[Daylan]

Hi everybody,
I am making an hydrolisis of TAG with pancreatic lipase. And I have some problems to yield the enough sample to get results in the GC determiantion of Fatty acids. I am starting with 5 mg of sample and I am afraid I do not yield enough MAG, because at the end the GC can not find anything.
 I do the hydrolisis, extract the products with diethyl ether, wash the solution with water, then I dry the solution with sodium sulfate, then evaporate the solvent, I add then 1 mL of diethylether to separate the fractions in a TLC plate, after that I scrape them out, and add hexane to dissolve them. I filtrate the solution to get rid of the silica, and then the products are transesterified to determined the fatty acid composition.

I have already determined the rate of hydrolisis by a GC, so I know that my hydrolisis work, and that I actually have a MAG fraction.

I am not sure what could be wrong, I run already the GC analysis with the product of 8 repetitions and this still does not work.
I am using a reference of MAG to determine the MAG fraction that I need to scrape. My MAG stays most of the time in the origin, according to literature, they will stay a bit forward in the plate. Now I am wondering that probably that could be a wrong. But of course, at this point I am thinking that everything could be wrong.

Has somebody done this procedure before? Do anybody have some ideas?

Thanks in advance

[OC pro]

I assume hydrolysis is under basic conditions (meaning pH > 7). This gives the fatty acids as salts which will poorly dissolve in organic solvents. That can be the point that you don´t detect any fatty acids. Therefore, you should acidify the solution to at least pH <4 with perhaps 1N HCl. Then you will get the protonated acids which can then be easily extracted with diethyl ether, dichloromethane or ethyl acetate.

[Daylan]

I did the hydrolisis at pH 8.
I am making a mild hydrolisis, I am reaching just 50% of hydrolisis. When I rich around 90% of hydrolisis I get salts.
I do not want to have migration in the monoacylglycerols  fractions this is why I cannot acidfy the solution.
I can actually dissolve the products in diethyl ether or in hexane. Cause when I did the GC analysis I diluted them in Hexane and I got a GC response, this analysis was to get the time in which I could get the desired hydrolisis rate.
And when I make the separation in TLC, I use diethyl ether and I get separeted fractions.

So, I guess this is not my case.
Any other idea?

[manishbiyani]

Daylan,

I never performed enzyme facilitated organic reaction. Just a thought, does the monoacylmigration takes place at pH 2-3 also. Have tried this or seen any literature precedence for this.