Purity is a profile, not a number
HPLC reports the target peak’s area as a percentage of total peak area. Everything else falls into "impurity" — a category that collapses several chemically distinct populations into one figure.
A chromatogram with a single small, well-separated secondary peak describes a different material from one showing a broad cluster of unresolved shoulders, even when both integrate to identical purity. Reading the trace rather than the summary line is what distinguishes them. The methods themselves are covered in HPLC vs mass spectrometry.
Synthesis-related impurities
Solid-phase synthesis adds residues sequentially, and each coupling step is slightly less than perfect. The by-products are predictable and structurally related to the target — which is what makes them hard to separate.
| Species | Origin | Analytical signature |
|---|---|---|
| Deletion sequence | A coupling step failed | Mass lower by exactly one residue |
| Truncated chain | Synthesis terminated early | Mass lower by a defined fragment |
| Incomplete deprotection | A protecting group remained attached | Mass higher by a known increment |
| Racemized residue | Stereochemistry inverted during coupling | Identical mass; resolvable only chromatographically |
Degradation-related impurities
These accumulate after synthesis and grow with time, temperature, and moisture. Unlike synthesis by-products, their level is not fixed at manufacture — it is a function of how the material has been handled since.
| Route | Chemistry | Mass shift |
|---|---|---|
| Deamidation | Asparagine or glutamine converts to aspartate or glutamate | +0.98 Da |
| Oxidation | Methionine, cysteine, or tryptophan gains oxygen | +16 Da per oxygen |
| Hydrolysis | Backbone cleavage, favoring labile bonds | Fragment masses |
| Disulfide scrambling | Cysteine bridges re-form incorrectly | No change in mass |
Because these routes are condition-dependent, they are the impurities that handling actually controls — the practical reason storage discipline matters. See peptide storage and stability.
Counter-ions are not impurities, but they are mass
Trifluoroacetate or acetate carried over from purification forms part of the powder’s weight without appearing as an HPLC impurity peak. It is not contamination in the analytical sense; it is simply not peptide.
This is why net peptide content is reported as its own line on a Certificate of Analysis, and why a vial’s gross weight overstates the peptide it contains. A lot can be 99.5% pure by HPLC and still be well under its label weight in actual peptide.
Aggregates
Aggregated material may not resolve on a standard reverse-phase run at all — it can elute as a poorly defined early feature or fail to elute in a quantifiable way, escaping the purity integration entirely.
Detecting it takes an orthogonal method such as size-exclusion chromatography or light scattering. For sequences prone to aggregation, a high reverse-phase purity figure alone is an incomplete description of the material.
What to ask a supplier for
The chromatogram, not only the percentage. The wavelength and method used for integration. Whether the method is capable of resolving closely related species, or merely of producing a high number.
A supplier that answers all three is describing a measurement. One that answers only with a figure is describing a claim — and the distinction becomes visible in the data, not in the marketing.
Chromatograms, not just numbers
Ethos Bio publishes the full independent analytical report for every lot, including the HPLC chromatogram behind the purity figure.
Frequently asked questions
What are the impurities in a 99% pure peptide?
The remaining fraction is typically a mix of synthesis by-products — deletion sequences, truncated chains, incompletely deprotected residues, racemized residues — and degradation products such as deamidation, oxidation, hydrolysis fragments, and scrambled disulfides. Counter-ions like TFA or acetate add mass without appearing as HPLC impurity peaks.
Can two peptides with the same purity be different quality?
Yes. Purity is a single number summarizing a profile, so two lots both reading 99.2% can differ substantially in what makes up the remaining fraction — one well-separated secondary peak versus a cluster of unresolved related species. Reading the chromatogram, not only the percentage, is what distinguishes them.
What is deamidation in a peptide?
Deamidation converts asparagine or glutamine residues to aspartate or glutamate, increasing mass by roughly 0.98 Da. It is one of the most common time- and moisture-dependent degradation routes, which is why lyophilized storage and low residual water matter.