Unlocking the Potential\nof Isopropanol as an Eco-Friendly\nEluent for Large-Scale Fractionation of Fulvic Acids via Preparative\nReversed-Phase High-Performance Liquid Chromatography and Multidimensional\nRP-HPLC: Evaluation of Molecular Diversity a

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