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The measurement
In reversed-phase HPLC the sample passes through a column (commonly C18) while the solvent shifts from water toward acetonitrile, usually with a little trifluoroacetic acid. Components elute at different times and a UV detector records them as peaks. At 214–220 nm the detector sees the peptide bond itself, so nearly every peptide-related species shows up.
The number
Purity is the main peak’s area divided by the total area of all peaks, as a percentage. A figure of 98% means 98% of what the detector saw at that wavelength was the main component.
Formula
Fig. 1 · RP-HPLC, UV 220 nm
Example values
Full scale
| Peak | Time, min | Area, % | Assignment |
|---|---|---|---|
| 1 | 8.22 | 0.22 | Impurity |
| 2 | 8.55 | 0.09 | Impurity |
| 3main | 9.75 | 99.38 | Main peak (purity) |
| 4 | 10.51 | 0.16 | Impurity |
| 5 | 11.43 | 0.15 | Impurity |
| Total | 100.00 | All integrated peaks |
Release minimum
A lot can’t be published here unless its certificate shows HPLC purity of at least 99.0% and an observed average mass within ±1.0 Da of the average mass calculated from its sequence.
What it can miss
Anything that doesn’t absorb at the chosen wavelength, or that co-elutes under the main peak, is invisible to the calculation. Mass spectrometry can reveal a co-eluting impurity of different mass, such as a deletion sequence; a racemized residue has the same mass and needs a chiral method to detect.
Purity is not peptide content
Lyophilized peptides carry water and counter-ions from purification. Net peptide content, the fraction of the powder’s weight that is peptide, is often well below 100% even for a very pure lot.
Note
For exact molar work, use net peptide content where the certificate reports it.