Solid-phase synthesis in one paragraph
Bruce Merrifield's 1963 idea, for which he received the 1984 Nobel Prize in Chemistry, was to anchor the first amino acid to an insoluble resin bead so that every subsequent reagent could be washed away by filtration. The chain grows from the C-terminus toward the N-terminus, one residue per cycle. When the sequence is complete the chain is cut from the resin, the protecting groups are removed, and the crude product is purified. Almost every synthetic peptide sold for research is made this way, today almost always with the Fmoc protecting-group chemistry.
One cycle, four steps
- Deprotect. The Fmoc group on the free end of the growing chain is removed with a mild base, exposing an amine.
- Wash. Excess base and by-products are filtered off the resin.
- Couple. The next amino acid, itself Fmoc-protected and with any reactive side chain masked, is activated and added. It forms a peptide bond with the exposed amine.
- Wash again. Unreacted amino acid and coupling reagents are removed, and the cycle repeats for the next residue.
A tripeptide like KPV needs three cycles. A 39-residue molecule like retatrutide needs thirty-nine, and its acylated lysine side chain needs additional steps to attach the fatty diacid. Each cycle is close to but not exactly 100% efficient, which is where the next section comes from.
Where impurities come from
- Deletion sequences. If a coupling fails on a fraction of chains and those chains are not capped, they continue growing without that residue. The result is a peptide one amino acid short, often hard to separate because it is so similar to the product.
- Truncated sequences. Chains that stopped growing altogether, usually because a coupling failed and the site was then capped.
- Incomplete deprotection. A protecting group that survived cleavage leaves an adduct with a characteristic extra mass.
- Oxidation. Methionine, cysteine and tryptophan can oxidise during synthesis, cleavage or storage.
- Racemisation. Some residues, histidine and cysteine especially, can flip stereochemistry during activation, giving a diastereomer that co-elutes with the product.
The longer the sequence, the more cycles there are for something to go wrong, and the harder the impurities are to remove. That is why a 99% purity figure on a 39-residue peptide represents more work than the same figure on a tripeptide.
Cleavage, and why TFA is in the vial
Cutting the finished chain from the resin and stripping the side-chain protecting groups is done with concentrated trifluoroacetic acid (TFA), usually with scavengers added to catch the reactive fragments released. The crude peptide is precipitated, then purified by reversed-phase HPLC using a mobile phase that also contains a small amount of TFA. The purified fractions are pooled and freeze-dried, and the peptide comes out of that process as a trifluoroacetate salt: every basic residue carries a TFA counter-ion. This is the counter-ion mentioned on the certificate guide, and it is why net peptide content is lower than the gross mass. Some manufacturers exchange TFA for acetate in an extra step; the certificate or the manufacturer will say if so.
Closing the loop: HPLC and mass spectrometry
Analytical HPLC separates the purified product from whatever survived purification and reports the main peak as a percentage of the total, which is the purity figure on a certificate. Mass spectrometry measures the mass of that main peak and confirms it matches the intended sequence. Together they answer the two questions that matter: how much of the material is one compound, and is that compound the right one. A deletion sequence shows up as a peak with a mass exactly one residue short; an oxidised methionine as a peak sixteen mass units heavier. The certificate is the record of that check for one lot.
What this means for the vial in your hand
- The powder is peptide plus counter-ion plus a little water. See why peptides are freeze-dried.
- Purity is per lot, because every synthesis run is a separate set of cycles. A certificate belongs to a batch, not a product.
- Sequence is what the specification says it is only if mass spectrometry confirmed it. Identity and purity are separate measurements and a good certificate reports both.
- The identity data for every compound we carry is on the reference table.