I have extracted fixed oil from seeds and wish to analyse for its fatty acid composition by GC. Is it possible to identify the fatty acids with out using MS?
You can analyse FAMEs using a FID detector. Supelco has a good column for that purpose - SP2340. They also have a FAME mix with 37 components that are well separated with that column and can help you finding out the RT.
To prepare FAMEs, a saponification with 0.1 M KOH followed by the methylation with 1 M H2SO4 at 60 °C will do just fine. At the end, you recover the FAME with n-hexane and they are ready to be injected.
Thanks Dr. Fiamononcini for your valuable information. Can you please send the detailed protocol for preparing FAME?
the reference is :Lab Pract. 1973 Jul;22(6):475-6 passim.
Rapid preparation of fatty acid methyl esters from lipids.
Hartman L, Lago RC.
I used a modified version:
Basically you get the total lipid extract (50 uL or so) and add 1,5 mL of 0,1 M KOH in a test tube with a screw cap. Leave it for 1 hour at 60 °C. Then let the tubes cool down and add 3 mL of 1 N H2SO4. Again leave it for one hour at 60 °C. When the tubes are cold again, add 1 mL n-hexane (or less, depending on your GC vials) and vortex the tubes and let it settled until 2 phases develop (quick centrifugation can help). Take the upper phase (n-hexane) to GC vials and it is ready to inject. You can express the results as percentage of each fatty acid in the tolat lipid extract.
Thanks Dr. Fiamononcini for your kind support. I will try the procedure in my lab.
FAME is used for identification of fatty acids in fixed oil. Dried seeds of plants
are cleaned and powdered. The powdered drug (50g) is then subjected to Soxhlet
Extraction with Petroleum Ether 60-80 (200ml). Extraction is further done till sufficient amount of oil is obtained . 1g of oil is taken in 250 ml RBF, to which 12ml of 0.5M
methanolic NaOH is added and subject to reflux for 45-50 minutes. 15ml BF3- Methanol is then add from top of the condenser and then again boil for 20-25 minutes. 4-5ml of Petroleum Ether 60- 80 is then added through the condenser, heated for 3-5 minutes, heating is stopped immediately and 12ml saturated NaCl is added. The mixture is shaken vigorously for 1 minute, additional NaCl is then added so that petroleum ether solution floats into the neck of flask. FAMEs are collected by means of syringe and keep in closely tight glass vial in refrigerator.
I agree with the researcher who proposed the Hartman method for FAME.This si the best artefact free technique I have found and I think Bill Christie once sanctioned this technique.You can do the reaction quickly in sealed tubes followed by hexane extraction in the same tube.Then clean up the preparation by passing though a llittle minicolumn of calcium chloride and silica to remove methanol and catalysts to help preserve you stationery phase/
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