Chromatography Forum

I'm developing a new method for measuring multiple compounds that are difficult to separate.

There is a report from Waters on the targeted FFA analysis where they were able to quantify those FFA despite the poor separation.

If the software (Analyst in my case) allows extracting a single peak from the overlaid mix, does this mean that one can neglect the selectivity of the LC column?

Ex. page 3
https://www.waters.com/webassets/cms/li ... 6464en.pdf

If your compounds are ionizable, mixed-mode columns will allow you to separate them based on small difference between hydrophobic/hydrophilic and ionic properties of your compound. Our columns are fully compatible with MS as they have ion-pairing reagent attaches to the surface . Contact me if you need help with your separation.

I'm developing a new method for measuring multiple compounds that are difficult to separate.

There is a report from Waters on the targeted FFA analysis where they were able to quantify those FFA despite the poor separation.

If the software (Analyst in my case) allows extracting a single peak from the overlaid mix, does this mean that one can neglect the selectivity of the LC column?

Ex. page 3
https://www.waters.com/webassets/cms/li ... 6464en.pdf

Are you doing it as full scan or MRM?

If there are unique masses or the MRM is unique to each analyte you can separate them on the MS side without separating them on the HPLC side. The nice thing about MS is the ability to separate different analytes that co-elute so long as they are not isomers of each other.

I'm developing a new method for measuring multiple compounds that are difficult to separate.

There is a report from Waters on the targeted FFA analysis where they were able to quantify those FFA despite the poor separation.

If the software (Analyst in my case) allows extracting a single peak from the overlaid mix, does this mean that one can neglect the selectivity of the LC column?

Ex. page 3
https://www.waters.com/webassets/cms/li ... 6464en.pdf

Are you doing it as full scan or MRM?

If there are unique masses or the MRM is unique to each analyte you can separate them on the MS side without separating them on the HPLC side. The nice thing about MS is the ability to separate different analytes that co-elute so long as they are not isomers of each other.

Thank you very much for the explanation! Yes, all my MRM transitions are unique and optimized for each compound.

If your compounds are ionizable, mixed-mode columns will allow you to separate them based on the small difference between hydrophobic/hydrophilic and ionic properties of your compound. Our columns are fully compatible with MS as they have ion-pairing reagent attaches to the surface. Contact me if you need help with your separation.

Thanks! I will look into that.

Yes, but with risks.
Yes, you can get a signal from the mass spec that is unique to your compound, and quantify this, without separation. However, if you're using electrospray you can get competition between coeluting compounds, where they affect one another's ionization efficiency. If A and B coelute, a large peak of A can reduce the apparent size of B.

Ideally you'd compensate for this by using an internal standard chemically identical to B (i.e. an isotopically labelled version), which will coelute exactly, and be suppressed by A to exactly the same extent. But often internal standards aren't available.

So the answer is yes, but be careful. If you can get better separation, it's a good thing. But if you're working on complex mixtures, you will always get coelutions, even if they're coelutions with components of no interest to you. The problem with MRM methods is that you don't necessarily know what's coeluting, because the MS will only show what you've asked it to show.

Yes, but with risks.
Yes, you can get a signal from the mass spec that is unique to your compound, and quantify this, without separation. However, if you're using electrospray you can get competition between coeluting compounds, where they affect one another's ionization efficiency. If A and B coelute, a large peak of A can reduce the apparent size of B.

Ideally you'd compensate for this by using an internal standard chemically identical to B (i.e. an isotopically labelled version), which will coelute exactly, and be suppressed by A to exactly the same extent. But often internal standards aren't available.

So the answer is yes, but be careful. If you can get better separation, it's a good thing. But if you're working on complex mixtures, you will always get coelutions, even if they're coelutions with components of no interest to you. The problem with MRM methods is that you don't necessarily know what's coeluting, because the MS will only show what you've asked it to show.

Thank you for the detailed explanation, much appreciated! I will try to improve my separation then so that I can be sure that nothing is suppressed.