Ion-exchange centrifugal partition chromatography: A methodological approach for peptide separation

This article presents the scope and optimization strategies employed in ion-exchange centrifugal partition chromatography (IXCPC). Both the weak and the strong modes were used to separate the constituents of a model mixture of dipeptides. Thus, the combined use of the quaternary biphasic solvent system, methyl-tert-butylether/acetonitrile/n-butanol/water (2:1:2:5, v/v) in the descending mode, of the lipophilic di(2-ethylhexyl)phosphoric acid (DEHPA) cation-exchanger, and of two displacers: calcium chloride and hydrochloric acid, has proven to be efficient for the preparative separation of the model mixture of five dipeptides (GG, GY, AY, LV and LY, in the order they were collected). The separation was optimized by splitting the stationary phase into two sections that differed by their triethylamine concentration. Moreover, the chemical nature of the exchanger/analyte entities that were involved in the chromatographic process was determined by 31P and 1H DOSY NMR experiments.