What the document is, and where it stops
A certificate of analysis (CoA) records the measurements made on a single production lot. Someone who has never held the vial should be able to tell from it what was tested, by which method, on what date and with what outcome. HPLC says how clean the sample is, and mass spectrometry says whether it is the right molecule. A certificate needs both.
The document is a snapshot: some changes develop in storage, so the figures hold for the day of analysis and not for ever. It is also silent on sterility and endotoxins, which call for separate tests. The sections below follow the usual layout: header, purity, mass, optional extras.
The header: name, formula, lot number, date, appearance
The top block ties the paper to the product, and a recycled certificate can often be recognised here.
- Product identity. Peptide name, sequence (or a pointer to it), molecular formula, theoretical molecular weight. Hold that weight against the published one, which every profile here lists; see BPC-157 or semaglutide.
- Lot or batch number, with the date of analysis. The number must be the one on your vial label. Otherwise the page describes some other batch.
- Appearance. White to off-white freeze-dried powder is normal. GHK-Cu and other copper peptides are blue.
- Laboratory and approval. The laboratory that did the work, the person who reviewed it and the signature that released the result.
The purity line: what an HPLC percentage means
The technique is reverse-phase high-performance liquid chromatography. A pump forces the dissolved sample through a steel column. The packing is silica beads coated with C18 chains, hydrocarbon tails long enough to make the surface repel water. The solvent mixture changes during the run: mostly water at first, then a rising share of acetonitrile, with a trace of trifluoroacetic acid to keep the peaks narrow. The more hydrophobic a component, the longer it sticks to the C18 layer and the later it emerges. Its time of emergence is the retention time.
A UV detector sits at the column outlet, normally set to 214–220 nm. The peptide bond absorbs at those wavelengths, so any peptide-like impurity is seen regardless of its sequence. Purity is reported as area-percent: main-peak area over the total area of every integrated peak, multiplied by 100. If the certificate says 99.2%, then 99.2% of the absorbance recorded came from the main peak.
Next to the number the certificate should print the trace itself, the peak table and a description of the method (column, gradient, detection wavelength). In the trace, look for three things.
- A main peak that is narrow and symmetrical.
- A baseline that stays flat.
- Minor peaks that were integrated and listed, not skipped.
The method has blind spots. The calculation treats impurities as if they absorbed as strongly as the target, a fair assumption at 214 nm. Water and salts do not absorb there and go unseen. An impurity that co-elutes with the main peak is hidden inside it.
Side peaks: the usual impurities and their signatures
Impurities in a synthetic peptide are mostly near relatives of the target. They therefore elute close to it, and mass spectrometry can often name them.
| Impurity | Origin | Mass shift | Detection |
|---|---|---|---|
| Deletion sequence | One coupling failed to finish, so a single residue is missing | Minus the missing residue, for example 57 Da (glycine) or 97 Da (proline) | Minor peak beside the main one |
| Truncated sequence | Chain growth halted early | Far lighter | Peak well separated from the main one |
| Incomplete deprotection | A protecting group survived the final cleavage | Plus a tell-tale amount, 56 Da in the case of tert-butyl | Mass spectrum |
| Oxidation | Methionine converted to its sulfoxide; tryptophan can oxidise too | Plus 16 Da for each oxygen | Mass spectrum. MOTS-c, with two methionines and one tryptophan, is a likely case |
| Deamidation | Asparagine or glutamine loses its side-chain amide and becomes acidic | Just under 1 Da | A low-resolution spectrum may not show it |
| Racemisation | During synthesis a residue flips from its L form into the D form | Zero | Invisible to mass spectrometry; needs a separation, normally HPLC |
The first three are by-products of synthesis that purification is supposed to remove. Oxidation and deamidation can also arise later, while the material is stored. Synthesis is outlined in Research Peptides Explained, and the conditions that slow the later changes in Storage and Reconstitution: A Bench Protocol.
The mass line: identity by mass spectrometry
One sharp HPLC peak is what a pure sample of the wrong peptide would give too, so identity is settled by weighing the molecule. Two ionisation methods dominate: electrospray (ESI), frequently attached to the HPLC itself as LC-MS, and matrix-assisted laser desorption/ionisation (MALDI), usually paired with a time-of-flight analyser.
Their spectra look different. In MALDI most ions carry a single charge, so you look for one signal at the molecular weight plus a proton, written [M+H]+. ESI loads several protons onto each molecule, [M+nH]n+, and the result is a ladder of peaks whose mass-to-charge (m/z) values lie far under the molecular weight. For semaglutide (4113.6 Da) the ladder includes about m/z 1372.2 for the ion with three protons and 1029.4 for the one with four. Deconvolution software collapses the ladder into a single mass.
Observed and theoretical mass should be printed side by side and, on a routine instrument, differ by no more than a dalton or so. Small gaps usually have one of two causes. A high-resolution instrument tends to report the monoisotopic mass, based on the most common isotope of every element; around 4 kDa this lies roughly 2.5 Da under the average mass quoted in our profiles. Alternatively the ion picked up sodium or potassium instead of a proton, which raises the reading by about 22 or 38 Da.
