Preparative reversed-phase HPLC is the established method for the purification of peptides, but has significant limitations. We systematically investigated the use of high-performance reversed-phase flash chromatography (HPFC) to rapidly purify laboratory-scale quantities of crude, synthetic peptides and chemically modified insulins. We demonstrated these methods for a diverse set of peptides, including short, medium, and long peptides. Depending on the purity profile of the peptide, HPFC can be used either as the sole purification method, or as a prepurification method prior to final HPLC purification. Furthermore, HPFC is suitable for the purification of peptides that are not fully in solution. We provide guidelines for the HPFC of synthetic peptides and small proteins, including the choice of columns, eluents, and gradients. We believe that HPFC is a valuable alternative to HPLC purification of peptides and small proteins.

The use of C‐terminal peptide thioesters and hydrazides in synthetic protein chemistry has inspired the search for optimal solid‐phase peptide synthesis (SPPS) strategies for their assembly. However, peptide thioesters are not directly accessible by conventional Fmoc‐SPPS owing to the nucleophilicity of the secondary amine required for Fmoc removal. Here, we report the mild and effective activation of the pGlu linker and a new safety‐catch linker that was used for the convenient synthesis of peptide thioesters and hydrazides via efficient amide‐to‐imide activation followed by nucleophilic displacement.