How separation works
The sample is injected onto a column packed with fine particles and carried through by a solvent. Components interact with the packing to different degrees: those that interact strongly move slowly, those that do not move quickly. They emerge separated in time.
Peptides are usually run in reversed phase, where the packing is non-polar and the solvent gradient becomes progressively less polar. More hydrophobic peptides elute later.
Reading a chromatogram
A detector measures absorbance as material leaves the column, producing peaks. Position identifies, area quantifies. Purity is the target peak area as a percentage of total peak area.
Peak shape is informative too. A broad or tailing peak can indicate a poor separation or interaction with the column, and can conceal a closely eluting impurity.
Why method matters
Column chemistry, gradient steepness, temperature and detection wavelength all affect the result. A shallow gradient resolves closely related species that a steep one merges into a single peak — reporting a higher purity for the same material.
This is why a COA states the method. A purity figure without one is not comparable to anything.
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