Analysis of Humic Substances by SEC

Humic and fulvic acid extracts prepared from brown forest soil (Danto, Japan) and crumbly black topsoil (Inogashira, Japan) standard samples (provided by the Japanese humic susbtances society) were analyzed by size exclusion chromatography. Under these conditions, humic acids were found have a wide size distribution, while fulvic acid is much less dispersed than humic acids, as show by the molecular weight profiles. OHpak SB-805 HQ is a polyhydroxymethacrylate based size exclusion column with an exclusion limit of 40,000 MW (determined with a pullulan standard). It supports a wide pH range (pH 3-10), is compatible with organic solvents such as acetonitrile and methanol, and is used for the analysis of water-soluble macromolecules.


Sample : Humic substances, 30micro-L
Humic acid　0.02mg/mL
Fulvic acid 0.02mg/mL

Columns      : Shodex OHpak SB-G (6.0mmID*50mm) + SB-805 HQ (8.0mmID*300mm)
Eluent　　   : 10mM NaH2PO4 + 10mM Na2HPO4(pH7.0) + 25% CH3CN
Flow rate    : 0.8mL/min
Detector     : UV(260nm)
Column temp. : 40deg-C

Data courtesy of Associate Professor Nobuhide Fujitake,
Student Affairs Section, Graduate School of Agricultural Science, Kobe University