We are currently trying to venture into trying to analyze Ricinoleic Acid which is a hydroxy fatty acid. We routinely analyze PUFA/SAT fatty acids. We are having a little trouble with developing a procedure for Ricinoleic Acid though. We are trying to to directly methylate iet using the BF3 methylation and using a DB-23 column on GC-FID. We found out that we do need to derivative with TMS or Ricinoleic Acid will hang up in the column. We are having a problem right now detecting the peak or having a very reduced area. We previously were able to detect the peak with a large area but now when we prep a standard using the same method we get a peak at 1/10 the area if we get one. Does anyone have any suggestions on what could be causing this issue or any fixes we could try?
Hi Jeremy,
Methanolic boron trifluoride is a strong lewis acid and will transesterify many saturated and unsaturated fatty acid though it is known to decompose fatty acids with particular chemical groups, such as hydroxyl fatty acids. The peak you have have detected before switching to silylation could of been an artifact.
As for the reduced peak area, is there any noticeable tailing?
Also, what reagent are you using for the silylation, and what are your derivitisation conditions?
Cheers,
Marcus
Hi Marcus,
Thanks for the reply. We do not have any tailing after silylation. As for how we are silylating we are taking the FAMES in the hexane layer (~100uL) drying the hexane layer down under N2, adding in 300uL of Chlorotrimethylsilane and then heating for 30minutes at 60C We then dry the Chlorotrimethylsilane off under N2 and then reconstitute back into 100uL of Hexane.
Hi Sandra,
We saw a few papers that used sodium methoxide but we currently do not have any at the moment so we are trying to alter how we analyze PUFA/SAT FAMEs. Isn't going to best so we might have to get some sodium methoxide in. Was your procedure basically as follow: Add NaOCH3, heat for ~10minutes, add some glacial acetic acid in, add saturated NaCl solution, add hexane, and then inject the hexane layer? Did you have to derivative?
What column where you using that you could analyze the FAMES of hydroxy fatty acids? We went with a DB-23 column (Agilent) since that is waht we found a few papers used and the only way to make the hydroxy fatty acids show up are to derivative with TMS, they wont show up as FAMEs.
Also, the acid is only used to neutralize the base and return the pH to about neutral correct?
If I am understanding your last statement correctly and we do the following:
Add sodium methoxide, heat for ~10minutes and then add saturated NaCl solution and hexane. All of the FAMEs would be in the hexane layer but the acid (in our case Ricinoleic Acid) would be in the bottom layer?
Thanks for clarifying, I just misunderstood that statement. Thanks for all the help and information. We will more than likely end up ordering sodium methoxide and going that route with a TMS derivation at the end. While we wait for that regent to come we might try substituting methanolic sodium hydroxide in (since we have some) and heating for extended period of time.
Our article published in Chemistry and Physics of Lipids 163 (2010) 685–688 may solve your problem. The oil (castor oil) was simple dissolved in methyl acetate or ethyl acetate. the mixture was then trans-esterified with NaOH catalyst. The carboxylic was converted to methyl or ethyl ester and the hydroxy was acetylated. And the acetylated alkyl riconoleate was easily eluted off the GC colume.
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