Separation and spectroscopic characterization of soil fulvic acid constituents by hydrophilic interaction chromatography and reversed-phase high-performance liquid chromatography with π-π interactions

Fulvic acids (FAs) are humic substances that are soluble in both alkali and acid, and are considered to be self-assembled supramolecular associations of small heterogeneous molecules bound together by weak bonds. In this study, hydrophilic interaction chromatography (HILIC) using an amide-bonded stationary phase and reversed-phase high-performance liquid chromatography (RP-HPLC) with pi-pi interactions using a pentabromophenyl-bonded stationary phase were conducted to separate soil FA constituents. Additionally, a two-dimensional (2D) HPLC combining these techniques was also employed to improve separation efficiency. When HILIC and RP-HPLC were applied separately, over 70 peaks were resolved in the soil FAs using either method. Performing 2D HPLC with HILIC as the first dimension and RP-HPLC as the second dimension revealed recognition of over 500 peaks by UV or fluorescence detection. Many sharp peaks were observed along with broad peaks in the HILIC fractions by RP-HPLC. Online measurements of the UV-visible absorption spectra revealed that UV-detected sharp peaks had absorption only in the UV region, with each peak displaying a characteristic absorption spectrum, whereas UV-detected broad peaks exhibited featureless spectra, with absorbance gradually decreasing from the UV region to the visible region. Excitation-emission matrix fluorescence spectra measured for the major fluorescence-detected peaks demonstrated varying excitation and emission wavelengths of the fluorescence among the peaks, indicating that the FAs comprised molecules with different fluorophores. This study highlighted the effectiveness of 2D HPLC combined with HILIC and RP-HPLC incorporating pi-pi interactions in separating soil FA constituents with diverse spectroscopic characteristics.