HPLC, plainly explained

High-performance liquid chromatography is how nearly every purity number on a Certificate of Analysis gets produced. The principle is simpler than the name suggests.

The idea

A sample is dissolved and pushed under high pressure through a column packed with fine particles. Different molecules interact with that packing to different degrees. Ones that cling travel slowly; ones that do not move through quickly. By the time the sample exits, its components have separated in time — arriving one after another rather than all at once.

A detector at the far end watches what emerges. For peptides this is usually ultraviolet absorbance, often at 214 nanometres, where the peptide bond itself absorbs. The detector's output plotted against time is the chromatogram.

Reading the chromatogram

Each peak is a component. Its position — the retention time — reflects how strongly it interacted with the column, and is characteristic of that molecule under those exact conditions. Its area reflects how much of it was present.

Purity is calculated from those areas: the target peak's area as a percentage of all peak area combined. A result of 98% means the main peak accounts for 98% of everything the detector saw.

The limitation worth knowing

That last distinction matters. Anything the detector cannot see does not appear in the calculation. UV absorbance at 214 nm responds to peptide bonds — so residual solvents, salts, and water are largely invisible to it. A peptide that is 98% pure by HPLC area can still be a substantially smaller fraction of the powder's actual mass.

This is why purity and mass content are separate entries on a well-formed certificate, and why a supplier reporting one without the other has told you less than it appears.