Chromatography Forum

Hi
In derivatization with BF3-methanol is it essential that fatty acid methyl ester extracted in organic solvent (hexane)?
Why we have to extract?
If we do not extract, is it possible that damage the column?


Sorry for my english...i'm a persian

Hi,

my experience in FAMEs is very limited but I think that extraction (with n-heptane) is necessary because the solubility of FAMEs in methanol is very low.
The method I know recommends to boil the sample with methanol-BF3 solution under a reflux condenser for 10 min, then add n-heptane and boil for other 10 min. Allow to cool and wash the diphasic solution with a saturated solution of NaCl. This procedure allows a better separation between FAMEs-heptane solution and methanol-BF3. Then dry the organic layer (over anhydrous Na2SO4) and inject in GC.

The injection of methanol solution gives, in my opinion, two problems:
- low sensitivity (as I said before FAMEs are not soluble in methanol)
- risk of damage the GC column for the presence of BF3 Lewis acid

Good luck

thanks for reply.
I need a procedure for extraction of fatty acid from the real sample(e.g fish oil).please help me

This is how we do it.

10 mg of sample(oil) is transferred to a test tube. 1,00 mL of sodium methylate is added and the tube is vortex mixed for 30 sec.

Place the test tubes in a 60°C water bath until the solution is clear. (apx. 10 min.) Then leave the samples for an additional 30 min.

Add 4,00 mL of saturated sodium Chloride and 1,0 mL pentane. Vortex mix for 30 sec.

Let the phases separate . The top phase will be pentane and contain the FAMEs.

Hope you can use it.

Hi Dear leadazide
I want preparation FAME with BF3 /methanol, so I should extract the fatty acids.
thanks

You must remove the BF3 excess from the analytes before you inject. If you inject the powerful Lewis acid BF3 onto your column you will destroy it. Doubting? You should have seen the hole I bore into a packed porous polymer column over two decades ago because my boss said "go ahead, I want numbers now". lol

Extract your esters into toluene, CH2Cl2, CHCl3, EtoAc, or some alkane.

best wishes,

Rod

hello
what is the temprature for store the FAMEs (samples and standards)?
Do the FAMEs need the anti oxidant (e.g BHT)?
thanks for reply

If you have volatile or unsaturated FAMEs it is recommended that you store them as cool as possible, freezer is best, but 4°C as a maximum temperature. You may wish to flush the air from the storage vessel with nitrogen or argon.

unsaturated FAMEs will turn yellow or orange with time as they oxidize, and will turn darker and darker. Avoid using such solutions on your column.

You must remove the BF3 excess from the analytes before you inject. If you inject the powerful Lewis acid BF3 onto your column you will destroy it. Doubting? You should have seen the hole I bore into a packed porous polymer column over two decades ago because my boss said "go ahead, I want numbers now". lol

Extract your esters into toluene, CH2Cl2, CHCl3, EtoAc, or some alkane.

best wishes,

Rod

thank you
my BF3/methanol is in a cool place. but there are solid particles in vial.
is it unsuitable for use?
How do I undrestand that it is undegradate?
thanks

I am uncertain what you are saying. Do you mean the free acids you wish to esterify are solids? You may esterify them if they can be dissolved into the BF3 in methanol or a mix of BF3 in methanol and another solvent like methylene chloride AT THE REACTION TEMPERATURE.

Organic material in your sample that is acid sensitive will be affected and possibly destroyed by the reagent. Do not heat poly unsaturated acids for more than 8 minutes with this reagent.

You normally heat the reagent to dissolve the free acid, for example benzoic acid salts, and to convert it to the methyl ester. Benzoic acid (or oxalic acid) usually requires a 30 minute reaction @ 110°C to give a high yield of ester. Organic free acids like lauric, myristic, palmitic, oleic, only require 8-10 minutes @ 110°C. The reagent/solvent is cooled to amibient and an aqueous saturated NaCL or some other salt solution and non-polar solvent are added to separate the non-polar layer containing the esters.

20mg acid, 1mL BF3 in methanol, 110°C for 10 minutes, 12 mL saturated NaCl aqueous solution with 1mL of hexane, is a common receipe.

Temperatures can be 50 to 110°C? and time from 5 to 30 minutes.

Determine the best parameters for your sample. And don't forget, you are creating the acid HF with this reagent when it contacts water. Very strong and toxic acid.

best wishes,

Rod

Hi rod
excuseme, my question is about BF3/methanol.
thanks for explanation

in vial, in bottle, or in ampule?

Some particles can be found in glass bottles when stored for a long time. This usually does not make the reagent unusable. But if there is a lot of particles I would get fresh reagent.

thanks
these are very helpful.
best regards.