Coming back to: Resolution and tailing factor: real acceptance criteria posts 31–60

Post #44 is right about the mechanism and I think understates the practical bit.

The strongest argument against my own position on Resolution and tailing factor, stated as well as I can state it, since nobody else has yet.

Reversed-phase separates on hydrophobicity. A peptide is retained on a non-polar stationary phase and eluted by increasing organic solvent. For peptides the mobile phase almost always contains an ion-pairing acid, typically 0.1% TFA, which suppresses secondary interactions and sharpens peaks.

The short answer was in the first line; everything after is the working.

Distinguishing three things in the Resolution and tailing factor discussion that keep getting used interchangeably: the observation, the proposed mechanism, and the recommendation that gets attached to both.

On post #46 — agreed on the reasoning, with one qualification.

What I would tell a new member reading about Resolution and tailing factor for the first time: the confident posts are not the reliable ones, and the reliable ones are longer.

Column temperature affects retention and selectivity and is omitted from most certificates. Two runs at twenty-five and forty degrees are not the same method.

Speaking for myself and not for anyone else who has posted here.

This is why a purity figure without the underlying chromatogram is weaker evidence than it appears. It is also why two competent laboratories can report different numbers on the same vial without either being wrong.

The variance between people here is larger than the effect being discussed.

Reading rather than contributing, but this is the most useful thread I have found on it.

Resolution and tailing factor: I would want to see the raw numbers rather than the summary before agreeing. Summaries lose exactly the information that would settle this.

Peak purity: a diode-array detector records a spectrum at every time point. If a peak contains two co-eluting species with different spectra, the spectrum changes across the peak. A passing peak-purity result says the spectrum is constant; it is weak evidence of homogeneity if the impurities have similar spectra.

I would put the burden of proof on the interesting explanation, not the dull one.

Where I part company with post #53, and it is a narrow parting.

The documentation on Resolution and tailing factor is better than this thread and I say that as someone who has posted in the thread.

Post #53 is the version of this I will quote in future. One addition.

Reframing Resolution and tailing factor slightly, because I think the disagreement is about the question rather than the answer. If the question is "does it happen", yes. If it is "how often", nobody here knows.

Detection at 214 nanometres sees the peptide bond and therefore sees almost everything peptidic. At 280 it sees aromatic residues, so a peptide without tryptophan or tyrosine will look very different or not appear at all.

I checked the source rather than the summary, and they differ.

Resolution and tailing factor has a well-known answer and a correct answer, and the interesting work is establishing that they are the same. Nobody has done that here yet.

Post #57 put the caveat in the right place and I want to underline it.

What I would check first on Resolution and tailing factor is whether the thing being measured moved or whether the way of measuring it moved. Those look identical in a graph.

Trifluoroacetic acid at 0.1 per cent is the near-universal ion-pairing additive for this work, and it also raises the baseline at 214 nanometres. That is why the same sample looks noisier at low wavelength.

The answer changed when I changed how I was measuring, which was informative.