Humic Substances, Part 5, B: Characterization Of

CHARACTERIZATION OF HUMIC SUBSTANCES: B

The formation of aggregates within solutions of humic acids was studied by capillary electrophoresis for the first time by Fetsch et al. (1998a, 1998b). Humic acids (HAs) aggregation has also been studied in aqueous solution by high performance size exclusion chromatography (Peuravuori and Pihlaja 1997), light scattering
(Manning et al. 2000), vapor pressure osmometry (VPO) (Marinsky et al. 1990), ultrafiltration (Aiken

and Malcom (1987), conductometry and spectrophotometry in combination with factor analysis (Peña-Méndez et al. 2004). HAs consist of a mixture of molecules with much lower molecular weight than proposed before in the literature. HAs components are low molecular weight compounds but they aggregate step by step to give higher molecular weight aggregates and supramolecules of higher molecular weight. Hosse and Wilkinson (2001), using fluorescence correlation spectroscopy observed that the aggregation of HAs is due to the formation of dimers and trimers at low pH.

The characterization of complex mixtures of FAs and HAs is considered to be one of the most important items in HS research nowadays. Thanks to the development of analytical techniques and computer technologies, great efforts have been made to elucidate the molecular structures of FAs and HAs. From the work of Stevenson (1982), Buffle et al. (1977) to more recent models of Shulten (2002, 2003), Kujawinski et al. (2002a, 2002b) and Stenson et al. (2002, 2003), several molecular structures describing the structure of humic acids have been proposed. For a long time, it has been suggested using different analytical techniques that HAs are high molecular weight compounds. However, five years ago, we proved by CE that HAs are low molecular weight compounds. This was announced for the first time at the 9th International Meeting of the IHSS, Adelaide Australia, September 20–25, 1998, IHSS, Atlanta (Havel et al. 2001) and in a subsequent publication. Mass spectra of HAs (Fig. 4) obtained by LDI-TOF MS shows the presence of low molecular weight molecules from low m/z values to higher. The analysis of the mass spectra obtained for different HAs showed that several m/z values are the same for all the HAs, suggesting that some compounds are the same and that they are present in HAs from very different origins and sources. The latest results from mass spectrometry (MS) combined with other analytical techniques have confirmed our previous results. As for fulvic acids, several thousand compounds have been identified, and are considered to be mostly derived from lignin (Kujawinski et al. 2002a, Stenson et al. 2003).

The chemical formulas of individual fulvic acids have been determined (Stenson et al. 2003). In the case of HAs, interesting results have been achieved recently applying Electrospray Ionization (ESI) (Kujawinski et al. 2002a, Brown and Rice 2002, Kujawinski et al. 2002b, Stenson et al. 2002, Stenson et al. 2003) coupled with Fourier Transform ICR mass spectrometry (FT-ICR) and Laser Desorption/Ionization time of flight (LDI-TOF) (Pokorná et al. 1999, Havel et al. 1999, Gajdošová et al. 2000, Gajdošová et al. 2003). Based on the latest experimental results obtained by LDI-TOF MS and also on the base of the isotopic patterns observed for compounds present in HAs (Fig. 4), Pacheco and Havel (2004) have suggested the latest empirical formula for HAs; the results from their model are in a good agreement with those obtained by ESI (Kujawinski et al. 2002a, Brown and Rice 2002, Kujawinski et al. 2002b, Stenson et al. 2002, Stenson et al. 2003).