Chromatography Forum

I am running marine oils by the AOCS method with a few minor amendments, maily that we are using 1% sulfuric acid in methanol instead of BF3 in methanol and BHT is added for use as an antioxidant. Evry so ofen all of our extracts start to go cloudy and a blob of jelly is formed, meaning these samples can't be injected onto the GC.

I am new to FAMES analysis so any comments would be appreciated.

Thanks

We also run quite a lot of FAME analysis.. And we see the same thing.. Once in a while a sample just goes cloudy.. We run on lipids either directly from oils or from from extraction from meat, cheese and lots of other stuff.. We haven't been able to find a reason.

We don't follow the AOCS method. Our method involves methylating the lipids with a sodium methylate solution and and treating the solution to a high conc off salt and pentane where after we just remove and inject the pentane phase. And it's the pentane phase that sometimes goes cloudy or has some sort of precipitate floating around in it.

Hope someone smarter then me can find a reason!

In my experience, the material in the organic phase is either free fatty acids ( if the reaction hasn't worked properly ), unreacted triglycerides/glycerol or, much more likely, polar lipids - especially if the sample is crude extract.

If the reaction hasn't worked properly ( sometimes presence of water ) and insoluble materials can be washed out, the solids are often FFA or glycerol, otherwise long chain MAGs, DAGs, TAGs from a poor reaction.

The esterification can also react with some of the polar lipid consituents and they precipitate out. The most common ones I've seen in marine oils are from diacyl glyceryl ethers and similar. In animal and plant oils it tends to be the phospholipids, sphingolipids etc.

As always, the best information on lipid analysis, and issues associoated with lipid sample prepartion is in William W.Christie's books.

Bruce Hamilton

Bruce is quite right. Especially about the web site. Highly recommended.

Free acids mono-glycerides and other non-polar material can carryover in the extraction step. When chilled these can form a suspension (or even crystalize) forming a cloudy solution.

Try TLC to characterize your extraction. Use simple silica plates, mobile phase suggestions are 100% hexane, 98% hexane-2% ether, 97% hexane-2%ether-1% acetic acid.

Use a simple sulfuric acid in methanol spray (or Iodine vapor) detection and heat in the oven @ 100°C.

You will see how effective your esterification reaction and your extraction procedures are.

Stahl's classic book is useful for mobile phase systems and detections.

best wishes,

Rod

Does anyone have any experience of how accurate these derivatisation methods are (in particularly the AOCS method) as we are only seeing recoveries of 90% while our precision is fantastic (RSD's of less than 1%).

Thanks

In my experience virtually all the derivatisation methods work well, and if you are seeing only 90% with a clean, pure, sample, then you need to follow the exact procedure specified to identify the cause of the loss.

Bill Christie's site has some information on the different derivatisations.
http://www.lipidlibrary.co.uk/index.html

Try to identify whether recovery loss is due to FAME fractionation in the GC or poor derivatisation/extraction. Consistently wrong data is usually considered better than randomly wrong, but not by much.

If necessary, review your protocols by using a couple of pure triglyceride oils, such as cod liver oil ( which usually isn't from cod ), corn oil, or even coconut oil. Ensure that you are consistently obtaining a good separation.

Good luck,

Bruce Hamilton.