Chromatography Forum

Hi there!
Can someone help me over here, please? I have a situation in which one of my analytes have a much lower area count than it's supposed to have. The chromatography has 2 components, but only the early eluter is the one that presented the problem, the last eluter is fine. Upon re-injection of the HPLC vial, the results are higher (what is expected). The rest of the parameters for syst suitability are ok.
Any ideas?

Thanks,
mochoa

How "early" is your "early eluter"? To be specific, what is the k' value?

I've seen this sort of thing happen with very low k' peaks running into issues with "t0 noise".

Hello Tom, thanks for replying. The k' is between 0.8-0.9. Please advise. Can you elaborate a bit more on the experience you've had similar to this scenario? Thanks, very much appreciated. Regards,
MOchoa

How "early" is your "early eluter"? To be specific, what is the k' value?

I've seen this sort of thing happen with very low k' peaks running into issues with "t0 noise".

My colleague John Dolan wrote up a similar problem in one of his LC-GC "LC Troubleshooting" columns:

LC-GC 13(9) 708-712 (1995)

I don't think LC-GC's on-line articles go back that far, so I'm sending you a copy via e-mail.

In that case, the early peak was occasionally significantly bigger, but same pattern as your problem: the anomaly ocurred only sporadically, and re-injection from the same vial looked OK. The early peak in that case was around k' 0.3, so earlier than what you're seeing. The problem with early eluters is that the system is "out of equilibrium" right around t0, and random artifacts are common. If you look at the FDA's "Guidance for Reviewers" document, they suggest that k' should be > 2.

For what it's worth, the actual cause of the problem in that article was never identified (I subsequently spoke to the reader who had submitted the problem). The early peak was an internal standard, which was actually counterproductive, so they went back to good old external standardization.

mochoa, how do you know that your first peak is your analyte and not a system peak?

In fairness: If one chose to have the external standard/analyte near "to" one should at least expect as much problems as with an internal standard near there.

HWMueller, this is a validated method that we use to quantify our pharmaceutical products. We know it's not a system peak. As far as k' for this analyte, our newer methods consider having k'>2 but this is a relatively old method.
Thanks for your comments,
MOchoa

mochoa, how do you know that your first peak is your analyte and not a system peak?

In fairness: If one chose to have the external standard/analyte near "to" one should at least expect as much problems as with an internal standard near there.

Tom,
Thanks again for your comments.
Best regards,
Mariela Ochoa

My colleague John Dolan wrote up a similar problem in one of his LC-GC "LC Troubleshooting" columns:

LC-GC 13(9) 708-712 (1995)

I don't think LC-GC's on-line articles go back that far, so I'm sending you a copy via e-mail.

In that case, the early peak was occasionally significantly bigger, but same pattern as your problem: the anomaly ocurred only sporadically, and re-injection from the same vial looked OK. The early peak in that case was around k' 0.3, so earlier than what you're seeing. The problem with early eluters is that the system is "out of equilibrium" right around t0, and random artifacts are common. If you look at the FDA's "Guidance for Reviewers" document, they suggest that k' should be > 2.

For what it's worth, the actual cause of the problem in that article was never identified (I subsequently spoke to the reader who had submitted the problem). The early peak was an internal standard, which was actually counterproductive, so they went back to good old external standardization.

When I was asking "how you know" I was expecting some unequivocal evidence.....