Why I ask for the gradient before I discuss the number

Building on post #12 rather than restating it.

A shoulder on the trailing edge is most often a closely related species rather than an artefact. The way to find out is to change the gradient slope, not to argue about the integration.

A chromatogram image at a resolution where you can see peak shape but not baseline detail is worth having and is not the same as the data. Ask for the integration table if the number matters.

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.

It is the sort of thing that seems obvious in retrospect and was not at the time.

If two laboratories disagree by more than two or three percentage points, work through method, integration, sample handling, whether it was the same lot and the same vial, and whether suitability passed. After all five, a gap needs an explanation.

That has been true for the cases I have seen and I have not seen many.

Purity by chromatography answers "what proportion of what I detected is the intended species". It does not answer how much is in the vial, which is a separate assay.

Where the baseline is drawn is the least documented and most consequential choice in the whole determination. Valley-to-valley, tangent skim and forced-to-zero can span a couple of percentage points on the same chromatogram.

The uncertainty is in the assumption, not in the calculation.

Change the wavelength and the proportions change even though the sample has not. That is the reason the wavelength has to be on the document for the number to mean anything.

The short version is the first sentence; the rest is why.

Before anything else: what was the gradient, and at what wavelength? Area percent at different wavelengths is not the same number even on the same sample because different species absorb differently at different wavelengths. With the method stated, I can tell you something useful. Without it, all I can say is that there is one large peak.

Posted with less confidence than the sentence structure implies.

Post #19 describes the usual case. This is about the unusual one.

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.

Adding the measurement that post #23 says would settle it.

Injection volume matters because column overload distorts peak shape, and an overloaded main peak can swallow a small neighbour. A certificate without injection volume is missing something load-bearing.

It is the kind of thing that is obvious once and never again.

Detection wavelength: 214 nm detects the peptide bond and is relatively insensitive to composition. 280 nm detects aromatic residues and is strongly composition-dependent. Area percent at one wavelength is not area percent at the other.

Reading it back, the second half matters more than the first.

Fine by me. I had wanted a stronger conclusion and there is not one available.

On post #23 — agreed on the reasoning, with one qualification.

Gradient delay volume differs between instruments and shifts the whole chromatogram. It is why a transferred method rarely reproduces retention times exactly on a different system.

A partial answer, offered because a partial answer beats none.

Particle size and column dimensions determine what resolution is achievable at all. A 5 micrometre 250 millimetre column and a sub-2 micrometre 100 millimetre column are different instruments in practice.

I would want to see it done twice before believing it once.

Retention time is only comparable within a laboratory on a given method. Quoting a retention time across two reports as evidence of identity is not a valid comparison.

Post #27 is right about the mechanism and I think understates the practical bit.

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.