How high-performance liquid chromatography separates a peptide from its impurities, how to read a chromatogram, and what peak area percentages mean on a certificate of analysis.
Always read the method with the number
Gradient steepness, column chemistry and run length all change the reported figure. Compare purity within a supplier and method, not across unknown methods.
The principle
High-performance liquid chromatography separates a mixture by pushing it through a packed column under pressure. For peptides the standard is reverse phase: a non-polar C18 stationary phase with a polar mobile phase, typically water and acetonitrile with 0.1% trifluoroacetic acid, run as a gradient. More hydrophobic species stick to the column longer and elute later, so components separate by retention time.
Reading a chromatogram
- X axis is retention time in minutes; Y axis is detector response, usually UV absorbance at 214 nm.
- The main product is normally the largest, sharpest peak; its retention time should be consistent between batches of the same compound.
- Peak area, not peak height, is used for quantification — a broad peak can carry more material than a tall narrow one.
- Small peaks close to the main peak are typically related impurities; peaks near the solvent front are usually unretained salts and solvent.
Why the method matters as much as the number
Two laboratories can report different purities for the same vial purely because of gradient steepness, column chemistry, temperature or run length. A steep, short gradient co-elutes close impurities into the main peak and inflates the number. A certificate that states the column, gradient and wavelength lets you judge whether the figure is meaningful; one that states only ‘99%’ does not.
For that reason, compare purity figures within a supplier and method rather than across suppliers using unknown methods.
Common signals worth investigating
- A shifted retention time versus previous batches of the same compound — possible identity or salt-form change.
- A shoulder on the main peak — often a diastereomer or deletion sequence co-eluting.
- A rising baseline late in the run — column contamination or carryover from a previous injection.
- Broad, tailing peaks — column ageing, overload, or secondary interactions.
References
- Mant, C.T. et al. (2007) ‘HPLC analysis and purification of peptides’, Methods in Molecular Biology, 386, pp. 3–55.
- European Pharmacopoeia — general chapter on liquid chromatography, European Directorate for the Quality of Medicines & HealthCare.
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