Reference masses for profiles on this site
Formulas and average molecular weights as the profiles give them, sorted by mass. The observed mass on a certificate should agree.
| Profile | Formula | Average molecular weight | What the mass settles |
|---|---|---|---|
| GHK-Cu | C₁₄H₂₄N₆O₄ (GHK) | 340.4 Da; ≈ 402 Da as the copper complex | Ask which form was weighed |
| Melanotan-2 | C₅₀H₆₉N₁₅O₉ | 1024.2 Da | Ends in a C-terminal amide |
| PT-141 | C₅₀H₆₈N₁₄O₁₀ | 1025.2 Da | Free acid; roughly 1 Da above melanotan II |
| BPC-157 | C₆₂H₉₈N₁₆O₂₂ | 1419.5 Da | Neither methionine nor tryptophan in the sequence, the usual oxidation sites |
| CJC-1295 | C₁₆₅H₂₆₉N₄₇O₄₆ (with DAC) | 3647.2 Da with DAC; 3367.9 Da without | Tells the DAC form from the DAC-free form |
| Semaglutide | C₁₈₇H₂₉₁N₄₅O₅₉ | 4113.6 Da | Fatty-acid side chain included |
| Retatrutide | C₂₂₁H₃₄₂N₄₆O₆₈ | 4731.3 Da | Around 82 Da lighter than tirzepatide |
| Tirzepatide | C₂₂₅H₃₄₈N₄₈O₆₈ | 4813.5 Da | C20 fatty diacid included |
| TB-500 (full-length Tβ4) | C₂₁₂H₃₅₀N₅₆O₇₈S | 4963.5 Da | Short fragments traded under this name are far lighter |
Three rows need a comment. TB-500 is a trade name: some suppliers mean full-length synthetic thymosin beta-4, others a short piece around the actin-binding motif, Ac-LKKTETQ for instance, at under 900 Da. Only the mass shows which you have. For CJC-1295 a reading close to 3647 Da proves that DAC is attached and one close to 3368 Da that it is not; the half-life is days in the first case and minutes in the second. PT-141 and melanotan II are about 1 Da apart, so distinguishing them takes a well-calibrated spectrum, preferably backed by the retention time.
Lines many certificates leave out: water, counter-ion, net content
A complete certificate carries three more entries: net peptide content, counter-ion content and water content, the last normally determined by Karl Fischer titration. Paperwork for research-grade material often omits them. When present they settle something HPLC cannot: what fraction of the powder is peptide.
Freeze-dried peptide is a salt. The free N-terminus and the lysine, arginine and histidine side chains are basic and positively charged, and every positive charge is paired with a negative counter-ion. Because TFA is used in synthesis and purification, that counter-ion is normally trifluoroacetate, although some manufacturers swap it for acetate or chloride. Water is present as well, since the powder is hygroscopic.
The net peptide content states which fraction of the powder weight is peptide. HPLC cannot measure it; amino acid analysis or elemental nitrogen analysis can. In practice the value is always under 100%, and lower still for sequences with many basic residues, which hold more counter-ions. An example: a vial contains 10 mg of powder, HPLC purity is 99% and net peptide content 80%. The peptide present is 8 mg, so a concentration based on 10 mg is 25% too high.
Labels are inconsistent about whether the stated quantity is powder or peptide. If neither the certificate nor the product page says, assume powder weight. Part D of the bench protocol applies the correction to a stock solution.
Red-flag checklist
Any one of the following is a reason to question the supplier before the vial is used.
- Lot number missing, or not the one on the vial. Paper and product are then unconnected.
- A generic certificate. An identical document, date or chromatogram appears for different lots, fill sizes or even different peptides.
- Purity without a trace. A percentage with no chromatogram cannot be audited.
- No mass spectrometry. Purity without identity proves little.
- Masses that disagree. Observed and theoretical mass lie more than a dalton or two apart, or the theoretical mass does not fit the sequence.
- Exactly 100.00%. Every real synthesis leaves some related impurity. A perfect figure suggests that minor peaks went unintegrated or were removed from the picture.
- No method, no laboratory. With column, wavelength and laboratory unnamed, nobody can repeat or dispute the result.
- HPLC purity under 98%. Below the usual floor for research material.
King Peptides, the shop that the order links on this site lead to, lists an HPLC purity of 98% or higher for every product on its lab reports page and 99% or higher for most of them; tesamorelin, GHK-Cu and MOTS-c are listed at 98% or higher and selank at 98.88% or higher. It supplies a lot-specific certificate with HPLC and mass spectrometry for every lot. Those are the shop’s statements, and this site has no laboratory in which to test them. The checklist is how a buyer does so for one vial. Supplier checks are covered in Buying Research Peptides in the Netherlands, Belgium and Luxembourg.
Quick answers
Is a 99% purity figure enough to accept a lot? No. It has to come with a confirmed mass, and for quantitative work with a known net peptide content. High purity attached to the wrong mass is worthless.
The observed mass sits 16 or 22 Da above the theoretical value. What does that point to? Plus 16 Da indicates an oxidation, and plus about 22 Da a sodium adduct. Hundreds of daltons mean a different compound.
Does it matter that the supplier ran the analysis in its own laboratory? That is normal practice in this trade. What counts is a certificate that is lot-specific, complete and consistent with itself. An outside laboratory adds accountability only when its document is complete as well.
Does a clean chromatogram say anything about sterility? No. Neither HPLC nor mass spectrometry detects microbes or endotoxins. Those need separate tests.
Research use only. Everything on PeptidenBenelux.com describes peptides for laboratory research. Nothing here is medical advice. Always comply with the laws that apply in your jurisdiction.