HPLC purity

What it measures

High-performance liquid chromatography (HPLC) separates the components of a dissolved sample. For peptides the detector is usually UV, often around 214–220 nm, and each component it sees appears as a peak. Area-percent purity is the target peptide's peak area divided by the total area of the integrated peaks, written as a percentage such as 99.2%.

Because it is a ratio of the peaks that method detects, purity leaves out anything that doesn't absorb at that wavelength, and it doesn't measure water, salts or counter-ions such as acetate or TFA.

An impurity that elutes at the same time as the main peak (co-elution) is counted as the target peptide, so a single purity figure can read higher than the true value. Results also depend on the column, gradient and wavelength used, so purity figures from different labs or methods aren't strictly comparable.

What it doesn't tell you

Purity is not quantity. A vial can show 99% purity and still hold more or less peptide than its label states; that amount is measured separately, usually as mg per vial against a reference standard.

Purity is not identity. HPLC on its own shows that there is a main peak; mass spectrometry (MS) checks that the peak's molecular mass matches the expected peptide.

Purity is not safety. Endotoxin, heavy metals, sterility and microbial contamination are separate tests.

Capsules and tablets

Oral products contain fillers and other ingredients, so a whole-product purity percentage isn't meaningful. Their certificates usually report identity and the amount of compound per capsule or tablet instead.