Reading time: ~2 min · Part of the free certification in peptide science
A purity of 99% means up to 1% of the chromatogram area corresponds to something else. Knowing what that «something else» usually is lets you read a COA with judgment and understand why two batches with the same purity may not be equivalent.
Synthesis-related impurities
Solid-phase synthesis adds one amino acid per cycle; no cycle has a 100% yield. Typical impurities are:
- Deletion peptides: a residue is missing because a coupling was incomplete. They are the hardest to separate because they closely resemble the product.
- Truncated peptides: the chain stopped before finishing.
- Diastereomers: a residue racemized (L → D) during coupling. Same exact mass, different shape: mass spectrometry alone cannot tell them apart.
- Residual protecting groups: remnants of the synthesis chemistry not fully removed.
Degradation impurities
- Oxidation of methionine (+16 Da), tryptophan or cysteine.
- Deamidation of asparagine or glutamine (+1 Da).
- Aggregates and hydrolysis products.
How they are detected
HPLC coupled to mass spectrometry (LC-MS) is the reference tool: HPLC separates and quantifies each peak, and MS assigns a mass to each one, which shows whether an impurity is a deletion (lower mass by a specific residue), an oxidation (+16) or a deamidation (+1).
The counterion: TFA versus acetate
Peptides are purified and isolated as salts. Trifluoroacetic acid (TFA) is the usual counterion after HPLC purification; acetate is obtained with an additional exchange step. The counterion is part of the powder's mass but not of the active peptide, and therefore:
- It contributes to net peptide content being below 100% of the vial weight.
- Residual TFA is considered undesirable in preparations intended for pharmaceutical use, which are usually converted to acetate or chloride.
A complete COA shows the chromatogram (not just a percentage), the observed versus theoretical mass, the batch number, the date and the issuing laboratory. A percentage without a chromatogram does not show how many impurities there are or how the peaks were integrated.
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