Total luminescence spectroscopy was applied to the fluorescence characterization of humic substances obtained from the International Humic Substances Society (IHSS). Results show that total luminescence spectra, represented as excitation−emission matrices (EEMs), may be used to discriminate between soil-derived and aquatic-derived IHSS humic substances and between humic and fulvic acids derived from the same source (soil or aquatic). Ionic strength in the range of 0−1 M KCl and humic substance concentration in the range of 5−100 mg/L had little effect on the fluorescence spectral characteristics of the humic substances, while pH had significant effects as expected. Absorbance correction was shown to be essential for accurate representation and comparison of the EEMs of the humic substances at high concentrations.

A new technique, total lifetime distribution analysis (TLDA), is described for rapid, sensitive, and accurate lifetime characterization of complex samples. Multiharmonic Fourier transform technology in a commercial, frequency-domain fluorescence lifetime instrument allows rapid acquisition of TLDA data. High sensitivity derives from the use of the entire fluorescence emission from the sample in the lifetime measurement. The maximum entropy method (MEM) provides a consistent basis for modeling of the lifetime data for accurate recovery of the total lifetime distribution of the sample. Because MEM is self-modeling, it is not subject to the same sources of bias that influence nonlinear least-squares fits of lifetime data to a priori models. These features make TLDA an effective tool for sample characterization and fingerprinting that is based on the responsiveness of fluorescence lifetime to the chemical composition and dynamic processes that contribute to the uniqueness of the sample. TLDA results are presented for coal liquids and a humic substance. The effect of signal intensity on lifetime recovery is investigated, and comparison is made between MEM and conventional nonlinear least-squares for data analysis.

Phase-resolved excitation-emission matrices (PREEMs) are shown to provide a unique visual representation of the intrinsic fluorescence properties of humic acids under a variety of solution conditions. The calculation of spectral peak ratios in PREEMs as well as steady-state excitation-emission matrices provides a convenient means for quantitating differences between the spectra with good precision. Absorbance correction is shown to be essential for accurate comparison among spectral features. Increased detail is available from PREEMs at various modulation frequencies that reveal the distribution of fluorescence lifetime contributions across the spectral surface. Direct measurement of fluorescence lifetime recovered three ranges of lifetime components in the humic substances, <1 ns, 2–5 ns, and 8–14 ns, that are consistent with previously reported lifetimes. PREEMs, which provide a concise “survey” of how the lifetimes change across the spectrum, may aid in pinpointing spectral regions that provide the best lifetime discrimination among samples.

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