1) Deprotection: Fmoc protected columns and monomers must be treated with a basic ion (piperidine) to remove the protecting group of the amino group.
2) Activation and cross-linking: The carboxyl group of the next amino acid is activated by an activator. The activated monomer reacts with the free amino group to form a peptide bond. In this step, a large amount of super-concentration reagent is used to drive the reaction to completion. Cycle: The two steps of the reaction are repeated until the synthesis is complete.
3) Elution and deprotection: The polypeptide is eluted from the column and its protecting group is eluted and deprotected by a deprotecting agent (TFA).