HPLC, explained without the chemistry degree.

What HPLC does

HPLC pushes a dissolved sample through a tightly packed column under high pressure. Different molecules interact differently with the column's chemistry and exit (elute) at different times. A detector records what comes out and when, producing a chromatogram — a series of peaks over time.

For peptides, the standard setup is reversed-phase HPLC: a C18 column (a surface the peptides stick to) and a water-to-organic solvent gradient that peels different species off in order of how strongly they bind.

Reading the chromatogram

• The main peak is the target peptide — it should dominate.
• Small peaks flanking or following it are impurities: truncated sequences, deletion products, oxidized or degraded material.
• Area % — purity is computed from the area under each peak as a share of total area. "≥99% by HPLC" means the main peak was at least 99% of total area. This is the purity number discussed in peptide purity.

What HPLC can't tell you

• What the impurities are. A peak is a shape, not an identity. Identifying peaks requires LC-MS or identity testing.
• Mass. HPLC says nothing about how much of the vial is peptide versus water and salts — that's peptide content.
• Safety. HPLC doesn't detect endotoxin (endotoxin testing) or confirm sterility (sterility testing).

Variants you'll see on COAs

• Analytical RP-HPLC — the standard purity method described above.
• Preparative HPLC — same physics, bigger column, used to purify the batch rather than measure it.
• HPLC at two wavelengths — some impurities show only at specific UV wavelengths; dual-wavelength runs catch more than a single trace.

What a good HPLC result looks like on a COA

A stated method (e.g., "RP-HPLC, C18, 214 nm"), a chromatogram or a clear area-percent result, batch- and date-specific to your material. For the document-level checks, see how to read a peptide COA.