Chromatography Forum

Hi all,

Iam doing a FAME analysis and iam using the standard of supelco 37 mix (10mgs/ml).
Iam using DB wax 10 meter column and iam getting some 30 peaks but unable to identify it with the reference chromatogram which is there with standard. I know i used some conditions different from what they mentioned like FID temp 280 deg, ramp temp started with 90 deg C, carrier gas is used is helium.
Please guide me how to quantify my samples which is omega 3 fatty acids.
please encourage me with your valuable suggestions

Checking in the Supelco catalogue I found two pieces of information of interest to you.

1-The Supelco 37 component FAME Mix is listed as a "qualitative" standard meaning it is meant for identification purposes only. It is not a "quantitative" standard.

2-You can contact technical services at techservice@sial.com for help with the chromatographic analysis.

Hope this helps

Thank you for your reply..

As skundek_once said, the Supelco 37-mix compound is a qualitative standard for identification purpose of FAMEs, although you can use it for quantitative analysis, since total wt is given, but it not a 'certified' quantitative standards. You'll have to buy the individual FAME standards if you want a more regulated standard for quantification (they have to use NIST traceable weights to prepare those standards).

Your column may be too short for the 37 FAME mix. Some labs use columns length as long as 100-m for an adequate separation. I've seen good separations with 15-m of EQUITY-1, probably just a couple of co-elutions (C22's). Most labs as far as i know use the cyanophase for FAME analysis with good results, or also WAX phases, but you have to be more careful with those, as any leak my degrade the columns quicker. Some of the new ionic liquid-phases are also good for FAME analysis, like SLB-IL 111 or SLB-IL 60.

But I'll probably change the column length first

We used this Supelco 37 mix on a 30M x 0.25mm x 0.25 um df column, with N2 as a carrier gas. It can produce all the peaks as indicated on the reference chromotograph.

Search Supelco/Sigma's website, they have chromatographs using different carrier gas (H2, and Helium, but not N2) on Supelco Omegawax column (I suppose they are similar to your DB WAX).

Hi all,

Iam doing a FAME analysis and iam using the standard of supelco 37 mix (10mgs/ml).
Iam using DB wax 10 meter column and iam getting some 30 peaks but unable to identify it with the reference chromatogram which is there with standard. I know i used some conditions different from what they mentioned like FID temp 280 deg, ramp temp started with 90 deg C, carrier gas is used is helium.
Please guide me how to quantify my samples which is omega 3 fatty acids.
please encourage me with your valuable suggestions

Thanks Tigeria,
The the thing is even i also got the peaks but quantification of the omega three fatty acids is the issue here.
How to quantify the omega three fatty acids with 37 mix as standard...

..... quantification of the omega three fatty acids is the issue here.

How to quantify the omega three fatty acids with 37 mix as standard...

THE ANSWER IS: YOU CANNOT DO THIS. As it is not a certified standard. Why?

The omega three fatty acids are not stable compounds. They oxidize very easily and degrade.

The easiest solution is to buy small neat amounts of certified individual fatty acids methyl esters you wish to measure and prepare your own standard solutions. Be aware that if you try to esterify these yourself it is easy to achieve partial recovery due to the reactivity of the unsaturated fatty acids. In other words, 100% going in may only give you 90% coming out.

Verify the relative response factors for each fatty acid against stearic acid methyl ester or some other fatty acid that is more stable.

Run this fatty acid standard against your samples daily or for every sample set.
Make new omega three standards weekly or monthly from NEW certified components and track your results.

If you ARE REQUIRED to furnish proof of your results then pay the price. It is the cost of doing business. If you only want answers, then determine the response factors of each acid against stearic acid or some other fatty acid that is more stable and just report answers. But don't expect the results will be accepted in a court of law as accurate.

Getting answers is easy. Getting legal accurate answers is not. That is why we are paid the big bucks.

best wishes,

Rod

is it possible to quantify the omega 3 acids by comparing their areas versus a known internal standard? Isn't FID an equimolar detector?

"Isn't FID an equimolar detector?"

No, it is not. An HID is closer however. For some CLASSES of compounds FID can be close.

But either it is or it isn't. That is why we have response factors, because it isn't.

Compare ethane and hexane on a equimolar basis and see the difference in response area.

best wishes,

Rod

is it possible to quantify the omega 3 acids by comparing their areas versus a known internal standard? Isn't FID an equimolar detector?

In principal, and with a few controls built in, it would be possible to determine what mass fraction of the total esters was due to the esters of the target acids by calculating what % of the total peak area was contributed by their peaks. This is not the same as determining the mass fraction of those acids in the original sample - which is what I think that the OP wants to do. In this scenario the only contribution that the standard makes is to determine the retention times of the target esters.

Peter