Chromatography Forum

When a target molecule shows no interaction with the column, quantification of the resulting void peak seems easy to threat as a regular peak. My question: is this allowed for quantification?

Context:
From several molecules, all are well separated based on their interaction, except one. Its structure is different and shows - as expected - no retention with the column and elutes at the dead time of column. The complete instrumentation shows no form of contamination: when running blanc injections of mobile phase, absolutely no baseline distortion or dead peak is seen (which could be a first pitfall).

Can I be confident of this method or does chromatographic etiquette subscribes a minimum retention for robustness?

Thanks for your experience and opinion.

My gut tells me that unretained species shouldn't be used for quantification unless you have a detector which offers some specificity to verify the peak is what you expect --an MS for the specific M/Z or a PDA with the UV-Vis fingerprint. Otherwise, you may be quantifying a contaminant or different species. Even with the specific detector, I'd try for some retention.

Can you do a 2D separation? Or mixed mode columns?

Thanks for your response.

Detector is specific (MS) with an absolute garantuee of identifying the specie. It is invisible when not injected, and appears steady (with acceptable peak shape) when injected. 2D separation is not possible (in my opinion), except when done off-line.

The question relates more to the chromatographic part. Where are the chromguru's?

If the analyte is not retained by the column you could just as well do away with the column and determine it by direct infusion to the MS - you then have a wider range of solution chemistries to play with to enhance detectability.

Peter

Can I be confident of this method or does chromatographic etiquette subscribes a minimum retention for robustness?

Of course it does. Non-retained compounds are per definitionem not part of the chromatographic analysis and should therefore usually excluded von the evaluation of the chromatogram (integration).

Particular for quantification with mass spectrometer the chromatographic separation is often necessary. Even when you are using a mass spectrometer, hands-on one will often observe many unforeseen compounds, which may also elute during the dead-time. But this other compounds (e.g. solvents) can have great influence to the ionization in the electrospray-process (I’m assuming that you use ESI) and this will falsify your results.

Even high-resolution MS cannot substitute a proper chromatographic separation since the electrospray process is very complex. Also with only UV-detectors I would really exclude peaks at dead time from the evaluation.

I guess I would disagree with most of what's been said. Certainly it is true that quantitating an unretained peak is generally not a good practise in chromatography. And the reason for this is that you have no selectivity between the unretained peak and whatever other unretained garbage may be coming out in the void. However:

- Anything that you can validate, and therefore prove that it works, can be used. So I might think it would be OK, even if you had just a UV detector (I say "might" because, there would still be a concern about "junk" showing up at some point in the future, and interfering)

- But since you have a mass spec detector, you have essentially solved the problem of poor selectivity. Because you have another mode of resolution built in.

In your situation, I would not hesitate to use the method.

Anything that you can validate, and therefore prove that it works, can be used. So I might think it would be OK, even if you had just a UV detector (I say "might" because, there would still be a concern about "junk" showing up at some point in the future, and interfering)

But for most of the possible applications, there will be a lot of work necessary to demonstrate the specificity of the method for an unretained substance. (We should remember, these compounds are par definitionem not a subject of the chromatographic method).


CDER Reviewer Guidance - Validation of Chromatographic Methods - 1994

Recommendations:
The peak should be well-resolved from other peaks and the void volume. Generally the value of k’ is > 2

But since you have a mass spec detector, you have essentially solved the problem of poor selectivity. Because you have another mode of resolution built in.

Sorry, but this demonstrates a (common) misunderstanding of the technique. Of course the mass spectrometer give some selectivity. But the ionization process in the spray chamber is of course influenced by coeluting substances especially solvents or ‘salts’ which are usually eluting also during dead time.