Hi,

i have some comments to your question. I undesrtand that you are analyssing fatty acids in plancetal tissue material. I also undesrtand that you are using and acid catalyzed direct transmethylation/esterification procedure. Well, in my experience your temperature/time is excessive and such conditions can alter the fatty acid profile (please see atteched work).

I suspect that droplets you are looking are water, methanol or oil from your sample. How do you stop reaction and dry you FAME?. I would need more details about your sample preparation. Contact me and I would be happy to help you.

Article Preparation of fatty acid methyl esters for gas-liquid chromatography

Thank you very much for your reply. I am sorry I wasn't clear with my question. We have been using transesterification protocol since a long time, only in recent days I am facing this error.I am personally trying to understand it but now I need expert help.The protocol I am following is as follows:

Collection of Sample: Within an hour the placenta is transferred to our laboratory. Chopped and washed with PBS to stored into cryo vails at -80 degrees Celsius.

Homogenization:0.2 gm of tissue is homogenized in glass homogenizer with 3 ml ice cold Lysis buffer(1X PBS 100 ml + Aprotenin 32 ul + Leupeptin 32 ul + PMSF 3.2 ul). Centrifuged at 4 degree Celsius and supernatant is removed. Cell membrane then reconstituted in 0.5 ml of lysis buffer.

Transesterification: 100 ul of cell membrane taken in 10 ml screw capped tubes and 4 ml of methanolic HCl (5% HCl + 0.1% BHT) is added. Vortexed well and sealed with Teflon tape and capped (to prevent evaporation). Tubes incubated at 80 degrees Celsius for 2 hours in water bath and then kept in ice for half an hour. Hexane is used to extract FAME (2 ml + 3 ml) in two steps using Pasteur pipettes. Then extrated hexane is dried in Argon gas stream directly using small nozzles.

GC Analysis: Dried vails are reconstituted in 50 ul chloroform out of which 1 ul injected in the GC for analysis.

I have read the paper you sent. Before completely changing the protocol I would like to know what is the error I am facing. I have tried extracting lipid through Folch method and then doing transesterification but unfortunately the oily thing still remains. That oily thing is not soluble in water and hexane but very much soluble in chloroform.

Dear Alka Rani,

Your protocol is ok. In my experience your time/temperature conditions are too much and despite you adding BHT PUFA can be altered. I think the residue in your samples may be water or methanol. So what you can do is dry hexane (previously to evaporation) with anhydrous sulfate (a pinch or two is enough). Of course, after that you will need to centrifuge your sample, collect hexane again and dry. You must also be careful with the interphase between aqueous phase and hexane when collecting: do not take all the hexane, leave a couple of microliters.

What if it is not water or methanol?. Then the residue are lipids. However these compounds are highly soluble in hexane (that´s why I think your drops are a polar solvent). Anyway, if are lipids that means that reaction is not complete for these kind of samples. When you derivatize triglycerides you perform a transmethylation but for free fatty acids is esterification. The problem is that acid-catalysed derivatization cannot yield FAME from triglycerides and other esterified compounds. You need base-catalysed reactions.

However I think that your problem is sweeping of water/methanol in the apolar solvent. In Folch method I always dry my samples with an anhydrous salt prior to evaporate, if not I will obtain drops like that you have.

I hope this help you. If not let me know. I will be happy to help!. Good luck!.