Laboratory quality
HPLC
High-performance liquid chromatography — the separation technique that generates most peptide purity numbers, and what its output actually says.
- Last updated:
- 2026-07-22
Overview
High-Performance Liquid Chromatography (HPLC) is a separation technique that pushes a dissolved sample through a packed column at high pressure. Different components in the sample interact differently with the column and elute (come out) at different times. A detector at the column outlet produces a peak for each component.
For peptide analysis, HPLC is typically run in reverse-phase mode with ultraviolet detection at around 214 nm (the wavelength at which peptide bonds absorb strongly). The result is a chromatogram: a graph of detector signal versus time, with one main peak (the peptide) and, ideally, small or absent peaks elsewhere (impurities).
Why it matters
Almost every peptide purity figure on a CoA comes from HPLC. Understanding what the number represents makes it easier to read a CoA as evidence rather than as marketing.
Key concepts
Area percent
The purity number on a peptide CoA is usually "area percent" — the area under the main peak divided by the total area under all peaks. "98% purity" therefore means 98% of the UV-detected material has the main peak's retention time.
Method dependence
Different HPLC methods can produce different purity numbers for the same sample. A well-drafted CoA specifies the column, gradient, and wavelength.
Detection limits
UV detection sees only what absorbs at the chosen wavelength. Impurities that do not absorb well at 214 nm may be present without producing a peak.
Common misconceptions
- "HPLC purity" is not chemical purity. It is the fraction of UV signal attributable to one peak under one method.
- "99% pure" is not the same as "free of impurities." The remaining 1% may contain related peptides (deletion sequences, oxidation products) with their own biology.
- HPLC does not measure sterility, endotoxin, or mass. It measures composition of the peptide-related material dissolved in the injection.
Practical interpretation
When a CoA reports HPLC purity, read the number alongside the method. A higher purity number under a more discriminating method is more evidence than a higher purity number under a less discriminating one.
Limitations
HPLC alone does not confirm identity. Identity is typically confirmed by mass spectrometry (see "LC-MS", "Identity testing").