I have been trying to quantify oleic acid by HPLC using this protocol derived from the www.cyberlipid.org website.
Dissolve oleic acid standard in 0.5ml methanol (concentration range from 2mg/ml to 62.5μg/ml). Add 5μl of dilute phenolphthalein solution and raise pH with 0.2M KOH in methanol until colour change (pH9.8). Evaporate solvent under nitrogen.
Add 0.1ml 2mM 18-crown-6 ether in acetonitrile and 0.1ml of 2,4'-dibromoacetophenone (cyberlipid didn't give a concentration: I used 20mg/ml). Heat at 80°C for 15 minutes. Carry out separation on C18 column using 80% acetonitrile. Detect at 242nm.
I get several peaks ranging from about 1 to 7 minutes, but none corresponded to the putative concentration of oleic acid, which makes me think the derivatisation method is not working. Can anyone tell me what I should be doing, please?
If you have derviatisred oleic acid with trimethylsily it form methyl derivative with silane group. Remove silane by heating it in oven and dissolve residue with methanol. Then check on Hplc and more specifically on GC or GCMS as oleic acid is polar
Unfortunately that's not the method I am using. I want to know how to do 2,4'-dibromoacetophenone derivatisation properly.
Hi!
If you want to quantify oleic acid then why are you stressing so much on HPLC. Its better if you can use GC/GC-MS and the methods are also simple.
Next, I would like to suggest you to look at the website of lipid library (http://lipidlibrary.aocs.org). All the detailed methods and protocols are mentioned there. Thal will certainly help you.
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