In this paper, we investigated the retention mechanisms of a reversed-phase/anion-exchange/cation-exchange column (Acclaim trinity P1, Thermo Fisher Scientific) for the separation of epinephrine (EPI) from norepinephrine (NOE). The impact of the acetonitrile (ACN) content, pH, and salt concentration on the retention of these two catecholamines was studied under an isocratic mode with a mobile phase mixture of ACN and ammonium formate or acetate (pH 3 to pH 5). To better understand the retention mechanisms, several retention models were explored, including linear solvent strength, adsorption, quadratic, and mixed-mode models, using various chemical compounds in addition to EPI and NOE. The quadratic and mixed-mode models were the most appropriate to explain the column retention mechanisms according to the Akaike information criterion (AIC). The research showed the importance of the ACN content on the retention of compounds according to the quadratic model, and satisfactory resolution between EPI and NOE (>1.4) was achieved with 50% ACN content. The most important retention parameters were integrated in the mixed-mode model, namely ACN content, pH, and salt concentration. Using a three-factor Box-Behnken design (BBD), other optimal conditions were obtained to separate EPI and NOE with a resolution Rs > 1.5. The ACN content and salt concentrations of the aqueous part of the mobile phase were the parameters with the greatest impact on the separation performance of the stationary phase for both catecholamines. Finally, a rapid and simple separation of a mixture of EPI, NOE, and tetracaine was obtained using a mobile phase composed of ACN/ammonium formate (pH 4; 10 mM) (60:40, v/v), with a satisfactory resolution (>1.5) between the analyte peaks.
Keywords: Box-Behnken design; Epinephrine; Norepinephrine; Retention models; Reversed-phase/anion exchange/cation exchange column- Mixed-mode chromatography.
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