Towards multimodal HPLC separations on humic acid-bonded aminopropyl silica: RPLC and LEC behavior

In the present study, metal binding property of humic acid (HA) was successfully adapted to the ligand-exchange concept, and metal-loaded immobilized humic acid was used as a ligand exchanger stationary phase for separation of some nucleosides. Humic-acid bonded aminopropyl silica (EC-HA-APS) was turned into ligand exchanger forms by loading aqueous solutions of Cu²⁺, and Co²⁺ to the column (4.6 × 150; as mm) packed with EC-HA-APS. Metal ion solutions were loaded to the column in a stepwise manner where the concentration of metal ion solution being loaded to the column was increased gradually between 5 and 100 mM. The progress of metal loading process was monitored via the breakthrough curves propagated stepwise. Ligand-exchange chromatography (LEC) studies were performed on an HPLC system, and chromatographic behaviors of the studied nucleosides (i.e. uridine, Urd; thymidine, Tyd; cytidine, Cyd; adenosine, Ado; and guanosine, Guo) were investigated on Cu²⁺ and Co²⁺ loaded forms of the EC-HA-APS (Cu-EC-HA-APS and Co-EC-HA-APS). Effect of mobile phase composition, temperature, and the type of metal ion loaded to the column on the retentive behaviors of the compounds was studied, in detail. The studied solutes exhibited mixed-mode RPLC/LEC behavior on the stationary phase. Metal-loaded column (M-EC-HA-APS) was easily regenerated into its original form, EC-HA-APS, with 98 ± 2% metal recoveries, by using aqueous mixture of EDTA + NH₃ at pH = 7.5. Thus, the stationary phase exhibited a high flexibility between RPLC and LEC modes. This property, also, made it possible to convert the stationary phase into various ligand exchanger forms by loading different metal ions. Hence, capacity and selectivity of the stationary phase towards the studied species was manipulated easily by loading different metal ions to the stationary phase. Baseline separation for the studied species was achieved on Cu-EC-HA-APS and Co-EC-HA-APS and some differentiations were observed in capacity and selectivity, depending on the type of metal loaded. Thus, being as the first endeavor on usability of immobilized HA as a ligand exchanger stationary phase, the present study is believed to be useful to understand multifunctional character of HA-based solid/stationary phases.