Hexane, ethanol, methanol and acetonitrile are polar that's way they are miscible. You have to use a nonpolar solvent which is immiscible, like benzene, for instance.

The extraction of polyphenols from seed oils can be performed by dissolving oil in n-hexane and adding methanol (that is not miscible with n-hexane) in 1:1 volume ratio for three times. At the end of liquid-liquid extraction methanol will be removed by using rotating evaporator; in this way you will apply Folin-Ciocalteau or HPLC on C18 column for the analysis of polyphenols.

Extractio of phenols:

First of all to extract the phenolic compounds in seed oils you should remove the lipids (oil) from your seed flour in order to obtained a deffated flour. This you can do with n-hexane (HPLC grade) under agitation at room temperature for at least 8 hours (dependinding on the lipid content of your sample this time can be longer). After that you can extract the phenols using ethanol solution 70% (HPLC grade ethanol) added of 0.1% acetic acid for 1 hour under agitation at room temperature. Normally you can use 4.0mL of the extractor solution (ethanol + acetic acid) for  each 100mg of deffated flour. For quantification you can use a HPLC C18 column for the analysis.

I think all the above techniques will not work. There are two things what you can do at the moment:

1. Do a liquid-liquid separation based on hydrogen bonding- Mix sample with Hexane and Methanol-Water solution. Centrifuge it, which will create two layers. One layer will be methnol-water and phenolics while the other layer will be hexane and lipids. This will help you to a great extent.

2. Do a solid phase separation using Amino propyl or any other suitable column. Prepare the sample by dissolving it within chloroform and pass it through a column preconditioned with hexane. Now pass methanol-water solution through the column, which will wash the total phenolics.

You can use second method in combination with first. The separation of total phenolics and other coloring substances from your oil total depends on the polarity and hydrogen bonding ability of the residual molecules. But these two methods will help you to a great extent.

I am working on chemistry of separation of lipids and non-lipid materials mainly from seed oils. If you encounter any specific problem, mail me on [email protected]

I would also like to know the results of these separation techniques when you apply it on your lipid system, this will help me a lot.

Thanks!

Aman Paul

Thanks a lot for your suggestions!

I can perform the way you suggested, but I want to compare extraction ability of different solvents, then i can choose the one that gives me the highest phenolic recovery by Folin-Ciocalteau method. i will use this solvent to extract phenolic compounds and analysis them by LC-MS or HPLC. 

I would suggest you:

Influence of pearling process on phenolic and saponin content in quinoa (Chenopodium quinoa Willd)

Ana Maria Gómez-Caravacaa, b, , , 

Giovanna Iafelicec, d, 

Vito Verardoe, 

Emanuele Marconic, d, 

Maria Fiorenza Cabonie, f

Thanks a lot Antonio Carlos de Souza Medeiros!