I want to make a standard plot for the oleate by using different concentrations without using derivitization. I want to calculate oleate `s uptake rate in e.coli . Derivitization would be tough because i have lot of samples, so please help me out to how to do.
Without derivitization you will have to use HPLC with an organic acid column. Methyl esterification allows the fatty acid (oleic acid in your case) to become volatile and detected by the FID.
I used methanol-bufferA and 2-propanol:hexane-bufferB both buffers in 1:1
Try looking at the Waters, Agilent, and Phenomenex websites for organic acid columns. It could be the concentration of the water buffer is too high in your mobile phase.
Hexane really is not miscible with water either.
Is sodium oleate can be detected by hplc? If yes, then please give suggestions.
Shikha,
Yes. But you will have to use a ELSD (Evaporative Light Scattering Detector) or a CAD (Charged Aerosol Detector) since fatty acids do not have a strong UV spectrum!
In that case you must use the FAME GC-FID method. However, because the fatty acid is derivatized to a volatile methyl ester it is a fingerprint method (not quantitative, but is close!). See AOCS (Association of Oil Chemists Society) and even their lipid library.
Do a liquid/liquid extraction with Hexane 1st, then air dry down and reconstitute with 1 mL Methanol. Syringe ultrafilter (0.22 um) and finally inject.
Don't forget with the GC you may have to perform the FAME procedure, to eliminate any interferences.
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