Phospholipid Extraction From TLC scrapings

[ilovepeace]

Hi all,

I am basically a molecular biologist. For one of our experiments, we had to measure the fatty acid composition of the individual phospholipids of the cell membrane. I extracted the lipids using folch method and separated the phospholipids using Silica gel H plates with the solvent system Chlorofom:Methanol: Acetic acid: Water (50:37.5:3.5:2). Visualized the individual phospholipids using 2,7 Dihydrofluroscein. I could see all the five bands very distinctly. Until this step, i didnt have any problem.

After this, I needed to quantify the fatty acid composition of the individual phospholipids. I tried to scrap the individual bands and methylate using typical Lepage method (4:1 Methanol:Benzene, Acetyl Chloride, Potassium Carbonate, Hexane) but nothing came up in the GC. I searched for articles and came across a different method. This method is, add 2 ml of 6% Methanolic HCl to the TLC scrapings, incubate it at 80 degree celsius for 16 hours, stop the reaction with 1ml water and add 1ml hexane. I ran the samples in GC. I couldnt detect anything in the GC this time too.

I am not sure what is wrong in my methods, for that matter, i am not sure whether i am following the correct protocol at all.

If anybody could give some input on this, i would greatly appreciate it.

M.V

[freemab222]

Am I correct in understanding that you're trying to measure fatty acids using gas chromatography? The "standard method" has been to make the fatty-acid methyl esters (FAMA's), as I expect you are trying to do. The problem is that unless you succeed in the esterification of those fatty acids, you're unlikely to see them elute from a GC column.

So, if I'm right so far, what you need do is to make test samples from known fatty acid sample. (Not rocket science. Don't waste time and money trying to get exactly the fatty acids you expect to find. Bar soap is fatty acid sodium salts and would probably do in a pinch.) Derivatize these by your procedure and see whether you get peaks in your chromatogram. If not, there's your problem. In this case, find a different derivitization procedure appropriate to your sample.

As a secondary check, you might buy some methyl stearate, or similar FAMA, and see whether you get a GC peak for that. This will confirm the GC system will detect FAMA's if they're present.

[krickos]

Agree with freemab that you should include a reference/standard/SST or so so you can seprate instrument issues from sample preparation issues.

Not familiar with your specific procedure, but from a chemical point of view you seem to have at least one issue.I do not know how 2,7 Dihydrofluroscein react or if its non destructive but also it is an acid which I gather it is added on plates in excess unless it is removed in sample preparation I suppose it will interfer with your esterfication.

If 2,7 Dihydrofluroscein is destructive there non destructive reagents such as iodine vapour or UV (perhaps not so good for FAMEs).

[Distortus]

Hi,

the procedure is correct,
use Boron trifluoride BF3-methanol kit (this is the best 100% sure method( 1ml BF3 + 0.5ml of hexane heat for 1h at 85oC)
if you use methanolic HCl (6%) and you do not do it the dry way (bubling of methanol with concentrated vapors of HCl) you might face a problem with water in the methylation mixture than the recovery of FAME's is low or none.

another thing is that you can see the bands but are you sure that those are really phospholipids?
I'm working with plant complex lipids galactosylglycerols + sulpho and phospholipids and what I encountered were "ghost bands" you visualise them but they contain no fatty acids.

hope this is usefull for you

D.

[Distortus]

one more comment to the visualisation

Iodine is also little destructive because it binds to double bonds (you may loose some)
I use 0.01-0.05% (w/v) of primuline in 80% acetone and 365nm UV light. let it dry and than check it under UV. You dont need to shine for a long time and you can visualize ug of lipids per spot.

also there are selective dyes for phospholipids try to look at these pages:
www.cyberlipid.org/
lipidlibrary.aocs.org/

there you get some more ideas

D.

[DSP007]

one more comment to the visualisation

Iodine is also little destructive because it binds to double bonds (you may loose some)
I use 0.01-0.05% (w/v) of primuline in 80% acetone and 365nm UV light. let it dry and than check it under UV. You dont need to shine for a long time and you can visualize ug of lipids per spot.

also there are selective dyes for phospholipids try to look at these pages:
http://www.cyberlipid.org/
lipidlibrary.aocs.org/

there you get some more ideas

D.

Hi
For phospholipide determinations we used HPLC & TLC - densitometry. TLC - visualisation - phospomolybdanic reagent . We have not Camag - used Umax scanner. Desity calculation program - Sorbfil videodensitometer sharevare version here http://www.sorbfil.com/dens_download.htm
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