Two numbers on a certificate of analysis are often read as one. They measure different things, and the difference decides how much peptide is really in the vial.
MVMichel van der VeenRegistered Nurse · Science Editor, Peptalis Sources checked via PubMed · 3 referencesReviewed 15 JULY 2026 · 7 MIN READAlmost every certificate of analysis for a research peptide carries a purity figure. It often reads something like "98%". The number looks reassuring, and it is. But it answers a different question than most people assume. Purity and content are two distinct quantities. Keeping them apart is how a certificate is read the way an analyst intends.
What HPLC measures
HPLC stands for high-performance liquid chromatography. It is a separation technique. A dissolved sample is pushed through a column under high pressure, and the different molecules in that sample emerge at the other end at different times. Each component produces a peak in the chromatogram: a plot of signal against time.
The purity that HPLC yields is a relative measure. The software calculates the area under the main peak as a percentage of the total area of all peaks combined. "98% pure" therefore means: of everything the detector saw with this method, 98% of the peak area belongs to a single component. It states how dominant the main peak is relative to the impurities the method makes visible: related peptides, degradation products, synthesis residues (Lian et al., 2021).
Two things follow. First, purity is always method-dependent: what the detector does not see does not count. Second, purity says nothing about the absolute mass in the vial. It is a ratio, not a quantity.
What peptide content measures
Peptide content, also called net peptide content, answers the other question: of the powder in the vial, how much is actually peptide? The rest of the mass consists of substances that are not peptide yet still register on the balance: bound water, salts, and counterions.
That last part is the largest unknown for synthetic peptides. Peptides are made by solid-phase synthesis and purified using trifluoroacetic acid (TFA) as an ion-pairing reagent. As a result the final product almost always emerges as a TFA salt, with TFA counterions bound to the basic side chains (Erckes et al., 2025). Those counterions, plus absorbed water, form a real part of the weighed mass: a part that is not peptide.
The reference method for determining content is amino acid analysis (AAA). The peptide is fully hydrolysed into its individual amino acids, and the amount of each is then quantified precisely. Because the amino acid composition of the sequence is known, the absolute amount of peptide follows from that measurement (Muñoz et al., 2010). AAA measures mass; HPLC measures ratio.
Why high purity does not mean high content
This is where the two numbers separate. A peptide can be 98% pure by chromatography (the main peak dominates, related impurities are minimal) and at the same time have a net peptide content of, say, 80%. That 18% gap is not made of contaminating peptides. It is water, salts and TFA counterions, which do not appear as a separate peak under common UV detection but do weigh on the balance.
The consequence is concrete. A vial with 10 mg of weighed powder of a 98% pure, 80% content peptide contains roughly 8 mg of peptide, not 9.8 mg. The purity is high and honestly reported; the content is a second number that stands apart from it. Anyone looking only at purity systematically overestimates how much peptide the vial holds.
What matters is what each number does not claim. High purity is not a promise about quantity, and a reported content is not a statement about biological activity. It says only how much peptide is present, not what that peptide does.
How Peptalis reads both numbers
Peptalis treats purity and content as two separate questions that both deserve an answer. Purity by HPLC shows how clean the profile is: how small the related impurities are relative to the main component. That is the question of identity confidence and synthesis quality.
Content is the question of how much peptide is actually present. Where a supplier certificate gives only a purity percentage, that is an incomplete picture: it shows the ratio, not the amount. Peptalis therefore states what has and has not been established, and shows the distinction rather than hiding both under a single percentage. What has been verified is shown; what could not be established is named as such.
What to expect from a certificate of analysis
A good certificate separates the two. It reports the chromatographic purity together with the method used, and it reports a content determination, or notes its absence. A certificate that states only "98%" without specifying whether that is purity or content leaves the most important question open.
Three questions help when reading a certificate. Is this figure purity or content? By which method was it determined? And is the counterion form (TFA salt or exchanged) stated, since it affects the weighed mass? Anyone who can answer these three knows what is really in the vial, and what the number on the label does and does not mean.
About the authorMVMichel van der VeenRegistered Nurse · Science Editor, PeptalisRegistered nurse with eleven years in psychiatry and founder of Peptalis. Writes the platform's knowledge layer: compound profiles, evidence reviews and the quality methodology. Works from primary literature (PubChem for chemistry, PubMed for studies) and states where evidence is absent.
Sources checked via PubMed · 3 references
Compound profilesEvidence reviewsQualityIN THIS ARTICLE- What HPLC measures
- What peptide content measures
- Why high purity does not mean high content
- How Peptalis reads both numbers
- What to expect from a certificate of analysis
- In DepthMass spectrometry: identity, not proof of everything
- In DepthCertificate of Analysis: what it does and does not say
Compounds in this article
- Epitalon · In stock · Batch PEP-EPI-2026-01 · HPLC 99.11% · released 2 Jun 2026 · Research profile · View in catalogue
- BPC-157 · In stock · Batch PEP-BPC-2026-01 · HPLC 98.96% · released 31 Aug 2026 · Research profile · View in catalogue
For laboratory research use only. Not for human use.
SCIENTIFIC REFERENCES
These references are provided for informational and research purposes only. They do not constitute medical advice.
- 01Review / MethodCharacterization of Synthetic Peptide Therapeutics Using LC-MS.Lian Z, Wang N, Tian Y, Huang L. J Am Soc Mass Spectrom. 2021;32(8):1852–1860.Methodological review of LC-MS characterisation for synthetic peptide therapeutics, including how the chromatographic purity readout is derived and what it does, and does not, capture.View on PubMed →
- 02Method / AnalyticalTowards a Consensus for the Analysis and Exchange of TFA as a Counterion in Synthetic Peptides.Erckes V, et al. Pharmaceuticals (Basel). 2025;18(8):1163.Consensus-oriented analysis of TFA counterions in synthetic peptides, quantifying how much of the weighed mass they contribute and how they can be exchanged or accounted for.View on PubMed →
- 03Method / ReferenceQuantification of protein calibrants by amino acid analysis using IDMS.Muñoz A, Kral R, Schimmel H. Anal Biochem. 2010;408(1):124–131.Reference-method paper on amino acid analysis (AAA) with isotope-dilution mass spectrometry: the traceable route for determining absolute peptide content rather than relative purity.View on PubMed →