Analysis of the green fraction of humic acids

The green fraction of humic acids (HAs), Pg, was fractionated by gel chromatography on Sephadex G-50. Repeated chromatography of the crude Pg obtained by the first chromatography of HA yielded a sharp peak (G₁) and two broad bands (G₂) of purified Pg. The recovery of Pg in G₁ and G₂ as percentages of the total Pg content of the HA were 16.2 and 14.5%, respectively. Distinct peaks were detected at 615, 570, 450 and 280 nm in the UV-visible spectra of both G₁ and G₂ in alkali solutions. Estimates of weight-average molecular weight were 9.7 * 10³ for G₁ and 1.23 * 10⁴ for G₂. In the ¹H-NMR spectra the percentage of hydroxyl groups of 4,9-dihydroxy-perylene-3, 10-quinone (DHPQ) nuclei, which are known to be the chromophore of Pg, was 4.1 and 4.5% of the total H, respectively. Based on these values and the H/C ratios, DHPQ-C was estimated as 28% of total C. ¹³C-NMR spectra obtained using inverse-gated decoupling yielded 40–41% aromatic C, which suggested the presence of aromatic rings other than DHPQ. All of the IR, ¹³C- and ¹H-NMR spectra indicated that the two purified Pg samples largely differed from each other in their contents of alkyl and polysaccharide components.