Typical impurities in synthetic peptides
Deletion sequences, racemisation, oxidation. Where by-products come from and why they can affect the reliability of experiments.
No synthesis method works perfectly. Each amino acid coupling has a yield just under 100 %, and over 15 or 30 steps small losses add up. The review by D’Hondt et al. (2014) divides the resulting impurities into three groups.
1. From synthesis
- Deletion sequences: an amino acid is missing because a coupling was incomplete
- Insertions: an amino acid has been incorporated twice
- Racemisation: an amino acid is present in the wrong spatial form (D instead of L)
- Incompletely deprotected side chains: protecting groups from the synthesis are still present
2. From degradation of the peptide
- Deamidation of asparagine and glutamine
- Oxidation of methionine, cysteine and tryptophan
- Aspartimide formation at Asp-Gly or Asp-Asp motifs
- Diketopiperazine formation at the N-terminus
3. From interactions with excipients
Counter-ions and residues from purification, such as trifluoroacetic acid (TFA), do not count as peptide impurities in the strict sense, but they do affect the peptide content.
Why this matters for research
An impurity of 3 % sounds small. But if it has its own biological activity, it can distort measurement results. Reproducible research therefore needs not only high purity but also documentation of which minor peaks are present.
How we handle this
Every batch report states the minor peaks in the chromatogram alongside the overall purity. Hover over a peak to see the retention time and area share.