Reversed-Phase Chromatography of Peptides and Proteins

RP chromatography is one of the standard methods for analyzing peptides and proteins. It stands out for a very good selectivity and performance. 

One of the most common methods is the use of trifluoroacetic acid (TFA) in water against acetonitrile gradients. TFA has the advantages that it reduces the pH value, the carboxyl groups are protonized, and it forms an ion pair with the amino groups. Thus, the peptide or protein has no more free charges and, therefore, it is as hydrophobic as possible. 

If none of the information that is important for chromatography is available, the starting point is normally a screening gradient of 5-95% 0.1% TFA against ACN 0.1% TFA. The following sections describe the different parameters for the development and adaptation of methods. 

---