Chromatography Forum

Dear all,

For a phD project i'll need to do a preparative separation of
unsaturated fatty acids from different sources (fish oil, sunflower oil etc..)

Thereby I'd rather not esterify my fatty acids as i'll need to feed those
fatty acids later on to cell cultures/homogenates.

Is anyone familiar with a kind of hplc separation technique who just does that?

Thank you for your input,

Hi

Maybe liquid-liquid-chromatography ( some people name it countercurrent chromatography) will be good for this problem.

In this case, you change the solid stationary phase to a liquid stationary phase, the seperation is basing on the polarity of the sample compounds.


Best regards
Chris

I have separated the C18 fatty acid by HPLC. I used a Purospher STAR C8 column with a Water/Acetonitrile gradient. Both solvents contained 0.1% TFA. The detector was an ELSD.
You can check the website from alltech, as they have a few applications for separating fatty acid.
Regards, Gilbert

Hi Gilbert,

Thank you for your reply.
I'll check the website from alltech

I was also thinking about the Aminex columns from Bio-Rad, but I wonder if they would do for higher molecular weight fatty acids (c10 to c18).

Best regards,

Yannick

Yannick,

We have two application notes addressing this separation. One shows the separation of acids (C6, C8, C10, C12, C16 and C18) the other one deals with the separation of isomeric C18 acids.

Not sure if they are actually on our website but if you contact me via e-mail I will sent them to you.

rhaefele@hamiltoncompany.com


cheers,

I was also thinking about the Aminex columns from Bio-Rad, but I wonder if they would do for higher molecular weight fatty acids (c10 to c18).

Probably not. That column works by a mixed mechanism of ion-exclusion and hydrophobicity (at the time I worked with it, we used the term "ion moderated partition", but that never caught on). Above about C5 or so, the retention times get unrealistically long; since the columns are resin-based, they are not a good candidate for ACN gradients.

Thanks all,

rhaefe, were those applications tried on complex mixtures like diatery food oils?

Tom, thank you for sharing your experience on Aminex.

Yannick

Unfortunately not. They are separations of standards to show feasabilty. What kind of sample prep are you looking at in order to extract the fatty acids from your matrix?

cheers,

Hi Rhaefe,

Thank you for pointing out to an important fact. This started me to think of 'in what form will my material be before injection' and 'how to avoid clogging'.

Here is partly an answer to your question:
The diatery oils will be fish oil, linseed oil and sunflower oil. For those oils we didn't yet decide on the fatty acid extraction mode.
We will also use plant and fermentation sources wich have already been extracted and resolved in hexane. The latter ones have successfully been analyzed with a GS-FID.

If I could refine what we want to do, it would sound like:
Finding a preparative separation method for poly unsaturated fatty acid mixtures.

Regarding what to put on the column:
It is our intention to get those complex FA mixtures in a hexane solution prior to further analysis.

I hope this refines and make our intention more clear.

My question about 'has the column been tested on complex mixtures' is mostly stressed around the fear that the column wouldn't give enough resolving power for the separation of those complex mixtures (especially when they contain different fatty acids with equal chain length but different saturation degrees).

Thank you for your valuable input.

Yannick