Modeling the Nonlinear Gradient Behavior of a Pharmaceutical Relevant Peptide in RP-LC

t 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. Solvent gradient programs in RP-LC have been used to model/optimize the separation of the two crude (=not purified) mixtures, by varying the slope of the gradient and the percentages of the organic fraction (0,02% TFA in ACN). Three C18 columns with different dimensions, pores size and particles size have been tested. Knowing the optimal separation conditions in a batch method is the first step towards the scale-up of the purification process at industrial level, where implementing processes in a continuous manner is of great importance. The crude mixtures have been used also for obtaining the adsorption isotherms of the peptides in isocratic conditions at different mobile phase compositions, chosen depending on gradient experiments. It is known that the organic solvent fraction affects considerably the retention factor of a peptide and consequently the isotherm parameters [4], so this dependence has been evaluated. To collect the experimental data and fit them to several adsorption models we have used the inverse method, which is a numerical approach to isotherm determination particularly suitable when one has very little amount of a compound. The shapes of the chromatographic profiles suggest that the isotherms should be convex upward, for example Langmuir model fits quite well the experimental peaks. 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.