When a vendor publishes a certificate of analysis showing "98% purity," that number almost always comes from high-performance liquid chromatography. It's a workhorse technique, used across pharmaceutical manufacturing, academic research, and third-party peptide testing labs. Knowing what HPLC measures, and what it can't see, lets you read those certificates more critically.
How HPLC Works
High-performance liquid chromatography separates the components of a mixture. It pushes a dissolved sample through a column packed with fine particles, under high pressure, using a liquid mobile phase. Different molecules travel through the column at different speeds, depending on how strongly they interact with the packing material. As each component exits the column, a detector records a signal proportional to absorbance. The most common is a UV/Vis detector set around 210–220 nm.
The result is a chromatogram, a graph of detector signal over time. Each peak corresponds to a distinct chemical species. The purity percentage is typically calculated as:
Target peak area ÷ total peak area × 100
So "98% purity" means the peptide of interest accounts for 98% of the UV-absorbing material detected in that run. It's an area-percentage, not a mass fraction. As Bachem notes in its quality-control guide, purity is "assessed by UV detection at 210–220 nm, where the area of the main peak in relation to the total area of all peaks reflects the peptide purity."
What the Percentage Means, and Its Limits
A high HPLC purity figure is meaningful. It also comes with caveats:
| What HPLC detects well | What HPLC may miss |
|---|---|
| Truncated sequences and deletion peptides | Impurities that do not absorb UV at the chosen wavelength |
| Oxidised or deamidated variants | Water, residual solvents, counter-ions (e.g. TFA or acetate) |
| Related peptide by-products | Inactive aggregates that co-elute with the main peak |
| Solvent-related carry-over | Endotoxins or microbial contamination |
The calculation is area-based. Substances that absorb weakly at 214 nm contribute little to the denominator, and that includes some counter-ions and many non-peptide process impurities. A sample could show 99% HPLC purity while still containing meaningful levels of material the detector simply cannot see. That's why pharmaceutical-grade testing pairs HPLC with other methods: mass spectrometry (MS) to confirm molecular identity, Karl Fischer titration for water content, ion chromatography for residual TFA or acetate counter-ions, and limulus amebocyte lysate (LAL) assays for endotoxins.
The column chemistry matters too. Reversed-phase HPLC on a C18 column is the most common setup for peptides, and it separates primarily by hydrophobicity. Two structurally similar impurities that differ only in charge might co-elute with the target peak and inflate the purity reading. Some labs run orthogonal methods such as ion-exchange or size-exclusion chromatography to cross-check.
Reading a Third-Party Certificate of Analysis
Independent labs such as Janoshik Analytical run HPLC alongside mass spectrometry and publish results as certificates of analysis. Janoshik prints a unique key on each report that can be checked at its verification portal. The portal returns the original HPLC purity figure, the mass-spec identity result, and the raw chromatogram, so a vendor cannot quietly alter or fabricate a published result. When evaluating a CoA, researchers commonly look at a few things:
- Retention time confirms the compound elutes where expected for that peptide.
- Peak shape. A sharp, symmetrical peak suggests a homogeneous compound; tailing or shoulders may indicate impurities co-eluting.
- Wavelength used. 214 nm is standard for peptide backbone absorbance; 254 nm is common for aromatic residues.
- Instrument and method disclosure. A credible CoA names the column, gradient, and flow rate used.
Aggregators sit a layer above the labs. Finnrick.com commissions independent HPLC testing, often on anonymised samples, and displays raw chromatogram images alongside numeric purity values. As of mid-2026 it reports having published several thousand tests across more than two hundred vendors, which allows a degree of cross-lab comparison. Peptigrity.com is a similar review platform. It does not run tests itself but maintains a directory of accredited third-party labs and a searchable database of their published results. Bachem and PolyPeptide Group, two contract manufacturers supplying pharmaceutical-grade peptides, publish method information showing how HPLC is used alongside MS and amino-acid analysis in regulated production.
Why It Matters for Research Peptides
Peptides sold for research use, not approved for human consumption, are typically characterised primarily by HPLC. A published purity figure is a starting point. It is not a complete quality picture. HPLC purity reflects UV-detected chemical homogeneity under one set of conditions; complementary data such as MS confirmation of molecular weight and endotoxin testing fills the gaps the chromatogram alone cannot address. Researchers working in laboratory settings are better positioned when they keep that distinction in mind.