Understanding HPLC Purity: What ≥99% Actually Means

When a peptide COA reports "HPLC ≥99%," what is actually being measured?

The measurement

High-performance liquid chromatography separates a sample's components by their affinity for a stationary phase (typically C18 reverse-phase). A UV detector at 220 nm — the wavelength at which peptide bonds absorb — quantifies each peak by integrated area. The "purity" figure is the area of the target peptide peak divided by the sum of all detected peak areas, expressed as a percentage.

What ≥99% does and does not tell you

It does tell you:

• The main peak dominates the chromatogram.
• Related peptide impurities (truncated sequences, deletion sequences, oxidized variants) are below approximately 1% of total absorbance.

It does not tell you:

• Identity — that is what mass spec is for.
• Endotoxin content, residual solvents, or counterion mass — those need separate assays.
• Whether the peptide is what the label claims.

Why ≥99% is the working standard

Below 98%, related impurities can start meaningfully perturbing biological readouts. Above 99%, the primary limitation on assay interpretation shifts to identity and contaminants outside HPLC's window. Most reputable research peptide suppliers therefore target ≥99% and disclose the raw chromatogram.