Although SPPS is the method of choice for synthesis of peptides in both research and industrial modes, it could be interpreted as a black box. This creates an important challenge in the knowledge about the process, such as the reaction kinetics and reaction efficiency. There are indirect methods to analyze some of the steps involved in, but they led very often to false positive/negative results and require in all cases the interruption of the process.

Having this in mind, we have developed for the first time a process analytical tool, PAT, based on the refractive index, RI, that allows real-time monitoring of the three SPPS steps, coupling, deprotection, and washing.

The RI is a very sensitive parameter that varies in the function of the amount of mass dissolved in the reaction solution. Thus, by RI measurements we can detect: i decrease of protected amino acid in the solution during coupling, ii the dibenzofulvene concentration increases during Fmoc removal and iii the remaining compounds in the solution during the wash-steps. Moreover, the analysis of RI plots over time provide valuable information regarding the reaction kinetics, failures on the coupling or deprotection, unexpected reactivity of some protected amino acids, and even the presence of some side-reactions.

Using RI, we were able to determine the initial loading of the resin, optimize the synthesis of some peptides and, very importantly, evaluate new technologies such as coupling reagents and green solvents. Definitely, RI is an excellent PAT for Greening the SPPS process.