Chromatography Forum

I need to get set up to analyze for CLA and other fatty acids, and specificly to quantify c9t11 CLA.

My preferred route would be to use HPLC (since I have one available), and of course minimum sample prep. I have a PAD detector, which I believe should be capable of detecting the acids without derivatization. So my main area of concern really is sample prep and separation. I'd be most grateful for any input or experiences anybody has in this area.

Thanks for your help in advance!

Lotus,

We need more information in order to help you out. What are CLA and C9t11 CLA? What fatty acids do you need to analyze? Short-chain, long-chain, free fatty acids? Do you mean PDA (photodiode array) detector?
As far as sample prep, what is the sample matrix?

CLA: Conjugated Linoleic Acid, an 18-carbon fatty acid, which has several positional and chiral isomers. The one that is the most common and of most interest, is the cis-9-trans-11.
My samples: I will have two types of samples, an animal feed, and a treated corn oil. I'm not sure what the overall fatty acid composition will be, but should be pretty close to corn oil. The matrix from the feed is going to be a mess... The samples will also have a combination of triacylglycerides and free fatty acids, but for my purposes, I think I want total fatty acids, so I could/should convert it all to free fatty acids.
What I need analyzed? I'm not even 100 % sure yet, if I need to get a full profile, but at minimum, I need to quantify CLA. I can compromise on the other fatty acids, if I just could nail CLA.
PAD: Pulsed Amperometric Detector from Dionex

I hope I answered all the questions!

Lotus,

You are right that you need to extract the fatty acids in the triglyceride form to get the total fatty acid content. This will involve maceration and Soxhlet solvent extraction using hexane or petroleum ether to extrect the oil from the feed. The oils need to subjected to saponification to release the fatty acids from the triglycerides. The fatty acids in free or derivatized form can be analyzed by HPLC but I don't know if the PAD will detect them. Most fatty acid analyses are done using gas chromatography with a flame-ionization detector (FID) after conversion to the methyl esters. There are several reagents for the methylation reaction. You can check with vendors such as Alltech and Supelco for applications for this analysis. Hope this helps!

Lotus,

The Pulsed Amperometric Detector can only detect a few acids as most are not electroactive. Simple aliphatic acids can't be detected this way. You need to derivatize first or better still, use GC.

A chiral linoleic? What does that look like? I guess I am getting rusty on FA, . . . . what do you mean by "positional isomers" of linoleic?

Thank you for your answers!

Chris, thanks for the info on the PAD! Can you tell me what kind of derivative would work with PAD? I'm reluctant to do GC, if I can find a way to use HPLC, but GC is not totally out of question. This is mostly just a personal preference, as I'm most familiar with HPLC, and would also have to buy a new GC for this purpose.

HW, sorry for my misuse of "chiral", I should have said geometric, i.e. cis-trans isomers. By positional I mean that the conjugated bonds can be in different places in the 18-carbon chain.

OK, but positional variations usually go by a different name, not so important. Anyway, if you can separate these with HPLC I certainly would like to hear about it. Some FA isomer separations had even "dogged" GC experts for a long time.

OK, but positional variations usually go by a different name, not so important. Anyway, if you can separate these with HPLC I certainly would like to hear about it. Some FA isomer separations had even "dogged" GC experts for a long time.

HW, I haven't even gotten started on this, just have read tons of litterature. That's where I adopted the word "positional", since they seem to go by that name. I have found hints of maybe being able to do the whole thing by HPLC, and thought I'd try to ask here, just in case I'd get lucky and find somebody who's at least tried it. Seems to me that you could do it by using two columns in seriers, maybe a C18 and a lipid column, such as Varian ChromSpher Lipids (on their web site Varian states: "Ideal for the separation of cis-trans isomers of fatty acids"). But what about eluents, gradients, detection...? I'm just trying my luck here!

This link lists references to performing fatty acid analysis using HPLC with an Evaporative Light Scattering Detector. I came across it when researching my Tween 80 project that I recently asked the forum about. I don't have any experience with this myself but I hope that you can find something useful here.

Good luck.

Karen J

http://www.cyberlipid.org/elsd/elsd0002.htm#3

Lotus,

Sorry, if I gave you the impression that I was aware of derivatization reagents suitable for use with electrochemical detection. While such derivatizing agents this may well exist, I was unable to find any in a quick check of readily available references. What I was referring to in my post above is commonly employed derivatives used in conjunction with UV detection. Probably the most commonly employed derivative is based on producing a p-bromophenacyl ester. You can find quite a few methods using this derivative in the published literature.