System suitability injections: if they failed, the run did not happen — the long version

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.

Gradient slope is the single biggest driver of apparent purity differences. A shallower gradient over a longer run resolves more impurities and gives a higher purity figure. A steep gradient produces a tidier-looking chromatogram with fewer visible peaks and gives a lower purity figure. Both are legitimate methods and they will not produce the same number.

post #2 is right about the mechanism and I think understates the practical bit.

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.

Thank you for the correction. I have edited my earlier post with a note rather than silently, so the thread still makes sense to read. The error was mine and it was the kind that comes from remembering a figure instead of looking it up.

On post #3 — agreed on the reasoning, with one qualification.

Method validation is the demonstration that a method can separate the compound from its degradation products and impurities reliably. A method that cannot resolve an impurity from the parent peak will not detect that impurity.