A Certificate of Analysis (COA) is the analytical paper trail for a peptide lot: the report a lab issues after running a set of standardized tests on a specific batch. Every field on it answers a narrow, specific question — and no single field answers all of them. Researchers who read only the "purity %" line and stop there are missing most of what the document is telling them. This guide walks through each section of a typical peptide COA, what the number actually measures, what it does not measure, and why the same lot can look excellent on one line and mediocre on another.
Reversed-phase HPLC (RP-HPLC) is the standard method labs use to report peptide purity. A sample is run through a chromatography column, and the peptide plus any related impurities elute as separate peaks at different retention times. Purity is then calculated as the area of the main peak divided by the total area of all detected peaks, expressed as a percentage. A COA showing "≥98% HPLC purity" means: of everything that produced a detectable signal in that run, 98% of the peak area belonged to the target peptide.
Two limitations matter for interpreting that number correctly. First, HPLC purity is a relative measurement of UV-absorbing material, not an absolute mass measurement — it says nothing about how much of the vial's total weight is actually peptide (that is a separate figure, covered below). Second, some impurities — such as deamidated variants or isoaspartate forms — have chromatographic behavior and even molecular weight nearly identical to the parent peptide, making them genuinely difficult to resolve on a chromatogram. This is why a serious COA is read alongside the actual chromatogram trace, not just the summary percentage, and why HPLC is paired with mass spectrometry rather than used alone.
HPLC tells you how clean a sample is; it does not tell you what the main peak actually is. That's the job of mass spectrometry (MS), typically LC-MS pairing chromatographic separation with mass detection, or standalone ESI-MS / MALDI-MS. The lab compares the peptide's measured molecular weight against its theoretical (calculated) mass for the stated amino acid sequence. A match confirms identity — that the vial contains the peptide it's labeled as, not a different compound or a synthesis fragment of similar polarity that happened to co-elute on HPLC.
Two mass conventions show up on COAs and are not interchangeable: monoisotopic mass uses the most abundant isotope of each element, while average mass uses the natural-abundance-weighted average across all isotopes. For a mid-sized peptide the two figures typically differ by roughly 1 Da. High-resolution instruments report monoisotopic mass; lower-resolution instruments often report average mass. Neither is "wrong," but a COA should specify which one it's using, and the reported mass should match the peptide's known sequence within the analyzer's error tolerance (modern high-resolution MS routinely resolves within a few parts per million).
This is the single most consequential distinction on a peptide COA, and the one most often skipped in casual reading. Purity (the HPLC number) compares the target peptide against other peptide-related impurities. Net peptide content compares the target peptide against everything in the vial — including residual water and counter-ion salts left over from synthesis and lyophilization. A lot can be 99% pure by HPLC and still be only 70–85% net peptide by weight, because the remaining 15–30% of the vial's mass is non-peptide material that HPLC purity simply doesn't account for.
Net content is typically determined by amino acid analysis or elemental (nitrogen) analysis, since water and counter-ions do not contribute peptide-bound nitrogen. It matters for anything quantitative: if a vial is labeled 5 mg at 99% HPLC purity but only 80% net content, the actual peptide mass is closer to 4 mg, not 5 mg — a 20% error in any molar concentration calculated from the label weight alone.
Trifluoroacetic acid (TFA) is used both as a cleavage reagent in solid-phase peptide synthesis and as an ion-pairing agent during RP-HPLC purification. As a side effect, TFA ends up bound as a counter-ion to basic sites on the peptide — the N-terminus, lysine and histidine side chains, and arginine residues. For peptides with several basic residues, TFA salt content can account for a meaningful share of the vial's total weight, which is exactly why net peptide content and HPLC purity diverge more for basic, TFA-salt peptides than for peptides with fewer basic residues. A COA that reports TFA content (often as a percentage or mg/mg) is giving you the specific reason net content is lower than purity, rather than leaving it as an unexplained gap.
Lyophilized (freeze-dried) peptides are hygroscopic — they readily absorb ambient moisture — and residual water is one of the two major non-peptide components (along with counter-ion salt) that separate net content from purity. Water content is standardly measured by Karl Fischer (KF) titration, which quantifies water based on the amount of reagent consumed in a reaction specific to water. Coulometric KF titration, a micro-scale variant, is generally preferred for lyophilized peptide products because typical residual moisture in a freeze-dried vial is very low (often well under 10% by weight) and coulometric methods resolve that small a quantity reliably. A COA that reports a water content figure is telling you how much of the vial's mass is simply moisture — again, mass that HPLC purity does not "see."
Bacterial endotoxins are components of the outer membrane of gram-negative bacteria that can trigger a pyrogenic (fever-inducing) response, which is why endotoxin testing is standard for anything intended for injectable or cell-culture-adjacent laboratory use. The standard method is the Limulus Amebocyte Lysate (LAL) test, commonly run in its kinetic chromogenic form, which produces a quantitative result reported in endotoxin units per milligram (EU/mg). Not every peptide COA includes an LAL result — it's most relevant for compounds intended for in-vitro cell-based assays, where endotoxin contamination can confound experimental results independent of the peptide itself. When present, the LAL figure is a contamination-control measure; it is unrelated to peptide identity or purity and doesn't substitute for either.
The batch or lot number ties a specific COA to a specific manufacturing run, not to the compound in general. This matters because purity, net content, water, and TFA figures can all vary somewhat between lots of the same peptide from the same source — a COA for one lot does not certify a different lot, even of the identical product. A trustworthy vendor makes it possible to match the lot number printed on the vial label to the exact COA for that batch, rather than publishing one generic certificate for a product line.
Any manufacturer can run its own HPLC and print a number on a label. The reason serious researchers look for independent, third-party lab testing is straightforward: an in-house result has no external check on the instrument calibration, method, or reporting integrity — the same organization that makes the product is grading its own work. A third-party lab has no commercial stake in the outcome, which is precisely why its result functions as a meaningful check rather than a marketing figure.
The credibility of a third-party lab itself is not automatic either. ISO/IEC 17025 is the international standard specifying general requirements for the competence, impartiality, and consistent operation of testing and calibration laboratories — it covers instrument traceability, method validation, and quality-management practices. A lab operating under ISO 17025 accreditation (or an equivalent recognized scheme) has been independently assessed against that standard, which is a meaningfully different assurance than a lab simply claiming to be "accredited." When evaluating a COA, it's worth checking not just whether testing was done by a third party, but whether that third party's competence has itself been externally verified.
Every batch we sell is tested by an independent third-party laboratory, and the resulting Certificate of Analysis is made available per lot on our Analysis / COA page. When you receive a vial, match the lot number on the label to the corresponding certificate before use.