Is 214 nm or 280 nm the right detection wavelength for a purity number?

The method section is present, which is unusual enough that I want to make use of it.

These are treated as interchangeable and I do not think they are.

If both are acceptable I would like to know that, so I can stop thinking about it.

Is there a defensible reason to prefer one, or is this a coin flip?

Put another way, peak shape carries as much information as peak area does, and a badly tailing peak or a shouldered peak is telling you something about the sample or the column that matters.

Reverse-phase stationary phases use C18 or C8 chains bonded to silica, and the pore size of the silica matters more for a peptide of this chain length than the ligand length does.

The relevant detail is that temperature affects both the viscosity of the mobile phase and the dynamics of molecular interactions, and a method developed at 25 degrees and run at 40 degrees will not behave identically.

One qualification: the limit of detection on a reversed-phase HPLC is set by the noise on the baseline, which for these molecules is usually in the range of a tenth of one per cent or less, and anything smaller is not reproducibly detectable.

The practical summary: specify the method, run the same method on every sample you compare, and use orthogonal techniques to confirm the result.

On the detail: coupling the chromatography to mass spectrometry adds identity information but trades chromatographic quality for ionisation efficiency.

Wider-pore phases — 300 Angstrom rather than 100 Angstrom — have faster mass transfer and narrower peaks for peptides above three kilodaltons, which is almost every peptide you will see.

Sample preparation is almost always under-appreciated — a reconstituted peptide in strong solvent will distort its own peak on the gradient.

The limitation is that co-elution is invisible — if two species happen to have the same retention time, they will report as a single peak at their combined area.

If two labs give different numbers, the method difference is the first hypothesis, not lab quality.

The most important parameter is the one that is almost never specified: the gradient slope during the region where your main peak elutes.

Acetonitrile is the organic modifier of choice because it has a good UV cutoff, a reasonable viscosity and a refractive index that minimises baseline noise.

Inter-laboratory studies using identical methods on identical material show precision well within half a per cent when the method is locked down, pointing to method variability as the primary source of disagreement.

I would caution against over-interpreting small differences between runs, because the noise floor of the method is larger than most people assume.

Ask for the chromatogram and the system suitability data, not just the number.

High-performance liquid chromatography is a separation technique, not an identification technique, and it answers the question "how much of the detected signal is a single species" not "which species is it."

Proline-rich sequences are particularly problematic because the isomerisation kinetics are in the same timescale as the separation, leading to split or broadened peaks at low temperature.

The practical summary: specify the method, run the same method on every sample you compare, and use orthogonal techniques to confirm the result.