Four steps on a plastic bead, repeated once per amino acid. The impurity that is hardest to detect is created by the same loop that builds the peptide.
Almost every research peptide is built the same way. It is assembled one amino acid at a time, on a tiny plastic bead. The method is called solid-phase peptide synthesis, or SPPS. Robert Bruce Merrifield published it in 1963 and won a Nobel Prize for it in 1984. The machines have got faster since. The steps have not changed.
The trick is the bead. It never dissolves. So the chemists can flood it with reagents, rinse everything away, and leave the growing chain behind. That is what makes the whole process repeatable.
Steps 2 and 3 repeat, with a wash between them, once for every amino acid in the sequence. A twenty-link peptide means twenty passes around that loop.
Coupling is never perfect. On each pass, a small share of the chains fail to accept the next amino acid. Those chains are not thrown away. They stay on their beads and carry on through the remaining cycles, permanently one link short.
The result is a deletion sequence: a copy of your peptide missing an amino acid from somewhere in the middle. This is the main impurity in most batches, and it is the hardest one to remove.
The reason is uncomfortable. A deletion sequence is almost the same weight as the target and almost the same shape. So it travels through a chromatography column at almost the same speed. It reaches the sensor at almost the same moment. So it tends to hide under the main peak instead of forming one of its own. That is exactly the blind spot described in what a 99% purity number does not tell you.
It also explains something that looks odd at first. Longer peptides are much harder to make cleanly than short ones. Every extra amino acid is another cycle. Every cycle is another chance for chains to fall behind.
This article explains published research and the rules around it. It carries no dose, no protocol, and no claim that any compound treats, cures or prevents a condition in anyone. Where the evidence is thin we say so. See our editorial standards.
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