Peptide content versus purity — the distinction most buyers get wrong
Purity is a ratio of areas. Content is a fraction of mass. A 99 % pure vial can contain 78 % peptide, and the difference is the whole ball game.
Content and mass
Reference standards, and what "area percent" means without one
The difference between an assay and an area percent, why most research peptides have no available standard, and what that costs the numbers on this site.
An assay quantifies an analyte against a material of known content. An area percent compares a peak to the other peaks in the same chromatogram. The first tells you how much there is; the second tells you what fraction of the detected material it was. They are frequently printed in the same box on a certificate under the same heading.
The hierarchy of standards
| Tier | What it is | Available for research peptides? |
|---|---|---|
| Pharmacopoeial reference standard | Ph. Eur. CRS or USP RS, content certified by the pharmacopoeia | Oxytocin, desmopressin, gonadorelin, leuprolide, triptorelin, glucagon, thymalfasin — and nothing else in this index |
| Certified reference material | Content certified by an accredited producer with a stated uncertainty | A handful of the older compounds |
| Working standard, characterised in-house | Content established by AAA or qNMR against a primary standard | Possible for any compound, at cost |
| "Reference standard" from the same supplier as the sample | Circular. Establishes nothing about content | Common |
| No standard | Area percent only | The overwhelming majority |
What the absence costs
Three specific things, in decreasing order of importance.
- Content is unmeasurable by HPLC. Without a standard of known content, an HPLC peak area cannot be converted to a mass. Amino-acid analysis or quantitative NMR is required instead, and both are far more work.
- Relative response factors are unknown. Purity by area percent assumes every impurity absorbs at 214 nm exactly as strongly as the parent, per unit mass. For an impurity that has lost a tryptophan, that assumption is wrong by a large factor. This is the largest term in the uncertainty budget.
- Retention cannot be confirmed by co-injection. The definitive identity check for a chromatographic peak is spiking the sample with an authentic standard and observing a single peak of increased area. Without a standard, identity rests on mass and calculated retention.
The small-molecule case is worse
For the non-peptide adjuncts in this index — MK-677, enclomiphene, 5-amino-1MQ, SLU-PP-332 — reference standards exist commercially for some and not for others, and the certificates rarely say which was used. An area-percent figure for a small molecule at a single wavelength is a weaker statement than the same figure for a peptide, because small-molecule chromophores differ far more from each other than peptide amide backbones do. Of the MK-677 reports in this index, roughly one in six names a standard.
Normative references
Cited because they are the documents the acceptance criteria above are taken from. The index applies them as written and states every deviation.
- USP <11> — Reference standards
- Ph. Eur. 5.12 — Reference standards
- ICH Q6A — Specifications
- ICH Q2(R2) — Validation of analytical procedures
Related methods
Amino-acid analysis for peptide content
Acid hydrolysis followed by chromatographic quantification of the liberated residues — the reference method for content where no standard of the intact peptide exists.
Content and mass
Detection wavelength and response
214 nm, 220 nm, 280 nm — what each one sees, and how the choice changes a purity figure.
Chromatographic purity