The difference between peptide purity and peptide content

The difference between peptide purity and peptide content

In the process of using peptides, we often encounter two indicators: peptide purity and peptide content. Many friends may have this question: why is our peptide purity 95%, but the peptide content is only 80%? Because these are two completely different concepts.

Peptide purity: The purity of a peptide refers to the content of the target peptide detected by HPLC analysis at 214nm (214nm is the absorption wavelength of the peptide chain);

Peptide content: refers to the percentage of peptide substances in the sample relative to non peptide substances;

The purity of peptides refers to the content of target peptides detected by HPLC analysis at 214nm (214nm is the absorption wavelength of peptide chains); In addition to the target peptide, impurities that can also be detected by HPLC include: missing sequences (target sequences with one or more amino acid residues missing), truncated sequences (sequences generated during the protection process), and incomplete deprotection sequences (sequences generated during the entire synthesis process or the final cleavage process); However, HPLC cannot detect water and residual salts such as trifluoroacetate and acetate. In the process of peptide synthesis, the peptide sequence is cleaved from the resin by trifluoroacetic acid, and a small amount of TFA is also produced during purification. Therefore, the free amino end of the peptide and other side chains such as Arg, Lys, His can generate a small amount of TFA impurities; Another factor is water. Peptides are usually dehydrated by freeze-drying, and even so, water exists to varying degrees due to different covalent binding abilities. Therefore, peptides delivered usually contain TFA salts and trace amounts of water, which cannot be detected by HPLC, resulting in differences in peptide content and purity.

We know that the purity of peptides is determined by HPLC analysis, so how is the peptide content determined? Usually, peptide content is determined by amino acid composition analysis or ultraviolet spectroscopy. This information is mainly important for calculating peptide concentration in experiments that are sensitive to peptide concentration.