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

## TL;DR

Researchers used a new chromatography method to study how benzophenone interacts with humic acid immobilized on a polymer column.

## Contribution

A novel frontal affinity chromatography method was developed using Cu(II)-coordinated monolithic columns to immobilize and study interactions with humic acid.

## Key findings

- Benzophenone binding to humic acid showed two interaction site classes with binding constants KL = 1.2×10⁶ and 6.7×10³ L/mol.
- The maximum sorption capacity of benzophenone was found to be 19.2 μmol/g with good reproducibility across three columns.
- Cu(II) on monolithic columns allowed reversible immobilization of humic acid, enabling exchangeable chemistries for different sources.

## Abstract

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 KL = (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.

Image 1

•Frontal affinity chromatography on humic acid-supported stationary phase.•Benzophenone retained by immobilized humic acid.•Concentration-dependent retardation enables computing binding affinity and capacity.•Cu(II) on monolithic support is a versatile platform to immobilize humic acid reversibly.

Frontal affinity chromatography on humic acid-supported stationary phase.

Benzophenone retained by immobilized humic acid.

Concentration-dependent retardation enables computing binding affinity and capacity.

Cu(II) on monolithic support is a versatile platform to immobilize humic acid reversibly.