Topic summary

Follow-up: Trifluoroacetate content and its consequences

Analytics › Impurities & related substances counter-ion content endotoxin testing HPLC data table Solved Closed

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a.zamoraTL2 Moderator10 Oct 2024#3

post #2 answers the question as asked. The question underneath it is different.

Related substances: compounds chemically related to the target peptide but not the target peptide itself. The standard method separates them and reports them as area percent. How related they can be before they exceed specification is a regulatory question.

17 likes 22mo

d.tammTL2 Moderator Solution11 Oct 2024 · edited#4

On post #2 — agreed on the reasoning, with one qualification.

Disulfide formation: if a peptide contains cysteine, it can form disulfide bonds with itself or with other molecules. Under oxidising conditions multiple species appear. Reducing conditions (like DTT) convert them back.

33 likes 22mo

n.norgaardTL2 Moderator15 Oct 2024#8

Aggregates: multiples of the monomer mass. May not elute at all under a standard reversed-phase method. A species that does not come off the column does not appear in the area percentage.

25 likes 21mo

k.okaforTL2 Moderator17 Oct 2024#11

b.nwosu, post #10: Coming back to post #8, because the follow-up matters more than the original answer. Dimer and higher-order multimers: two or more peptide molecules bonded together. They appear at double the mass and higher. They may or may not separate from the monomer on HPLC depending on the method. Go to post

Truncation products: fragments from incomplete synthesis or from degradation. They elute quite differently from the intact peptide because they are much smaller and have different hydrophobicity. They are usually well separated.

23 likes in reply to #10 21mo

n.serranoTL2 Moderator20 Oct 2024#15

ni.stanescu, post #13: Coming back to post #11, because the follow-up matters more than the original answer. Deletion sequences (incomplete coupling during synthesis): lower in mass by one residue. Chromatographically they usually elute earlier or later depending on the residue's hydrophobicity. They are the most common impurity in solid-phase synthesis. Go to post

31 likes in reply to #13 21mo

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Second pass at: Where impurities in solid-phase peptide synthesis come fromAnalytics › Impurities & related substances · 51 replies
Deamidation and the close-eluting pair it produces — the long versionAnalytics › Impurities & related substances · 3 replies

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