At the bench, a peptide is only worth having if you know what it is and how pure it is. Get either of those wrong and your results stop being repeatable. This guide walks you through how the purity and identity of a research peptide are confirmed, what a certificate of analysis (CoA) is really telling you, and how to keep freeze-dried peptides in good shape. It is written for qualified researchers, and everything mentioned is for in-vitro research only.
How purity gets measured with HPLC
Purity is usually pinned down with reversed-phase high-performance liquid chromatography (RP-HPLC). The sample travels through a hydrophobic column, normally a C18, on a water and acetonitrile gradient with a little acid in the mix, and a UV detector reads it as it comes off. That reading is usually taken at 214 nm, where the peptide bond shows up strongly, and sometimes at 280 nm to catch aromatic residues.
What you end up with is the slice of the total peak area that belongs to the main peptide. So a spec of ≥99% by HPLC is telling you the main peak makes up at least 99% of the UV signal, and whatever is left over is related impurities. Those are usually deletion or truncated sequences where a residue got dropped during synthesis, bits that were not fully deprotected, and the odd oxidation product.
Pinning down identity with mass spectrometry
HPLC can tell you how much of one thing is in the vial, but not what that thing actually is. For that you need mass spectrometry, usually electrospray (ESI-MS) or MALDI-TOF, which puts the measured molecular mass up against the mass you would expect from the sequence on paper. If the two line up within the instrument's tolerance, you have the right molecule. If they do not, something in the sequence is off, or the peptide has picked up a modification.
What you should find on a Certificate of Analysis
A CoA worth its salt is tied to one specific batch, and it usually spells out:
- Appearance: what it looks like, for instance a white freeze-dried powder.
- Identity: the measured mass next to the expected mass, from MS.
- Purity: the HPLC percentage, shown alongside the chromatogram.
- Net peptide content: how much of the powder is genuinely peptide. This one matters, because synthetic peptides soak up water and hang on to counter-ions, so the real peptide mass is normally less than the weight of the powder. It is worked out by amino-acid analysis (AAA) or nitrogen analysis.
- Water content: usually found by Karl Fischer titration.
- Counter-ion content: peptides made by solid-phase synthesis usually end up as acetate or trifluoroacetate (TFA) salts, and the counter-ion gets measured by ion chromatography or HPLC.
- Batch number and test date: so everything stays traceable.
Net peptide content is the one people skip over most: two vials both marked "5 mg" can actually hold different amounts of real peptide if their salt and water levels are not the same.
Why peptides turn up as a freeze-dried powder
Nearly all research peptides arrive as a freeze-dried (lyophilised) powder. Taking the water out under vacuum puts the brakes on the hydrolysis and enzyme activity that would otherwise chip away at a peptide sitting in solution, so you get a steady, long-lasting solid that also happens to be lighter and hardier to post.
Looking after freeze-dried peptides
Treated well, freeze-dried peptides keep for a long time. The basics are simple:
- Keep the sealed powder cold, usually around -20°C, or -80°C if you are storing it long term.
- Keep it dry and away from light, and let a sealed vial come up to room temperature before you open it, so moisture does not condense inside.
- Try not to put it through repeated freeze and thaw rounds, since those wear a peptide down over time.
(This is only about storing the material as supplied. Vosberg does not give any preparation, handling-for-use or dosing guidance, because our products are reference materials for in-vitro research and are not for human or veterinary use.)
Asking us for a certificate of analysis
Every batch we list gets its own traceable batch number and an HPLC and MS certificate of analysis, yours on request. See quality & testing for how we work, browse the catalogue, or read the companion piece on what HPLC purity testing means.
Research use only. Everything mentioned here is supplied strictly as laboratory reference material for in-vitro research. It is not a medicine and is not meant for human or veterinary consumption, or for diagnostic or therapeutic use.
Also worth a read: How to read a Certificate of Analysis.
