Methylation patterns of aquatic humic substances determined by13C NMR spectroscopy

¹³C NMR spectroscopy is used to examine the hydroxyl group functionality of a series of humic and fulvic acids from different aquatic environments. Samples first are methylated with¹³C-labeled diazomethane. The NMR spectra of the diazomethylated samples allow one to distinguish between methyl esters of car?ylic acids, methyl ethers of phenolic hydroxyls, and methyl ethers of phenolic hydroxyls adjacent to two substituents. Samples are then permethylated with¹³C-labeled methyl iodide/NaH.¹³C NMR spectra of permethylated samples show that a significant fraction of the hydroxyl groups is not methylated with diazomethane alone. In these spectra methyl ethers of carbohydrate and aliphatic hydroxyls overlap with methyl ethers of phenolic hydroxyls. Side reactions of the methyltion procedure including carbon methylation in the CH₃I/NaH procedure, are also examined. Humic and fulvic acids from bog, swamp, groundwater, and lake waters showssome differences in their distribution of hydroxyl groups, mainly in the concentrations of phenolic hydroxyls, which may be attributed to their different biogeochemical origins.