Chloroform‐extractable green fraction (CEGF) was detected in the supernatant obtained by alkali precipitation from the HCI‐dimethylsulfoxide (DMSO) extract of Pg‐rich soil. In the alkaline solution, the color of CEGF was green and CEGF showed strong Pg‐like absorption bands. Ultraviolet and visible (UV‐VIS) spectral analysis and gel chromatography on Sephadex G‐50 were performed to compare several properties between CEGF and Pg. CEGF, which was purified by gel chromatography on Bio‐Beads SX‐1, displayed strong absorption bands at 609, 562, 445 and 280 inn in the alkaline solution. These absorption bands were almost similar to those of Pg. Furthermore, the UV‐VIS spectrum of CEGF in the organic solvents showed a similar characteristic pattern of 4,9‐dihydroxyperylene‐3,10‐quinone (DHPQ), which was considered to be a chromophore of Pg. Based on the results of gel chromatography on Sephadex G‐50, CEGF mainly consisted of two fractions, corresponding to the G2 and G3 fractions of Pg. These results indicated that the method for extracting CEGF from Pg‐rich soil in the present study was easy and selective and that CEGF was one of the components of, or a closely related substance to Pg. A colorimetric method for the estimation of the CEGF content in soils was developed. The calibration curve of CEGF was linear over a wide range of contents from 2.75 to 220 mg L−1. The CEGF content in twelve samples of various soils was examined. CEGF was detected in all the soil sampled (5 orders) including three samples (3 orders) where Pg was not detected, and the content ranged from 0.07 to 1.66 g kg−1 (dry soil). Therefore, the method for estimating the CEGF content in soils developed in the present study was found to be suitable for various soil orders and it was assumed that CEGF occurred in various soil orders.

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The chemical composition of the hydrophobic acid fraction, which is adsorbed to XAD‐8 resin at low pH and desorbed with high pH solution, of water‐extractable organic matter in soil probably influences its environmental role and, therefore, it is important to recognize the differences in its chemical composition with season and soil horizon. Hydrophobic acid fractions were collected seasonally in 2004 from Oi, Oe/Oa, A and B horizons of Cambisol and were investigated using elemental analysis, high‐performance size exclusion chromatography and 1H nuclear magnetic resonance (NMR) spectroscopy. Atomic ratios of H/C and O/C decreased with increasing soil depth in winter and summer, suggesting that the hydrophobic acid fraction from lower mineral horizons had lower saturated bond content and carbohydrate content than the fractions from upper organic horizons. Based on high‐performance size exclusion chromatography, the molecular weight at peak maximum of the hydrophobic acid fraction decreased significantly with increasing soil depth. The 1H NMR spectra of the hydrophobic acid fraction showed remarkable changes. The signal intensities of carbohydrate protons (δ 3.0–4.3 p.p.m.) in the hydrophobic acid fraction from organic horizons decreased from winter to summer and increased in autumn, while the signal of aliphatic protons (δ 0–3.0 p.p.m.) increased and decreased. In all seasons of the year, the signal intensities of carbohydrate protons were greater in organic horizons and decreased significantly with depth in mineral soil, but the signal for aliphatic protons increased. Our results demonstrate that season and soil depth influence the chemical composition of the hydrophobic acid fraction of water‐extractable organic matter.

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A method of high-performance size-exclusion chromatography (HPSEC) for a wide variety of soil humic acids (HAs) was developed. Two types of soil HAs (Cambisol and Andosol HAs), which have substantially different chemical properties, showed different effects of salt and organic solvent concentrations in the eluent on chromatograms. A Shodex OHpak SB-805 HQ column with 10 mM sodium phosphate buffer (pH 7.0) containing 25% of acetonitrile (v/v) was found to be applicable for different HAs, and showed high reproducibility and recovery (87.0 -94.5%). The Cambisol HA was fractionated into five fractions using an ultrafiltration with different molecular-weight cut-offs. The order of the molecular weights of the five fractions calculated from the HPSEC analysis corresponded to that defined by ultrafiltration. This supported the reliability of the method.