How to read an HPLC chromatogram (peptides) — a quick QC guide

This plain-English QC guide explains what a peptide HPLC trace shows, how purity (area%) is calculated, why results are method-dependent, and the common artifacts to watch for. It also outlines exactly what Tide Labs publishes on batch pages inside the QC Hub.

Key takeaways

Chromatogram basics: x-axis = retention time; y-axis = detector signal (e.g., UV at 214–220 nm).
Purity (area%) = main peak area ÷ total peptidic area under that method. It’s an impurity profile, not identity.
Method matters: column, gradient, wavelength, flow, temperature and injection solvent all move peaks.
NPC ≠ purity: Net Peptide Content accounts for water/counter-ions; purity does not.

Anatomy of a chromatogram

Retention time (Rt): when a component elutes. Useful for comparing runs on the same method.
Peaks: features formed as components elute; the area under each peak is proportional to amount detected.
Baseline: the “zero” line. A stable, flat baseline helps separate real peaks from noise.

Purity (area%) vs Identity vs NPC

Purity answers “what fraction of UV-absorbing peptidic material is the target peak on this method?” Identity requires orthogonal evidence (e.g., MS/sequence). NPC estimates how much of the powder’s mass is peptidic (subtracting water, counter-ions, residual solvent, excipients).

Purity is about the peaks. NPC is about the powder.

What changes a trace (why labs include method notes)

Column chemistry & dimensions (e.g., C18, particle size, length/ID).
Gradient (solvent A/B ramp, % organic, run time) and flow rate.
Detection wavelength (peptides commonly 214–220 nm) and temperature.
Injection solvent & concentration (can cause broadening or fronting if too strong).

Common artifacts & how to interpret them

Tailing/fronting: asymmetry from column overload, strong solvents, or interactions.
Shoulders/co-elution: partially resolved species; may affect purity if shoulders overlap the main peak.
Baseline drift: temperature/gradient effects or late-eluting background; check method blanks.
Noise spikes: electrical or injection transients; true peaks repeat across injections.

Reading checklist (practical)

Confirm method basics (column, gradient length, wavelength, flow, temp).
Locate the main peak and note retention time.
Scan for shoulders or nearby co-eluting peaks.
Check purity (area%) and whether total area excludes non-peptidic background where applicable.
Cross-reference any reported NPC (%) and how it was measured (e.g., elemental N + moisture).
Download the chromatogram PDF/image for your records.