Investigations of metal-humic complexation using ion-pair reversed-phase HPLC coupled with fluorescence quenching titrations

Binding characteristics of separated fractions of humic acid (HA) and fulvic acid (FA) were studied using an ion-pair reverse-phase HPLC coupled on-line with apparatus for performing fluorescence quenching (FQ) titrations. Metal ion reagent solution was introduced post-column using a mixing "T" and was mixed with separated fractions eluting from the column by means of a post-column reaction coil. In this way, it was possible to compare the binding characteristics of the separated fractions as a function of polarity.; Binding parameters were calculated by fitting the FQ data to a 1:1 binding model using non-linear regression. Conditional stability constants (K) calculated for the binding of Cu{dollar}sp{lcub}2+{rcub}{dollar} by SFA decreased with decreasing polarity (log K values of 6.05, 5.85 and 5.36, respectively), suggesting decreased metal-binding ability with decreased polarity. An increase in the concentration of binding sites was observed with decreased polarity (C{dollar}rmsb{lcub}L{rcub}{dollar} of 19.7, 29.3 and 50.5 {dollar}mu M{dollar} for peaks 1, 2 and 3, respectively), possibly indicating different types and quantities of sites for fractions with different polarities.; Aldrich humic acid (ALHA) and Soil fulvic acid (SFA) were separated into fractions by ion-pair reverse-phase HPLC at pH 6.0 and 6.5 and metal-binding characteristics were studied using on-line fluorescence quenching titrations. As expected, the averaged log K values were approximately two orders of magnitude lower for Ni{dollar}sp{lcub}2+{rcub}{dollar} than for Cu{dollar}sp{lcub}2+{rcub}{dollar} for both ALHA and SFA at a given pH, due to Cu{dollar}sp{lcub}2+{rcub}{dollar} forming stronger inner-sphere complexes, while Ni{dollar}sp{lcub}2+{rcub}{dollar} forms complexes with its hydration shell intact. SFA exhibited increased binding ability with increasing pH for Cu{dollar}sp{lcub}2+{rcub}{dollar}, showing increased log K with increased pH (5.75 at pH 6.0 versus 6.10 at pH 6.5), presumably due to decreased competition for binding sites from protons at the higher pH. However, ALHA appears to bind more weakly at the higher pH, with a log K of 7.96 at pH 6.0 and 5.22 at pH 6.5.