Molecular size fractionation of soil humic acids using preparative high performance size-exclusion chromatography

• Published in: Journal of Chromatography A Vol. 1218; no. 37; pp. 6448 - 6453
• Main Authors: Asakawa, Daichi, Iimura, Yasuo, Kiyota, Takuro, Yanagi, Yukiko, Fujitake, Nobuhide
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 16.09.2011; Elsevier
• Subjects: Agronomy. Soil science and plant productions; Biological and medical sciences; Chemical properties; Chemical, physicochemical, biochemical and biological properties; Chromatography; Chromatography, Gel - methods; Chromatography, High Pressure Liquid - methods; Eluents; Fractionation; Fundamental and applied biological sciences. Psychology; gel chromatography; Humic acids; Humic substance; Humic Substances - analysis; Hydroxyapatite; Molecular size separation; Molecular Weight; Organic matter; Physics, chemistry, biochemistry and biology of agricultural and forest soils; Reproducibility of Results; Separation; Size-exclusion chromatography; sodium phosphate; soil; Soil (material); Soil humic acid; Soil science; Tailings; Time Factors; Fractionation; Humic substance; Size-exclusion chromatography; Soil humic acid; Molecular size separation; Organic matter; Molecular weight distribution; Molecular weight determination; Preparative chromatography; Organic acids; Gel permeation chromatography; Soils; Humic acid; Weight average molecular weight; Ultraviolet visible spectrometry
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2011.07.030

High performance size-exclusion chromatography (HPSEC) is useful for the molecular size separation of soil humic acids (HAs), but there is no method available for various HAs with different chemical properties. In this paper the authors propose a new preparative HPSEC method for various soil HAs. Three soil HAs with different chemical properties were fractionated by a Shodex OHpak SB-2004 HQ column with 10 mM sodium phosphate buffer (pH 7.0)/acetonitrile (3:1, v/v) as an eluent. The HAs eluted within a reasonable column range time (12–25 min) without peak tailing. Preparative HPSEC chromatograms of these HAs indicated that non-size-exclusion effects were suppressed. The separated fractions were analyzed by HPSEC to determine their apparent molecular weights. These decreased sequentially from fraction 1 to fraction 10, suggesting that the HAs had been separated by their molecular size. The size-separated fractions of the soil HA were mixed to compare them with unfractionated HA. The analytical HPSEC chromatogram of the mixed HA was almost identical to that of the unfractionated HA. It appears that the HAs do not adsorb specifically to the column during preparative HPSEC. Our preparative HPSEC method allows for rapid and reproducible separation of various soil HAs by molecular size.