Analytical methods
No single test proves a compound. Each method answers one question, and knowing which question is the difference between a supplier’s claim and a verified batch.
Ten methods, ten questions
| Method | Question it answers | When | Limit |
|---|---|---|---|
| HPLC with UV detection | How pure is it, as main peak area against all peaks? | Every batch | Does not see water or salts, and does not confirm identity |
| LC-MS | Is the molecular weight right, and what are the impurities? | Every batch | Does not show how much peptide is in the powder |
| NMR | Is the structure right, and did the unusual building block go in? | Novel compounds, small molecules, short peptides | Not a blanket test; less practical for long peptides |
| qNMR | How much is really in there, without a reference standard of the compound? | Novel compounds with no standard | Needs a certified internal calibrant and a competent laboratory |
| Amino acid analysis or nitrogen analysis | Peptide content, the established way | Alternative to qNMR for content | Does not confirm structure |
| Counter-ion content by ion chromatography | How much of the powder is TFA or acetate? | When content matters | None worth stating |
| Water content by Karl Fischer | How much of the powder is water? | When content matters | None worth stating |
| Chiral purity | How much D-isomer formed during synthesis? | Sequences prone to racemisation | Requires hydrolysis in deuterated acid, then chiral LC-MS or GC-MS |
| Gas chromatography, GC-MS | Residual solvents and volatile impurities | Small molecules, and solvents in any batch | Peptides themselves are not volatile |
| Endotoxin, LAL or recombinant | Bacterial contamination | Material used on cells | Says nothing about purity or identity |
Four points we stand on
- Purity is not content. HPLC purity is the main peak relative to everything that absorbs UV; it ignores water and salts. The amount of desired peptide is content multiplied by purity.
- Purity and mass are necessary, and not sufficient. HPLC and LC-MS come first on every batch. For a novel compound they do not settle structure or content on their own.
- Content can be measured without a reference standard of the compound. qNMR does this. Amino acid analysis and elemental analysis are the traditional alternatives.
- NMR where it is warranted. For novel compounds and unusual building blocks it is the real confirmation of structure. Established compounds with a proper reference standard do not always need it.
Sources
- Bachem: Quality control of amino acids and peptides: HPLC purity, counter-ions, water, residual solvents, identity by mass spectrometry
- Bio-Synthesis: Peptide purity, peptide content and absolute amount: purity against content
- Bruker: Validation and implementation of qNMR for oligonucleotides and peptides: qNMR for content
- L- and D-amino acid determination with deuterated hydrolysis (PMC7143715): chiral purity by hydrolysis in deuterated acid and chiral LC-MS
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