Where the integrator drew the baseline, and how much it moved the number

Where the integrator drew the baseline, and how much it moved the number — setting out what I have, and where I think it stops being reliable.

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

• Column: C8, 4.6 x 150 mm, 3.5 um
• Mobile phase: 0.1% TFA in water / 0.1% TFA in acetonitrile
• Gradient: 13% to 57% organic over 22 minutes
• Detection: 214 nm
• Injection: 20 uL
• Sample: tirzepatide, reconstituted to 0.5 mg/mL, injected within an hour

The main peak integrates at 98% 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.

Column chemistry and particle size: smaller particles (1.7 μm) give better resolution and higher efficiency than larger particles (3.5 μm or 5 μm), at the cost of higher back pressure. Newer methods increasingly use smaller particles.

System suitability testing: injections run before and during the sample run to establish whether the instrument, column and method were performing when the sample was analysed. If suitability did not pass, the sample results from that run are uninterpretable.

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.