Purification of naturally occurring peptides by reversed-phase HPLC

Reversed-phase high performance liquid chromatography (HPLC) has become the method of choice for the purification of peptides and small proteins (M_r < 10,000 Da) from natural sources. The technique combines high resolution and recovery with ease and speed of operation and is applicable to a wide range of peptides with different physicochemical properties. This protocol describes procedures for (1) the extraction of a biologically active peptide from animal tissue, (2) concentration of the extracts and partial purification on Sep-Pak cartridges, and (3) purification to near homogeneity on a range of silica-based HPLC columns. Standard operating procedures involve acetonitrile as organic modifier, trifluoroacetic acid as ion-pairing reagent and sequential chromatographies on octadecyl (C₁₈), butyl (C₄) and diphenyl wide-pore (300 Å) columns under gradient elution conditions. The limiting factor in the time taken to isolate a peptide is usually the speed at which assays to detect the peptide can be performed, but purifications can generally be accomplished within 1 or 2 weeks.