Unlocking the Potential of Isopropanol as an Eco-Friendly Eluent for Large-Scale Fractionation of Fulvic Acids via Preparative Reversed-Phase High-Performance Liquid Chromatography and Multidimensional RP-HPLC: Evaluation of Molecular Diversity and Element Composition

Humic substances are organic mixtures of extreme complexity, which significantly complicate their analysis by any method. Fractionation into more homogeneous mixtures seems to be almost the only way to overcome these difficulties. Preparative high-performance liquid chromatography (HPLC) provides almost any amounts of substances required for both further fractionation and studies by other methods. For the first time, isopropyl alcohol (IPA) is proposed for these purposes; its advantages are shown by the example of groundwater fulvic acids (FA). IPA is much safer than conventional solvents and elutes the most nonpolar compounds that are nonelutable by methanol and acetonitrile. The isolated fractions differ significantly in their molecular composition, which is confirmed by ultrahigh-resolution mass spectrometry and molecular spectroscopy. Stepwise IPA-gradient HPLC-UV analysis of each preparative fraction demonstrates the possibilities of multidimensional HPLC for FA. The isolated fractions were studied for contents of a broad range of elements, and the relationships between the molecular and trace-element compositions of the fractions were revealed. Our data reveals the existence of numerous and different organometal compounds in FA composition; thus, the proposed approach can be used for a more in-depth study of the mechanisms of element migration in the environment.