Coming back to: Column chemistry choices for a 40-residue peptide

On the subject in the title: Column chemistry choices for a 40-residue peptide Working notes rather than a conclusion.

Posting the method first, because I know what the first three replies will otherwise be.

• Column: C18, 3.0 x 150 mm, 2.6 um
• Mobile phase: 0.1% TFA in water / 0.1% TFA in acetonitrile
• Gradient: 6% to 55% organic over 29 minutes
• Detection: 280 nm
• Injection: 14 uL
• Sample: semaglutide, reconstituted to 2.0 mg/mL, injected within an hour

The main peak integrates at 97.3% of total area. There is a small feature on the trailing edge that I cannot decide is a shoulder or a baseline artefact, and that is what I am actually asking about.

Coming back to the opening post, because the follow-up matters more than the original answer.

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.

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.