What impurities are found in synthetic peptides, and how does a COA show them?

In Fmoc SPPS, the peptide is built one amino acid at a time on a resin bead. Each cycle removes the Fmoc group from the growing chain and couples the next protected amino acid. At the end, a strong acid (usually trifluoroacetic acid) cuts the peptide off the resin and strips the side-chain protecting groups, and the crude product is purified by reverse-phase HPLC and freeze-dried.

Every one of those steps runs at less than 100 percent. A long sequence goes through dozens of cycles, so even a small per-cycle loss leaves a population of closely related chains. A 2014 review of peptide-related impurities groups them into synthesis-related impurities and degradation products, and notes that both can change the outcome of early functionality studies. That is the practical reason a research lab cares about them.

Deletion sequences, missing one residue, come from a coupling or Fmoc removal that did not go to completion. Insertion sequences, with one residue added twice, come from excess activated amino acid. Both differ from the target by exactly one residue mass, which makes them easy to spot by LC-MS and hard to remove by HPLC because they behave so much like the target.

Racemization changes one residue from its L form to its D form. The result, a diastereomer, has the same mass as the target, so mass spectrometry alone cannot see it; only a chromatographic method that separates the two can. Incomplete side-chain deprotection leaves adducts such as a tert-butyl group still attached (+56 Da).

Aspartimide formation is a well-known side reaction at aspartic acid residues, especially in Asp-Gly sequences. The ring-closed aspartimide is 18 Da lighter than the target, and when it reopens it gives alpha- and beta-aspartyl peptides of the same mass as the target. A 2025 review in ChemBioChem summarizes the protecting-group strategies developed to suppress it.

Oxidation of methionine (+16 Da) and tryptophan can happen during synthesis, cleavage or later storage. Degradation that happens after manufacture, such as deamidation or pyroglutamate formation, is covered in our guide to peptide degradation pathways.

A lyophilized peptide is never 100 percent peptide by weight. Basic residues carry counter-ions from cleavage and purification, most often trifluoroacetate unless the material was exchanged to acetate or another salt. Our TFA vs acetate guide covers why the salt form changes the weight of peptide in a vial.

Freeze-dried peptides also hold water, because the powder is hygroscopic, and can retain traces of the solvents used in synthesis and purification. ICH Q3C, published by FDA, classifies common solvents by hazard: acetonitrile, N,N-dimethylformamide and dichloromethane are Class 2 solvents, and diethyl ether and tert-butyl methyl ether are Class 3. Water is measured by Karl Fischer titration and solvents by headspace gas chromatography; neither appears in an HPLC purity figure.

HPLC purity is the main peak's share of the total UV peak area, usually at 214 to 220 nm where the peptide bond absorbs. It ranks the peptide-related impurities that the column separates. It does not count water, salts or solvents, and an impurity that co-elutes under the main peak is counted as product.

That is why two certificates with similar purity figures can describe very different vials. Net peptide content, or a measured quantity in milligrams, is the result that accounts for the non-peptide mass. The validation of the method itself, its specificity, accuracy and precision, is described in ICH Q2(R2).

Five checks that use only what is printed on the certificate.

1. Find the identity result: an LC-MS observed mass that agrees with the mass calculated from the sequence rules out most wrong-sequence and missing-residue problems in the main peak.
2. Look at the chromatogram, not just the number: small peaks close to the main peak are often deletion, insertion or isomer impurities; a report with no chromatogram cannot be checked.
3. Note the detection wavelength and method: HPLC purity is a UV area ratio, so it reflects only peptide-like material that absorbs at that wavelength and separates on that column.
4. Look for a counter-ion or salt form: trifluoroacetate or acetate content is part of the powder weight but is invisible to HPLC purity.
5. Look for quantity or net peptide content: this is the result that accounts for water, counter-ions and other non-peptide mass.

This page describes analytical chemistry for in-vitro laboratory research use only. It makes no claim about the purity of any particular lot; a published lot report, where one exists, is the only source for that. Vitality Chems publishes its lot reports on the testing page and in the COA dataset.