Chromatography Forum

Hi everybody,

we have to quantificate the content of tryglyceride (mainly composed by C8 and C10 saturated fatty acids) in a product composed by gelatin and glycerol.
We think to dissolve the product with water saturated by NaCl, add internal standard (methyl octanoate) and extract the tryglycerides with hexane. After the extraction we'll take the hexane layer to dryness under N2. After this we will make an hydrolysis and esterification with sodium hydroxide in methanol and boron trifluoride. At the end we will quantificate the FAMEs with standards of methyl caprylate and methyl caprate.
What would be the best solvent to extract the tryglycerides?
Do you see any problem in our procedure that we don't see?
Do you know better methods that could be used to analyse this sample?

Thank you very much,
Have a good day,
Davide

Your procedure of dissolving the matrix in saturated salt water is good.

Extracting with hexane is also good. I would add glyceride as an internal standard you would not expect to find in your sample, like tristearin.

You might try adding 25% to 50% methylene chloride to the hexane for better extraction efficiency. I would perform three extractions and combine them for the evaporation step.

Evaporating to dryness with N2 is fine. Be careful not to evaporate too quickly or you can lose part of your extraction. Keep the hexane extraction cold during evaporation (in a 15mL culture tube).

Then I would just add 1mL of 14% Boron Trifluoride in methanol to the culture tube, add a small amount of methylene chloride without a saponification, and heat for 10 minutes @ 100°C.

Add some additional hexane and salt water and extract your FAMEs.

best wishes,

Rod

Hi,

Without knowing more about the composition of your sample, I would be tempted to take a shot at simplifying it by taking the sample as is and transesterifying with methanol/sulphuric acid then extracting as Rod describes. Backwash the the hexane extract with potassium bicarbonate soln then dry it with anhydrous sodium sulphate. If it fails you haven't lost anything and you might save a lot of time.

Regards,

Ralph

If your goal is to determine how much total triglyceride is present, I'd extract with hexane and determine gravimetrically or by high-temperature capillary GC. If your goal is to determine chain-length distribution, I'd saponify first using NaOH or KOH in CH3OH, then add BF3-CH3OH or H2SO4-CH3OH to acidify and esterify by heating on steam bath five minutes, then cool, add some hexane, add saturated NaCl to float the hexane layer, then GC the hexane layer.