Chemical characterization of a chelator-treated soil humate by solution- state multinuclear two-dimensional NMR with FTIR and pyrolysis-GCMS

A California forest soil used for contaminant bioavailability studies was extracted for humic substances (HS) and then treated with 4,5-dihydroxy- 1,3-benzene disulfonate ('Tiron') to remove exchangeable metal ions. This yielded HS that was readily water-soluble at neutral pH without residual Tiron contamination, and the gel electrophoretic pattern was very similar to an international reference HS. The improved solubility facilitated analysis of HS by solution-state 1-D and 2-D ¹H, ¹³C, ³¹P, and ¹³C-¹H NMR. The amino acids Gly, Ala, Leu, Ile, Val, Asp, Ser, Thr, Glu, and Pro were identified in intact HS as peptidic from scalar coupling in TOCSY and dipolar interactions in NOESY and then confirmed by acid digestion of HS with 2-D ¹H NMR and GCMS analysis. The presence of peptides was also corroborated by FT- IR and pyrolysis-GCMS results. Carbohydrates containing α- and β-pyranoses, methoxyphenylpropanyl structures, phosphate mono/diesters, polyphosphates, plus phosphatidic acid esters were also evident. Furthermore, the ¹H NOESY, TOCSY, and HSOC together indicated that the peptidic side chains were mobile, whereas aromatic groups were relatively rigid. Thus the peptidic moieties may be more readily accessible to aqueous contaminants than aromatic groups.