High-performance liquid chromatography separates a sample into its components by pushing it through a packed column under pressure. Different molecules travel at different speeds, so they exit the column at different times and register as separate peaks on a detector trace. Purity is reported as the area of the main peak divided by the total area of every peak.
Why the number alone is not enough
Purity by area percent depends entirely on what the detector can see. A UV detector at 214 nm sees peptide bonds well and sees residual salts, solvents, and water not at all. That means a peptide can be 99% pure by HPLC area and still be, by mass, substantially acetate salt and bound water. This is normal and expected — but it is why net peptide content is a separate figure from chromatographic purity.
- Chromatographic purity — the area-percent number, typically ≥98% or ≥99% for research-grade material.
- Net peptide content — the fraction of the vial mass that is actually peptide, usually 70–90% for a TFA salt.
- Mass confirmation — MS data proving the main peak is the sequence you ordered, not a similar-mass impurity.
What to look for in a chromatogram
Ask for the trace, not just the summary. A clean chromatogram shows one dominant, symmetrical peak with a flat baseline. Shoulders on the main peak often indicate deletion sequences — chains missing a single residue — which are the hardest impurity class to remove because they behave almost identically to the target on a column.
Two vials can both say 99%. One was measured against a full trace at 214 nm; the other was measured against a window that happened to exclude the impurity peak.
Third-party versus in-house testing
In-house HPLC is standard and legitimate — most manufacturers run it as a release test. Third-party testing adds an independent party with no commercial interest in the result. For high-value or long-running research, periodic third-party verification of a batch you have already been using is a cheap insurance policy against a silent process change upstream.
On Likwid, every supplier publishes the same data points: testing method, whether a COA is on file, whether batch traceability is available, and typical lead time. The point is not to declare a winner. It is to make the comparison take ten seconds instead of an afternoon.