How to Read a Peptide Certificate of Analysis

A peptide certificate of analysis (COA) is a report from a testing service that records which tests were run on a sample, by which method, and what each test found. For a synthetic peptide the core lines are identity by mass spectrometry and purity by reversed-phase HPLC, often joined by appearance, net peptide content, counter-ion content and water content. Before reading any number, check three things: that the certificate was issued to the company supplying you, that it carries a report reference you can quote to the testing service, and that every result states its method.

This guide walks through a generic certificate field by field; the example entries are illustrative only.

What a certificate of analysis is, and what it is not

A certificate reports measured results for a sample, analysed on a stated date by a named testing service for a named client. It is evidence about that sample and the material it was drawn from.

Two other documents are easy to confuse with it:

• A specification states the limits a material is meant to meet, such as a minimum purity or an appearance. It is a target that applies to the product in general, not a measurement.
• A technical data sheet describes the compound itself: sequence, molecular formula, molecular weight, storage. It carries no test results.

A good certificate puts specification and result side by side: one column saying what was required, the next saying what was found. A result without a method, or a specification without a result, tells you much less.

The header: who, what and when

The top of a certificate identifies the document, the material and the parties.

The "issued to" line matters more than it looks. A testing service issues a certificate to the client who submitted the sample. If that client is a different company from your supplier, the document describes someone else's sample.

Identity: the mass spectrometry line

The identity line compares the mass measured by a mass spectrometer with the mass calculated from the peptide's molecular formula. A typical entry reads:

Three details decide whether the line means anything:

• Which mass convention. Average mass and monoisotopic mass differ by about 1 Da for a peptide of this size and by about 3 Da for a peptide near 5 kDa. The certificate should say which one it quotes.
• Which ion. A figure 1 Da above the expected neutral mass is often the singly protonated ion, [M+H]⁺, reported without conversion rather than a mismatch.
• What tolerance. The acceptance window depends on the instrument's resolution and mass accuracy. A high-resolution measurement is usually judged in parts per million; a low-resolution one within a tolerance stated in daltons.

A consistent offset can point to a specific problem: about +16 Da for an oxidised residue, about −18 Da for the loss of water, or the mass of one residue for a deletion sequence. Mass spectrometry and peptide identity explains ionisation, charge states and these offsets in detail.

Purity: the HPLC line

The purity line reports a reversed-phase HPLC analysis. A typical entry:

On a genuine certificate, that number is what you compare with the specification.

When you read the line, check that the detection wavelength is stated. The peptide bond absorbs strongly below about 220 nm, so detection between 210 and 220 nm sees every peptide. At 280 nm only aromatic residues absorb, and a peptide without tryptophan or tyrosine would barely register. A good certificate also reproduces the chromatogram, often with a table of the integrated peaks, their retention times and their areas, so the figure can be read back against the data.

Remember what the figure is: the main peak's share of the UV-absorbing material that the method separates. It is not the fraction of the powder that is peptide, and it does not confirm identity. HPLC and peptide purity covers columns, gradients, integration and the method's blind spots.

Content lines: appearance, net peptide content, counter-ion and water

A fuller certificate describes the solid as well as the molecule. Typical entries:

Synthetic peptides are usually isolated as salts. Basic groups such as the N-terminal amine and the side chains of lysine, arginine and histidine carry positive charges, and these are balanced by anions left from synthesis and purification, most often trifluoroacetate or acetate. Freeze-dried solids also hold some water. Net peptide content is what remains once those are accounted for. It counts peptide-related impurities along with the target sequence, so it measures something different from HPLC purity and cannot stand in for it.

The content figures matter whenever a mass of powder is converted into an amount of peptide. In the example, 10.0 mg of material at 81.5% net peptide content contains 8.15 mg of peptide. The molarity calculator works from the molecular weight you enter, and peptide purity vs net peptide content explains how the two measures relate.

Checks before you rely on a certificate

Work through these in order.

1. Issued to the seller. The client named on the certificate should be the company that supplied you.
2. A report reference. A unique report number you can quote to the testing service, which can confirm the report against its own records.
3. Method for every result. Mass spectrometry should name the technique and the mass convention; HPLC should give the detection wavelength; water should name Karl Fischer titration or another stated method.
4. Analysis date. The date the sample was analysed, with the received and issue dates in a logical order.
5. Issuer named. The testing service that performed the analysis is named on the report.
6. Internal consistency. The expected mass should match the compound's molecular formula, which you can check against a public record such as PubChem.

How certificates are published at British Peptide Labs

Our compounds undergo independent third-party testing by PeptideVerify, with reports published in the COA Library. Each published report is prepared for British Peptide Labs and listed with its issuer, report reference, analysis date and methods: HPLC for purity and LC-MS for identity. Our research compounds are supplied at ≥99% purity (HPLC). The quality page sets out what each product page documents, and the glossary defines the terms used on certificates.