Depends on your matrix and the fatty acids you are trying to separate.  You can use purification (not analytical) HPLC.  Check the Waters, Agilent, and Phenomenex websites.

Dear Sir,

Many thanks for the response.

I went through some HPLC methods for separation of fatty acids using HPLC. What I observe is that the peaks for various fatty acids are so close that separating them will be a hurdle.

Are there any other methods?

My specificity is for alpha Linolenic Acid only. Other acids are not important to me currently.

Regards

Many methods are patent protected, but you can look at this article.

https://www.google.com/patents/CN101402558A?cl=en

Thanks a ton sir... I will surely go through it.

Regards

If you are simply doing analysis and not preparative, a very sensitive way is preparing p-bromophenacyl esters of your mixture. This makes the fatty acids neutral at any pH and the UV chromophore on the phenacyl group is strong and makes for sensitive UV detection. The free carboxyl/carboxylate group is not good for any chromatography. With  5µm C18 you should get good results.  Argentation (silver) chromatography is also a very efficient technique to separate FA by not only the # of double bonds but by their cis/trans content, and to some degree, by their position. I think it's Varian that sells that HPLC column, it's called Chromspher LipidsTM, Varian-Chrompack, or you can make your own by silver ion impregnating an anion-exchange HpLC column... I've made my own it works very well.http://lipidlibrary.aocs.org/content.cfm?ItemNumber=40349

Dear Dean Cuebas Sir,

Many many thanks for this wonderful idea. I will go through it in detail and will get back to you in future queries.

Regards

Sachin

An optional way is to try ion exclusion chromatography (ICE), which can provide much better separation. If your purpose is to separate them, then it should be easy to achieve. If you want to measure it sensitively, then this method might not be a good one. Currently, my group developed a very sensitive ICE method for volatile fatty acids (see attached), but not alpha-linolenic acid. 

Dear Prof. Liao,

Commendable work it is.

I will refer is and look for separation of alpha linolenic acid particularly.

Many thanks

Sachin