Peptide purity: 98% vs 99% in practice

What does a peptide purity percentage measure?

The purity on a peptide certificate is almost always chromatographic purity. The laboratory runs the sample through an HPLC column, records a UV trace at one wavelength (214 nm on our BPC-157 certificate, where the peptide bond absorbs), and divides the area of the main peak by the total area of every peak it integrates. 98% means the main peak is 98% of the peak area the method could see.

Three limits follow from that. It is a ratio of UV signal, not of mass, so an impurity that absorbs weakly at that wavelength is undercounted and one that absorbs strongly is overcounted. Anything that does not show up as a peak, such as water, salts or a compound that never leaves the column, is outside the figure entirely. And it describes the lot that was tested, on the day it was tested. How to read a peptide certificate of analysis covers the rest of the document; this page is about that one number.

What is the other 1–2% in a 98% or 99% pure peptide?

In a peptide made by solid-phase synthesis, most of what is not the target is closely related to it. A review of impurities in peptide medicines groups them by origin. Synthesis gives deletion sequences, where a coupling step failed and one residue is missing; truncated sequences, where chain growth stopped early; insertions from excess reagent; side chains that kept a protecting group; and diastereomers, where one residue racemized from the L to the D form. Storage and handling add degradation products: oxidized methionine or cysteine, deamidated asparagine or glutamine, pyroglutamate and succinimide forms.

These are hard to see precisely because they are so similar to the main compound. A D-isomer has the same mass and nearly the same polarity as the intended peptide, and a review of enantiomeric purity testing describes its separation as a real analytical challenge. A deamidated form differs by about one dalton. Some co-elute with the main peak and are counted as product. When a reference laboratory characterized a synthetic C-peptide material by high-resolution LC-MS, it identified more than 65 related impurities in a single lot. This is why HPLC vs mass spec: what each test proves matters: HPLC tells you how clean the main peak looks, and mass spectrometry tells you what is in it.

When does the difference between 98% and 99% purity matter?

The difference matters when an impurity can do something the main compound does not, in quantitative work, and when lots are compared. For many experiments it does not. If a peptide is used at a concentration well above where it acts, and the related impurities are inactive variants of it, the difference between 98% and 99% is a small shift in effective concentration, smaller than the error in weighing a lyophilized powder that picks up moisture from the air.

It matters when the impurity can do something the main compound does not. A truncated or racemized sequence may bind the same target with different affinity, or act as a partial agonist or antagonist. The review of peptide impurities warns that impurities can distort early functionality studies enough to lead to wrong conclusions. It matters in quantitative work where the peptide is a calibrant or a standard, because every impurity becomes a bias. And it matters when lots are compared: a 98% lot and a 99% lot can differ in which impurities they carry, not just in how much, so a change in behaviour between lots may come from the impurity profile rather than the headline number.

If your experiment is sensitive to any of that, the useful question is not which figure is higher but what the impurity peaks are. A certificate that lists the impurities, or includes the chromatogram, answers it better than one more decimal place.

Is peptide purity the same as net peptide content?

No: purity is a share of HPLC peak area, while net peptide content is the share of the powder's mass that is peptide, and it is typically lower. A vial labelled 10 mg holds 10 mg of powder, and not all of that powder is peptide. Lyophilized peptides retain water, and they are salts: the basic residues carry counter-ions, most often trifluoroacetate left by the cleavage and purification steps, sometimes acetate or chloride. Net peptide content is the share of the powder's mass that is peptide, and it is typically lower than the purity figure.

HPLC purity does not see any of this, because water and counter-ions do not appear as peptide peaks. A sample can read 99% pure and still carry a substantial share of water and salt by mass, with more counter-ion in sequences with many basic residues. A recent study on exchanging trifluoroacetate for chloride found that counter-ion content varies with sequence and argued that it should be measured and stated for any assay using synthetic peptides. If you are making solutions to a precise molar concentration, net peptide content matters more than the difference between 98% and 99% purity.

Where our certificates list their tests, none lists a net peptide content assay; the three from North South Precision Testing do report moisture. how our testing works shows what each certificate reports.

Why is the same "≥99%" on every product a warning sign?

A catalogue that shows the same "≥99%" on every product is describing a target or a sales claim, not a set of measurements. Purity varies by peptide and by lot. A short, easily purified sequence and a long one with several cysteines do not come off the same purification at the same figure, and two lots of the same peptide rarely match to the decimal. Red flags in a peptide COA: spotting a fake covers other signs worth checking.

A minimum also hides the information you want. "≥99%" is satisfied by 99.0% and by 99.9%, and it says nothing about which lot it applies to. A measured value, tied to a lot number, a laboratory and a date, can be checked against the vial you receive and compared with the next lot. That is the standard we try to meet, and it is one of the things to ask of any supplier (How to vet a peptide supplier: a 10-point checklist). Even so, one of our own certificates, for Selank, reports ">99.80%" rather than a point value, and we publish it as printed.

What purity range do Aion's certificates report?

We hold 14 certificates. Their reported purities run from 98.00% to 99.98%. The lowest is our IGF-1 LR3 lot ABL-IGF-2605, assayed at 98.00% by Vanguard Laboratory, and that is the figure its product page shows. The highest single figure on file is 99.98%. The other twelve fall between 99.0% and 99.9%, and each figure belongs to one lot only.

Several products in the catalogue have no certificate yet, including AOD-9604. Their pages make no claim about purity, and the COA library lists them as in progress. We do not describe any product, or the catalogue, with a single purity figure.

References

1. D'Hondt M, Bracke N, Taevernier L, et al. (2014). Related impurities in peptide medicines. J Pharm Biomed Anal. [REVIEW] PubMed record for reference 1, D'Hondt 2014
2. Li M, Josephs RD, Daireaux A, et al. (2018). Identification and accurate quantification of structurally related peptide impurities in synthetic human C-peptide by liquid chromatography-high resolution mass spectrometry. Anal Bioanal Chem. [IN VITRO] PubMed record for reference 2, Li 2018
3. Badgujar D, Paritala ST, Matre S, Sharma N. (2024). Enantiomeric purity of synthetic therapeutic peptides: A review. Chirality. [REVIEW] PubMed record for reference 3, Badgujar 2024
4. Erckes V, Streuli A, Chamera Rendueles L, et al. (2025). Towards a Consensus for the Analysis and Exchange of TFA as a Counterion in Synthetic Peptides and Its Influence on Membrane Permeation. Pharmaceuticals (Basel). [IN VITRO] PubMed record for reference 4, Erckes 2025