Spectroscopic approach for elucidation of structural peculiarities of Andisol soil humic acid fractionated by SEC-PAGE setup

A Andisol humic acid (HA) was fractioned by preparative size exclusion chromatography and polyacrylamide gel electrophoresis in the presence of denaturising agent (SEC-PAGE setup). Three fractions exhibiting high, medium and low molecular size (MS) and showing well-defined and distinct electrophoretic mobility were obtained. These fractions were analysed by UV–visible, FTIR and 3-D fluorescence spectroscopies. UV–visible absorbance and A465/A665 ratio of fractions increased as the MS decreased. FTIR measurements coupled with HCl treatment of samples revealed that aliphatic moieties such as polysaccharides and peptides were mainly localized in high MS fractions while carboxylic functions in low MS fractions. Excitation-emission matrix spectra showed that long-wavelength emissions were almost exclusively observed upon excitation of low MS fraction. These data indicate that fractionation by SEC-PAGE setup yielded pools of soil HA macromolecules showing distinct structural characteristics.