Zr (IV) immobilized polymeric microspheres for selective adsorption and chromatographic separation of adenosine analogues

The efficient separation of adenosine analogues (e.g., Ado, AMP and dA) is crucial for clinical diagnosis, drug discovery and basic research. However, their structural similarities pose significant challenges to the separation and purification. PS@PGMA-IDA-Zr 4+ (PPIZ) microspheres were synthesized as IMAC stationary phases in this study. Efficient separation of Ado, AMP and dA was achieved by leveraging differential interactions including Zr 4+ -phosphate coordination, Zr 4+ -diol coordination and hydrogen bonding. Static adsorption experiments identified the PPIZ40–0.7 microspheres at pH 6 with optimal selectivity for the adenosine analogues (α (Ado/dA)  = 4.46, α (AMP/Ado)  = 41.40), while material characterization and adsorption kinetics elucidated the separation mechanism. Elution experiments demonstrated that Ado and AMP could be eluted utilizing PBS solutions with phosphate concentrations of 0.1 mM and 10 mM, respectively. Moreover, in chromatographic separation tests, PPIZ40–0.7 microspheres as packing material achieved excellent resolution for the adenosine analogues (Rs (Ado/dA)  = 5.39, Rs (AMP/Ado)  = 3.91), along with robust mechanical stability and reusability. Therefore, the PS@PGMA-IDA-Zr 4+ microspheres demonstrate significant promise for high-efficiency chromatographic separation of adenosine analogues.