Journal ArticleChromatographic‐Based Binding and Thermodynamic Studies of Antibiotic Micropollutants with Humic Acid Using Affinity MicrocolumnsSharmeen, Sadia [Department of Chemistry University of Nebraska‐Lincoln Lincoln Nebraska USA]; Kyei, Isaac [Department of Chemistry University of Nebraska‐Lincoln Lincoln Nebraska USA]; Poddar, Saumen [Department of Chemistry University of Nebraska‐Lincoln Lincoln Nebraska USA]; Iftekhar, Sazia [Department of Chemistry University of Nebraska‐Lincoln Lincoln Nebraska USA]; Sajeeb, BK [Department of Chemistry University of Nebraska‐Lincoln Lincoln Nebraska USA]; Graham, Lillian M [Department of Chemistry University of Nebraska‐Lincoln Lincoln Nebraska USA]; Snow, Daniel D [Water Science Laboratory and Nebraska Water Center University of Nebraska‐Lincoln Lincoln Nebraska USA]; Hage, David S [Department of Chemistry University of Nebraska‐Lincoln Lincoln Nebraska USA]High‐performance affinity microcolumns with entrapped humic acid were utilized to investigate interactions between this natural carrier agent and several classes of antibiotics that are common emerging environmental contaminants, or micropollutants. Aldrich humic acid was used as a general model for this type of binding agent. Chromatographic studies under various temperature and mobile phase conditions were used to characterize interactions of the humic acid with the antibiotics sulfadiazine and sulfamethoxazole (sulfonamides), clarithromycin (a macrolide), and lincomycin (a lincosamide). It was determined by this approach that sulfadiazine and sulfamethoxazole had moderate affinities for the humic acid at pH 7.0 and 25°C, with distribution equilibrium constants (KD) of ∼2–3 × 10^1 L/kg and global affinities (nK’a) of ∼0.8–1.0 × 10^3 M^−1. Lincomycin and clarithromycin had stronger binding, with KD and nK’a values of 3.8–7.5 × 10^2 L/kg and 1.3–2.6 × 10^4 M^−1. All the antibiotics had a negative for this binding, representing spontaneous reactions, and a negative change in enthalpy; however, the change in free energy due to entropy was positive in some cases but negative in others. The binding strength decreased in each case as the ionic strength increased. A change in pH also affected binding, as was consistent with the presence of significant electrostatic interactions from some of the antibiotics. These experiments demonstrated how affinity microcolumns could be employed to study such interactions quickly and with only small amounts of binding agent. The fundamental information obtained through this analytical technique should be valuable in characterizing the transport and activity of these antibiotics in the environment and in adapting this approach to the study of other binding agents and micropollutants that may be found in water.Wiley2026-01-0110668282Journal of Separation Science4911615-9306https://doi.org/10.1002/jssc.703452404209; 2320239AntibioticsMicropollutantsHumic acidBinding studiesHigh-performance affinity chromatographyAffinity microcolumnsNational Science Foundation