Peptide Purification

Bio-Synthesis offers several type of peptide purification methods to remove truncated or deletion peptide sequences, isomers or other side products occur at any step during peptide synthesis. The peptide purification strategies we use are usually bases on the combination of separation methods based on the physiochemical characteristics of peptides, including size, charge and hydrophobicity. Purification techniques include: 1) size-exclusion chromatography, 2) ion exchange chromatography (IEC), 3) partition chromatography, 4) high-performance liquid chromatography (HPLC).

Reverse-phase HPLC Peptide Purification

Reverse-phase chromatography (RPC) is the most versatile and most widely used method of peptide purification. With traditional methods of HPLC, the stationary phase captures polar, hydrophilic molecules that are then differentially eluted by increasing the concentration of polar solvents in the mobile phase. In RPC, as the name implies, hydrophobic molecules from aqueous solutions are instead captured by the stationary phase using hydrophobic C4, C8 or C18 n-alkyl hydrocarbon ligands, and their retention time is a function of the hydrophobicity of the molecule and that of the mobile phase.

RP-HPLC Peptide purification separates the target peptides from impuries such as isomers, deletion sequences, peptide products from side reactions with free coupling and protecting groups or peptide that have undergone side-chain reactions. Peptide purity is analyze as a percentage of the main peptide sequence to impurities that absorb at the peptide bond absorption wavelength (210-220nm), and varying levels of purity are commercially available based on the application in which the peptides will be used:

• >95% – Quantitative studies such as NMR, receptor-ligand binding studies, ELISA and RIA, monoclonal antibody production, in vivo studies
• >80% – High-throughput screening, non-quantitative blocking in immunohistochemical (IHC) and Western blot analyses, non-quantitative enzyme-substrate studies, antibody affinity purification and plate coating for cell attachment
• >70% – ELISA standards, ELISPOT assays and polyclonal antibody production