Personally, I would not recommend silylation (whatever silylation reagent one might chose to use) as a derivatisation method for free fatty acids (fFAs). From a GC separation point of view you would be better served to turn fFAs into their corresponding fatty acid methyl esters (FAMES) using BF3/methanol (14%) or Methylate Reagent (DMFDMA). Methylation using BF3/methanol is an easy to use and effective way to derivatise free FAs into FAMES. Formed FAMES can be extracted into n-hexane. Journal of Liquid Research, 1965. 5: p. 600-608 For your GC method you should consider using internal standards, e.g. 1 or 2 odd numbered FAs such as C11:0, C13:0 or C15:0. Last but not least, your sample prep protocol needs to reflect your research question. Are you interested in the free FAs profile or total FA profile of your extracts? If the latter, prior to FA derivatisation you will need to hydrolyse the lipid extact (such as triglycerides) to liberate bound FAs.

Refer Folch et al (1957) and Metcalfe et al. (1966)methods for lipid extraction and fatty acid derivatization. That would be helpful.

Hello, use hexane for extraction of fatty components from reaction mixture.

1.Prrapare sodium methylate solution by adding 4g of metallic sodium in to 100ml of pure methanol (solA).

2. Take 100ul of hexane fraction of sample add 300ml pure hexane(you can change this dilution step depends from total fat content in extracted hexane fraction ) so 100ul sample+ 300ul hexane +250ul SolA. Mix well or vortex at room temperature and use upper layer for direct injection to GC.

Hope you have GC with FID detector and FAME or equivalent GC column.

Good luck!

A very simple explanation is that the derivatization with BSTFA is basically a silanization of your target compound (s). Thus, it requires OH groups to happen and will happen if they are present.

Maybe you should consider instead esterifying the acids to analyze them by GC? You have good suggestions for this from Jannathulla and Karen.

Personally, I would not recommend silylation (whatever silylation reagent one might chose to use) as a derivatisation method for free fatty acids (fFAs). From a GC separation point of view you would be better served to turn fFAs into their corresponding fatty acid methyl esters (FAMES) using BF3/methanol (14%) or Methylate Reagent (DMFDMA). Methylation using BF3/methanol is an easy to use and effective way to derivatise free FAs into FAMES. Formed FAMES can be extracted into n-hexane. Journal of Liquid Research, 1965. 5: p. 600-608 For your GC method you should consider using internal standards, e.g. 1 or 2 odd numbered FAs such as C11:0, C13:0 or C15:0. Last but not least, your sample prep protocol needs to reflect your research question. Are you interested in the free FAs profile or total FA profile of your extracts? If the latter, prior to FA derivatisation you will need to hydrolyse the lipid extact (such as triglycerides) to liberate bound FAs.

Wolfram Meier-Augenstein

Thanks for the suggestions sir. I have tried with this method(BF3), but didn't get any results for my reactions.The work of P450 is to hydroxylate the substrate.

This does surprise me. Methylation of free fatty acids is a straight forward esterification that hardly if ever goes wrong. Does your extract actually contain any free fatty acids? If it mainly (only) comprises lipids you will need to hydrolyse (saponify) the lipid extract first to turn lipids such as triglycerides into free fatty acids (and glycerol).

@Wolfram.Actually I got a peak of pure fatty acid by this method,but not for sample

P450 is essentially the final step in an oxidase enzyme system. It is not possible to use P450 to hydrolyse ester bonds of lipids. One of the main function of CYPs is detoxification of xenobiotics. They are involved in the breakdown of e.g. phenacetin, paracetamol or caffein by transforming (oxidising) an R-H bond into R-OH.

There is your answer. If methylation of pure fatty acids works the problem is with your sample not the methylation method. Your sample does not contain any free fatty acids, either because it just doesn't or because your prepraration method does not work. As mentioned in my earlier post, it is impossible to use P450 to hydrolyse ester bonds of lipids. Saponification of lipids to yield free fatty acids is best done under alkaline conditions though acidic hydrylosis is also an option.

Wolfram Meier-Augenstein ...Thank you for the explanation sir. This helps me alot to design my experiments.