12 minutes, cooling to ambient and extracting with 7mL of saturated NaCl (aq).

Esterification of glycerides in organic base is also preferred rather than acid, although 37C overnight in ether will slow down your work.

best wishes,

Rod

Actually, I've never tried HCl to esterify fatty acids or soaps in methanol. We used to use BF3-methanol purchased about 12% in methanol. As a cost-savings we later switched to buying 50% BF3 in methanol from Aldrich, then diluting that down.

Then we started making up concentrated H2SO4 into methanol. Now I think we'd mainly just purchase, like http://www.sigmaaldrich.com/catalog/pro ... &region=US

http://www.sigmaaldrich.com/etc/mediali ... _h2so4.pdf

If our sample is triglycerides or esters, we saponify first, then add the sulfuric-methanol and esterify. Several ways to get there...

OK then 22 baume 11.4N HCl won't work because by definition it is in water as HCl is a gas. I'd need special HCl dissolved in methanol.

It looks like the 50% BF3 in methanol is my best bet. just $47 for 208ml after dilution.

Store in several bottles if possible with minimal headspace.

Make sure you have DRY methanol. After a bottle of methanol is opened it may easily pick up 1% or more water from the air.

good luck,

Rod

Perhaps store the methanol in a bottle with some anhydrous Na2SO4 in it.

When we esterify soap bars, liquid soaps, or "neat soaps", there is 10 to 80% water in the sample, and we do not dry the samples first.

How sensitive is the method to water? I had a derivitized HPLC method a while ago for Ivermectin where we derivitized with methyl imidazole and during the winter it was a cake walk and the summer it was a total nightmare of failed sample sets despite nitrogen blanketing the reagents and storing them in a desiccator.

I only know that when there was water in the 14% BF3/methanol the esterification reaction was incomplete.

With water present the formation of Carbonate salts may have been the problem? ?

It was a big problem especially when esterifying aromatic acids, but that is another topic.

To troubleshoot the reaction, test samples of pure oleic acid and then run TLC on silica gel for residual free oleic acid after extraction. The ester will easily separate from the free acid.

Detect the free acid using iodine vapors or sulphuric acid char method.

best wishes,

Rod

I'm afraid I have another question I came in this morning and regulatory threw a bombshell on me. They need to know the % trans fats. That mean just using a wax column is out the window. Which do you guys recommend a HP-88, SP-2330 is 30m enough?

Also is the basic protocol sufficient?

HP-88 100 m

ah darn that is a $1k column

The standard column for trans fat

is the SP-2560, 100 m x 0.25 mm I.D., 0.20 μm (24056)

from Supelco.

Review the AOCS Official Method Ce 1k-09 .

best wishes,

Rod

Supelco is reporting pretty good trans resolution using a .10mm ID column only 15m 2380, though they are using hydrogen as a carrier and I don't really want to setup my GC for hydrogen and I heard reports of some hydrogenation of fatty acids in the GC with hyrdrogen carrier. Also I assume with such a narrow column a pressure pulse is necessary to get it into the column from the injection port.

http://www.sigmaaldrich.com/technical-d ... c-and.html