Advanced TMAH and TMAAc thermochemolysis–pyrolysis techniques for molecular characterization of size-separated fractions from aquatic dissolved organic matter

The structural similarities and differences between the original DOM and the eight size fractions separated were studied in detail with the pyrolysis technique in combination with gas chromatography and mass spectrometry (Py–GC–MS) using two alkylating reagents: TMAH (tetramethylammonium hydroxide), to find both esterified and free carboxylic acids; and TMAAc (tetramethylammonium acetate), to specify only free carboxylic acids. A statistical analysis of the original multidimensional TMAH and TMAAc pyrograms disclosed that the overall structural compositions of the five most important size fractions, accounting for 84% of the original DOM, greatly resembled each other. The remaining three minor size fractions were not classified as homogeneous associations, but they also contained the same total, covalently bound and free carboxylic acid species as the other size fractions and the original DOM mixture, thus representing some kind of intermediate forms. This fundamental outcome strongly supports the opinion that the native dissolved humic-like macromolecules resemble supramolecular associations of smaller molecular size moieties with similar structural functionalities. The concentrations of free aliphatic and aromatic dicarboxylic acids in the DOM solution were so low that their effects on the potential formation of multiply charged ions in electrospray ionization–MS (ESI–MS) studies are likely insignificant.