Purification of two peptides crude mixtures with gradient elution RP-LC and study of their adsorption isotherms by means of inverse method

It is well-known that some peptides are able to trigger physiologic reactions, interacting with receptors in the human body. In the past, these biomolecules have been widely studied for their potential use in pharmaceutical field [1]. Peptides are obtained mainly by mean of solid phase synthesis, which unfortunately lead to very complex mixtures. RP-LC is one of the main techniques employed to separate single target molecule from the impurities that must be removed [2,3]. The focus of this research is centered on two peptides containing 8 and 10 amino acids respectively. The purification of crude (=not purified) mixtures has been studied with RP-LC using solvent gradient programs. The time gradient and the percentages of the mobile phases (MPA: 0.02% TFA in water; MPB: 0.02% TFA in ACN) have been varied to enhance separation efficiency. We tested also the behavior of two C18 columns with different particle size (1.9 um; 5 um) and dimensions (100x3 mm; 150x4.6 mm). Knowing the optimal separation conditions in a batch method is the first step to streamline the scale-up of the purification process at industrial level, where implementing processes in a continuous manner is of great importance. The finished (=purified) products have been used for obtaining the adsorption isotherms of the peptides in isocratic conditions at different mobile phase compositions. It is known that often the organic solvent percentage affects the retention factor considerably and consequently the isotherm parameters [4], so this dependence has been evalueted. Inverse method has been used to collect the experimental data and fit them to several adsorption models. The shapes of the chromatographic profiles suggest that the isotherms should be convex upward. References [1] de Castro R. J. S. et al., Food Research International 74 (2015) 185-198. [2] Bernardi S. et al., Journal of Chromatography A 1283 (2013) 46-52. [3] Asberg D. et al., Chromatographia 80 (2017) 961-966. [4] Marchetti N. et al., Journal of Chromatography A 1079 (2005) 162-172