Frontal affinity chromatography to investigate the interaction of benzophenone with humic acid supported on microbore monolithic columns

The principles of frontal affinity chromatography were used to determine the sorption constants and sorption capacities of benzophenone on immobilized humic acid. Poly(glycidyl-co-ethylene dimethacrylate) monoliths were constructed inside microbore capillaries (12 cm long × 1.016 mm internal diameter) and further aminated with ethylenediamine. The free amine groups coordinated Cu(II), which served as an intermediate ligand to immobilize about 27.2-28.7 mg of humic acid per gram of polymer skeleton (or 93 ± 4 μg per cm of column). The reversible nature of the interactions with Cu(II) allowed to leach and reload humic acid, thus suggesting that a single Cu(II) modified column may be further explored to immobilize humic acids from different sources using the concept of exchangeable chemistries on a stable monolithic platform. Frontal affinity chromatograms were obtained by injecting 1000 μL of benzophenone solutions of various concentrations (1.11-112 μmol L^-1) at 25 °C and pH 7.00 ± 0.1. The concentration-dependent elution volume enabled the construction of sorption isotherms that were fitted to Langmuir and Freundlich equations and the linearized Scatchard plot. The binding of benzophenone to the humic substance was ruled by two classes of interaction sites with <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:mrow><mml:msub><mml:mi>K</mml:mi> <mml:mi>L</mml:mi></mml:msub> </mml:mrow> </mml:math> = (1.2 ± 0.2) × 10⁶ and (6.7 ± 0.8) × 10³ L mol^-1 and a maximum sorption capacity of 19.2 ± 1.2 μmol g^-1. The results correspond to an average of duplicate injections in three columns, thus demonstrating the acceptable reproducibility and stability of the proposed methodology.