What is HPLC?

HPLC separates the components of a liquid sample according to how strongly each is held by a packed column, then records them one after another as they leave it. Size-exclusion, ion-exchange and reversed-phase modes are all used for peptides ^[1], but the purity figure on a peptide certificate of analysis almost always comes from reversed-phase HPLC (RP-HPLC), which sorts molecules by hydrophobicity.

How reversed-phase HPLC separates peptides

The column is packed with silica particles carrying bonded C18 hydrocarbon chains. The sample is loaded in a mostly aqueous mobile phase, so peptides adsorb to the hydrophobic surface. A gradient then raises the proportion of acetonitrile, and each peptide is released once the solvent is organic enough to compete for it. More hydrophobic sequences elute later, which is why the time a peak appears, its retention time, is characteristic of a molecule under a fixed method.

The role of TFA

Both solvents normally contain 0.1% trifluoroacetic acid, which works out at about 13 mM. TFA acts as an ion-pairing reagent: NMR experiments have shown it forming ion pairs with peptides in acetonitrile–water, and at low TFA concentrations peptide peaks develop a spike at the front and a long tail ^[2]. Because the purified fractions are dried from this mobile phase, HPLC-purified peptides are often recovered as trifluoroacetate salts ^[3].

A typical analytical method

A method sheet that omits the gradient or wavelength makes the purity figure impossible to compare with another laboratory's.

Why 214 nm rather than 280 nm

At 214–220 nm the detector reads the peptide bond itself, so every peptide-related species in the sample gives a signal, with aromatic side chains adding to it. At 280 nm only tryptophan and tyrosine absorb strongly. BPC-157 (GEPPPGKPADDAGLV) contains neither, so a 280 nm trace would barely show the main peak, and any method run there would over-weight impurities that happen to carry aromatic residues while missing those that do not.

Beyond a single purity number

Reversed-phase HPLC is one tool among several. Size-exclusion HPLC separates by size and can assess whether a peptide is present as monomer or oligomers ^[1], which a reversed-phase run under denaturing, acidic conditions may not reveal. HPLC also cannot see what does not absorb at the detection wavelength or elutes unretained: water, inorganic salts and counter-ions do not appear in the purity calculation, which is why net peptide content is a separate measurement.

HPLC does not identify anything either. A peak at the expected time is consistent with the target, but identity comes from mass spectrometry, often coupled directly to the column as LC-MS. How the purity percentage is calculated from the trace is shown under chromatogram, what the impurities usually are under peptide purity, and the HPLC testing guide puts the method in the context of batch documentation.