High-Performance Liquid Chromatography (HPLC)

High-performance liquid chromatography pumps a liquid mobile phase at high pressure through a column packed with small stationary-phase particles. Components partition between the two phases to different extents and so leave the column at different times, and a detector records response against time to give the chromatogram. Purity by this method is an area percentage: the area of the main peak divided by the total area of all integrated peaks. Ultra-high-pressure systems use sub-two-micron particles to sharpen peaks and shorten runs, on the same principle.

Peptide work is almost always reversed-phase, on a C18 column with a water and acetonitrile gradient acidified with trifluoroacetic acid, and detection is normally near two hundred and fourteen nanometres because that is where the amide bond of the backbone absorbs. The alternative near two hundred and eighty nanometres sees only tryptophan and tyrosine, so a peptide lacking aromatic residues is nearly invisible there. That single setting can change a reported purity dramatically without anyone falsifying anything.

What the technique answers is narrow: of the ultraviolet-absorbing material that eluted within this run, what fraction was the main peak. It is not a quantity, not an identity, and not a statement about stability. Coupled to a mass spectrometer it becomes an identity method; run against a characterised reference standard it becomes an assay for content.

The near-universal error is reading area percent as percent by weight of the vial contents. Counterions, residual water and inorganic salts do not absorb in the ultraviolet, so a vial that is a fifth salt and water by mass can honestly report ninety-nine percent purity. The second error is accepting a chromatogram with no axis labels, no gradient, no wavelength and a run so short that late-eluting hydrophobic impurities never appeared.