Synthetic peptide impurities: what the small HPLC peaks are
Quality · 4 min read · Updated November 5, 2026
The small peaks beside the main peak in a peptide chromatogram are mostly near relatives of the target, made during synthesis or storage. The common ones are deletion sequences missing one residue, chains capped early, residues still carrying a protecting group, and forms that are oxidized, deamidated or contain a D-amino acid. Most can be recognized by how far their mass sits from the target's.
How are synthetic peptides made?
Most research peptides are made by solid-phase peptide synthesis (SPPS), introduced by Merrifield in 1963. The chain is built one residue at a time from the C-terminus to the N-terminus while anchored to a resin bead. Each cycle removes the temporary Fmoc group from the chain's end, then couples the next protected amino acid. Reactive side chains carry permanent protecting groups, such as tBu on Ser, Thr, Tyr and Glu, Boc on Lys and Pbf on Arg, which come off with the resin in a final acid cleavage.
No step is perfectly efficient, and the shortfalls add up. If each coupling succeeds 99% of the time, a 20-residue peptide needs 19 couplings and only 0.99 to the 19th power, about 83%, of the chains are full length before any other side reaction. At 98% per step that falls to about 68%. That is why crude peptide is purified, usually by preparative reverse-phase HPLC, before it is tested and released.
What are deletion and truncated sequences?
A deletion sequence is missing one residue because a coupling failed and the next one succeeded on the same chain. It is written des-Xaa, or n-1 for the general case. It is the hardest impurity to remove, because a peptide one residue short often behaves almost like the target on the column. Its mass is the target's minus that residue's mass. For an invented decapeptide, GAYSLKTEVR, with a monoisotopic mass of 1,122.60 Da, des-Gly appears at 1,065.58 Da, des-Leu at 1,009.52 Da and des-Lys at 994.51 Da.
A truncated sequence stopped growing partway through. Many syntheses cap unreacted chains with acetic anhydride after each coupling, so a failed chain ends in an acetyl group and never grows again. Capped chains are several residues short and usually far from the main peak, which makes them easier to remove than deletions. Insertion sequences, with one residue added twice, also occur, though less often: an extra Gly in the example would appear at 1,179.62 Da.
What does an incompletely deprotected peptide look like?
If the final cleavage does not remove every side-chain protecting group, part of the material keeps one. These forms are more hydrophobic than the target, so they usually elute later, and their masses rise by a fixed amount: about +56.06 Da for a remaining tBu group, +100.05 Da for Boc and +252.08 Da for Pbf. For the example decapeptide, those would appear at 1,178.67, 1,222.66 and 1,374.69 Da.
Which impurities form after synthesis?
Some impurities form later, during cleavage, purification or storage, and they change the mass only slightly. Methionine and, less readily, tryptophan oxidize, adding 15.995 Da per oxygen. Asparagine and, more slowly, glutamine deamidate to aspartate and glutamate, adding only 0.984 Da, which is easy to miss on a low-resolution instrument. An N-terminal glutamine can cyclize to pyroglutamate, losing 17.03 Da. Moisture, warmth and solutions speed up all of these, which is one reason storage conditions matter.
Aspartic acid causes its own problem during Fmoc synthesis. The base used to remove Fmoc can make an Asp side chain close into a ring, called an aspartimide, which loses 18.01 Da. The ring then reopens, either to the normal α-linked peptide or to a β-aspartyl peptide, which has the same mass as the target but a different backbone. It can also react with piperidine to give a piperidide, about 67 Da heavier. The risk is highest when Asp is followed by Gly, Ser or Asn.
Which impurities does a mass spectrum miss?
Impurities with the same mass as the target pass an identity check by mass. They include β-aspartyl forms, sequences with two residues swapped and diastereomers, where one L-amino acid has turned into its D-form. That change, called racemization, happens most readily at cysteine and histidine during coupling. HPLC separates some of these variants from the main peak and not others. An unresolved one is counted as target in the purity figure.
What does a purity figure say about impurities?
HPLC purity is the main peak's share of the UV signal at 214 nm, so a lot at 99.3% has 0.7% of that signal in other peaks. A certificate usually reports the total rather than naming each impurity. To identify a particular side peak, compare its mass from LC-MS with the shifts above. A difference of about 16 Da suggests oxidation, a whole-residue mass suggests a deletion or insertion, and a fixed protecting-group mass suggests incomplete deprotection.
Every SCALE Performance lot is tested for purity by reverse-phase HPLC and for identity by LC-MS before release, and released lots are 99% or higher. These are the manufacturer's own results. The article on HPLC and mass spectrometry explains both methods in more detail.
Questions
What is a deletion sequence in peptide synthesis?
A peptide missing one residue because a coupling step failed and the next coupling succeeded on the same chain. Its mass is the target's minus the missing residue's mass, and it often elutes very close to the target in HPLC.
Why does peptide oxidation add 16 Da?
Oxidation adds one oxygen atom, 15.995 Da, most often to the sulfur of methionine. Each oxidized residue adds that amount again.
Can an impurity have the same mass as the peptide?
Yes. β-aspartyl forms, sequences with swapped residues and D-amino acid diastereomers all have the target's mass, so only a separation such as HPLC can tell them apart, and not always.
Is 99% HPLC purity free of impurities?
No. It means about 1% of the UV signal comes from other peaks, and any impurity hidden under the main peak is counted as target. Water and counter-ions are not counted at all.
References
- Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. Merrifield RB. Journal of the American Chemical Society 85(14):2149–2154, 1963.
- Amino Acid-Protecting Groups. Isidro-Llobet A, Álvarez M, Albericio F. Chemical Reviews 109(6):2455–2504, 2009.
- Related impurities in peptide medicines. D'Hondt M, Bracke N, Taevernier L, et al. Journal of Pharmaceutical and Biomedical Analysis 101:2–30, 2014.
- The aspartimide problem in Fmoc-based SPPS. Part I. Mergler M, Dick F, Sax B, Weiler P, Vorherr T. Journal of Peptide Science 9(1):36–46, 2003.
- Protein Identification Tools: mass values used (FindMod). SIB Swiss Institute of Bioinformatics, Expasy.