The column is packed with a stationary phase, and a solvent mixture carries the sample through it. Molecules that interact more with the stationary phase move more slowly, so components emerge separated in time.
A detector at the outlet records absorbance as material passes. For peptides this is usually ultraviolet, at a wavelength where the peptide bond or aromatic residues absorb. The output is a trace of absorbance against time, with a peak for each component.
The area under a peak is proportional to how much of that component passed the detector; the height is not, because peaks broaden as they travel and a broad peak can be low and still large.
Purity is therefore target peak area over total peak area. Deletion sequences from synthesis — chains missing a residue — are the characteristic impurity, and they appear as peaks close to the main one, which is exactly what the technique is good at resolving.
The figure only accounts for what absorbs at the wavelength used. Anything that does not — water, many salts, some counter-ions — is invisible to it and is reported separately if at all.
This is the specific reason a purity percentage and net peptide content are different numbers, and why a certificate that gives only the first is giving less than it appears to.
The column is packed with a stationary phase, and a solvent mixture carries the sample through it. Molecules that interact more with the stationary phase move more slowly, so components emerge separated in time.
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