How to read a Certificate of Analysis
A COA can show an impressive purity figure and still leave important questions unanswered. Here is what to check line by line.
What a COA is — and is not
A Certificate of Analysis is a document issued by a testing laboratory (or, less reliably, by a manufacturer) that records the analytical results for a specific production lot. It is not a guarantee of identity on its own — it is a snapshot of what was measured, for that batch, on that date, by that laboratory.
The critical word is batch-specific. A COA that does not carry a lot or batch number cannot be linked to the product you have received and is essentially meaningless for quality verification.
Section 1
Header fields to verify first
Product name & CAS number
Confirm the IUPAC name or peptide sequence matches your order. The CAS number should correspond to the exact compound, not a related analogue. For novel research peptides without a CAS number, the amino acid sequence (single-letter or three-letter code) must be stated.
Batch / Lot number
This number must appear on both the COA and the product label. If they do not match, the COA does not apply to your product. Reputable suppliers print the batch number on every vial and box.
Testing laboratory
Look for ISO/IEC 17025 accreditation — the international standard for analytical testing laboratories. An in-house COA produced by the manufacturer carries an inherent conflict of interest. Third-party accredited testing is the baseline expectation for research-grade material.
Analysis date & retest date
Peptides are stable compounds when lyophilised and stored correctly, but the COA reflects conditions at the time of testing. Most suppliers assign a retest date (typically 24–36 months from manufacture for lyophilised peptides stored at −20 °C). Material beyond its retest date should be re-analysed before use in critical experiments.
Section 2
Purity — the HPLC section
HPLC (High-Performance Liquid Chromatography) purity is almost always reported as area percentage — the proportion of the total UV-absorbance area attributed to the main peak. This is not mass percentage.
What to look for in the HPLC section:
- Method: Reverse-phase C18 column at 214 nm or 220 nm is the most common and reliable for peptides. Detection at 214 nm captures the peptide bond absorbance and is largely compound-independent.
- Specification vs result: The spec column states the minimum accepted purity (e.g., ≥ 95.0%). The result column must meet or exceed it.
- Chromatogram: A good COA includes the actual chromatogram trace, not just the number. A single sharp main peak with well-resolved minor peaks indicates a clean preparation.
- Gradient & run time: A 20–30 minute gradient on a 150–250 mm C18 column is long enough to resolve most peptide impurities. Very short runs (< 10 min) can mask co-eluting impurities.
HPLC purity alone does not confirm identity. A sample can be 99% pure by HPLC and still be the wrong compound if the synthesis went wrong at the sequence level. This is why mass spectrometry is essential.
Section 3
Identity — mass spectrometry
Mass spectrometry (MS) confirms that the compound has the correct molecular weight, providing direct evidence of chemical identity. The two methods most commonly used for research peptides are:
ESI-MS (Electrospray Ionisation)
The most common technique for peptides below ~5,000 Da. Produces multiply-charged ions ([M+H]⁺, [M+2H]²⁺, etc.). The calculated molecular weight is derived from the observed m/z values. The COA should state the theoretical MW and the observed MW — they should agree within ±0.5 Da for small peptides.
MALDI-ToF
Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight. Typically used for larger peptides and proteins. Less routine for smaller research peptides but equally valid for identity confirmation.
Red flag: COA without MS data
A COA that reports HPLC purity only — without any mass spectrometric confirmation — provides no evidence that the material is the stated compound. This is particularly important for peptides with similar molecular weights or sequences.
Section 4
Additional tests — what else to look for
Bacterial Endotoxins (LAL / BET) — USP ⟨85⟩
Endotoxins (lipopolysaccharides from gram-negative bacteria) are a critical concern for any material used in cell-based assays. Even trace endotoxin levels (≥ 0.1 EU/mL in many assay systems) can trigger innate immune responses and confound results. The Limulus Amebocyte Lysate (LAL) test is the standard method. Specification is typically ≤ 5 EU/mg for research materials.
Residual Solvents — USP ⟨467⟩ / ICH Q3C
Peptide synthesis uses solvents including DMF, DCM, acetonitrile and TFA. ICH Q3C classifies solvents by toxicity (Class 1–3). Class 2 solvents have permitted daily exposure limits. Residual solvent testing by headspace GC confirms that synthetic solvents have been adequately removed during lyophilisation.
Moisture content — Karl Fischer / USP ⟨921⟩
Lyophilised peptides typically have a moisture content below 5–8%. Elevated moisture accelerates degradation and reduces effective yield. Karl Fischer titration is the reference method.
Appearance
Most lyophilised peptides are described as a white to off-white powder or fluffy lyophilisate. Significant discolouration (yellow, brown) can indicate oxidation or degradation and should be investigated before use.
COA evaluation checklist
- Batch/lot number matches product label
- Testing laboratory is ISO/IEC 17025 accredited
- HPLC purity ≥ specification, chromatogram included
- Mass spectrometry data confirms molecular weight
- Endotoxin result within specification (if cell work planned)
- Retest date is current
- Appearance consistent with expected lyophilisate