"≥99% HPLC" is the most quoted number in the peptide market. It is a useful number — if you know exactly what it measures.
How the test works
Most peptide purity tests use reverse-phase HPLC:
- A small amount of the sample is dissolved and injected into a column packed with a hydrophobic material (often C18).
- A gradient of water and acetonitrile, usually with a little trifluoroacetic acid, pushes the sample through the column.
- Different molecules leave the column at different times, depending on how strongly they stick to it.
- A UV detector, usually set at 214–220 nm where the peptide bond absorbs, records each compound as a peak.
What the percentage means
The software measures the area of every peak. Purity is the area of the main peak divided by the total area of all peaks:
purity (%) = main peak area ÷ total peak area × 100
So "99.2%" means that 99.2% of the UV signal at that wavelength came from the main peak.
Which impurities it finds
HPLC separates the target peptide from related impurities formed during synthesis or storage, for example:
- deletion sequences — peptides missing one amino acid;
- truncated sequences — chains that stopped growing early;
- oxidised forms — such as oxidised methionine;
- deamidated forms — asparagine or glutamine changed during storage.
What HPLC purity does not tell you
- Identity. A pure peak can still be the wrong molecule. Identity needs mass spectrometry.
- Quantity. Purity is a ratio, not an amount. The powder also holds water and counter-ions, so a 10 mg vial of 99% pure material does not contain 9.9 mg of peptide automatically. Look for an assay or net peptide content.
- Endotoxin or microbial quality. These need separate tests.
- Anything the detector cannot see. Substances that do not absorb at the chosen wavelength do not appear as peaks.
Method details matter
Purity depends on the column, gradient, wavelength and integration settings. A good report states the method and shows the chromatogram, so the result can be checked.
How R3TA reports it
Each R3TA batch is tested for purity by HPLC and for identity by mass spectrometry at an independent laboratory. The report for every batch is on the lab reports page. See also how to read a certificate of analysis.