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.
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