Purification Of Peptides

The Purification Of Peptides Using Chromatography Techniques

Peptides are used in many applications across varied industries, but when used they need to be present in the purest form. Thus to employ these short-chain amino acids in their best quality, you need to first separate them and perform purification of peptides using any chromatography technique.

You can employ any chromatography technique to obtain pure peptides, like Ion exchange chromatography, Affinity chromatography, Size exclusion chromatography, or one of the most popular techniques used to purify the peptides, which is Reverse Phase Chromatography (RPC). Here, the choice of method depends on the specific peptide being purified from the mixture.

When implementing reverse phase chromatography, you can follow the usual routine of passing the synthetic peptide mixture through the stationary phase and a mobile phase. The stationary phase can make use of a good-quality silica adsorbent material, which can separate the peptide mixture according to its constituent amino profile, hydrophobicity, and charge.

In the RPC technique, the different amino acids have varying amounts of aquaphobic levels to the silica gel adsorbent in the stationary phase. Thus, when passing through the column, they are adsorbed at different levels and thus can be separated efficiently. In the mobile phase, you can use any organic solvent such as alcohol for the purification of peptides on reversed-phase resins.

In the reversed phase resin columns, the shape and size of the silica gel adsorbents make a lot of difference, you need to make sure the silica grades used are 10-16 microns to achieve better purity results in the end product.

RPC technique is compatible with both aqueous and organic solvents, thus making it the best choice for the purification of peptides. The technique is also comparatively easy to set up and operate for the purification of peptides in various applications.

You can also combine RPC with mass spectrometry if you want to analyze the peptide and protein output from the column chromatography. The output you achieve will have different levels of purification of peptides, from smaller peptides to large amounts of proteins, and thus is also considered a highly scalable technique. When you check the purity of the peptide output using mass spectrometry, you can repeat the chromatography process multiple times till you get the desired level of purity