Combination of a Copper-Ion Selective Electrode and Fluorometric Titration for the Determination of Copper(II) Ion Conditional Stability Constants of Humic Substances

Abstract

A fluorescence quenching model using copper(II) ion (Cu²⁺) ion selective electrode (Cu-ISE) is developed. It uses parallel factor analysis (PARAFAC) to model fluorescence excitation–emission matrices (EEMs) of humic acid (HA) samples titrated with Cu²⁺ to resolve fluorescence response of fluorescent components to Cu²⁺ titration. Meanwhile, Cu-ISE is employed to monitor free Cu²⁺ concentration ([Cu]) at each titration step. The fluorescence response of each component is fit individually to a nonlinear function of [Cu] to find the Cu²⁺ conditional stability constant for that component. This approach differs from other fluorescence quenching models, including the most up-to-date multi-response model that has a problematic assumption on Cu²⁺ speciation, i.e., an assumption that total Cu²⁺ present in samples is a sum of [Cu] and those bound by fluorescent components without taking into consideration the contribution of non-fluorescent organic ligands and inorganic ligands to speciation of Cu²⁺. This paper employs the new approach to investigate Cu²⁺ binding by Pahokee peat HA (PPHA) at pH values of 6.0, 7.0, and 8.0 buffered by phosphate or without buffer. Two fluorescent components (C1 and C2) were identified by PARAFAC. For the new quenching model, the conditional stability constants (logK₁ and logK₂) of the two components all increased with increasing pH. In buffered solutions, the new quenching model reported logK₁ = 7.11, 7.89, 8.04 for C1 and logK₂ = 7.04, 7.64, 8.11 for C2 at pH 6.0, 7.0, and 8.0, respectively, nearly two log units higher than the results of the multi-response model. Without buffer, logK₁ and logK₂ decreased but were still high (>7) at pH 8.0 (logK₁ = 7.54, logK₂ = 7.95), and all the values were at least 0.5 log unit higher than those (4.83 ∼ 5.55) of the multi-response model. These observations indicate that the new quenching model is more intrinsically sensitive than the multi-response model in revealing strong fluorescent binding sites of PPHA in different experimental conditions. The new model was validated by testing it with a mixture of two fluorescing Cu²⁺ chelating organic compounds, i.e., _l-tryptophan and salicylic acid mixed with one non-fluorescent binding compound oxalic acid titrated with Cu²⁺ at pH 5.0.