High-performance liquid chromatography (HPLC) is the standard method for measuring peptide purity. It pushes a dissolved sample through a packed column under pressure. Different molecules travel through the column at different speeds, so they come out separately and can be measured one by one.
The purity percentage
The headline number on most COAs is purity, such as 99.5%. This means about 99.5% of the material the detector measured is the target peptide. The remainder may be closely related sequences, incomplete chains or synthesis by-products.
Purity is measured by peak area, usually with a UV detector at a set wavelength (often 214 or 220 nm). It describes the peptide content relative to other detected material, not the total weight of the vial.
Reading the chromatogram
The chromatogram is the chart on the report. The horizontal axis shows time, and the vertical axis shows signal strength. Each peak represents a substance leaving the column.
A clean sample shows one large main peak with few or very small side peaks. The time when the main peak appears is called the retention time. Many sizable side peaks suggest lower purity.
Mass spectrometry confirms identity
HPLC tells you how pure a sample is, but not exactly what it is. Mass spectrometry (MS) fills that gap by measuring molecular weight. The report will usually list an expected (theoretical) mass and an observed mass. Values that closely match confirm the peptide's identity.
A strong COA reports both HPLC and MS, so purity and identity are each confirmed.
Quantity vs. purity
Purity and quantity are different measurements. A vial can be very pure but contain slightly more or less material than labeled. Some labs report a measured amount in milligrams, which lets you compare it to the label. Small variation is normal in peptide manufacturing.
Putting it together
When reviewing a report, check that the lot matches your vial, purity is reported with a method, mass spec confirms identity, and the measured amount is close to the label. Our COA Library lists each of these where the lab provides them.