With co-funding from the Hydrolgic Sciences Program in the Division of Earth Sciences, the Analytical and Surface Chemistry Program supports Prof. Alexandra Stenson of the University of South Alabama to investigate development and use of micro-preparative chromatography followed by ultra-high resolution multi-dimensional mass spectrometry for elucidating the structural composition of humic substances of environmental importance. The most immediate stumbling block to molecular characterization of humic substances is the immense complexity of such samples. Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) has been demonstrated to resolve individual humic ions and to provide information on the molecular level (e.g. molecular formulae). More detailed structural (vs. merely compositional) data will require multidimensional MS experiments on individual ions, an approach that is currently hindered by sample complexity. Thus, the first goal of this research is to reduce sample complexity through tailored "micro-preparative" chromatography. Metal-complexation and in-cell reactions are employed to garner further functional group information.
The lack of structural information on humic substances is presently an issue for such important environmental considerations as solid and liquid waste-management (including radioactive waste), as well as the precise detailing of metal-, carbon-, and nutrient-cycles. This work promises to lay the groundwork for complete structural characterization, which, once achieved, would be transforming to a plethora of environmentally important fields. Undergraduates are thoroughly involved in the work, providing extraordinary research opportunities.
Humic substances are complex mixtures of environmental, agricultural, industrial, toxicological, and pharmacological importance. They are degradation products of slowly decaying organic matter such as leaves and bark. Humic substances are studied for their role in the sorption and transport of radioactive metals. In aqueous environments, they are studied for their effect on limiting nutrients in the world’s oceans, their own role as organic nutrients, and their contribution to carbon cycling and sequestration against the backdrop of global climate change. The ubiquitous presence of humic substances in drinking water reservoirs is of concern because of their ability to interact with disinfection reagents such as chlorine to produce harmful byproducts. Finally, humic substances and their synthetic analogues are studied for antiviral, antibacterial, and anti-cancer properties. Some over the counter medicines (Activomin, Weinböla GmbH, Switzerland) and veterinary drugs (Natürliche Huminsäuren WH67, Weinböla GmbH, Switzerland) in Europe list natural humic substances as the active ingredient. However, most pharmacological research involving humic substances is focused on synthetic analogues, such as the work by Bruccoleri et al. looking at humic derived compounds which demonstrate antiretroviral and antibacterial properties. Against the backdrop of rapidly mutating diseases, the existence of a large pool of non-toxic compounds with antiviral and antibacterial properties (i.e., humic substances) is of critical importance. Crippling to all these lines of investigation is how little is known about the molecular structure of humic substances. The challenge is that humic substances exist in nature as extremely complex mixtures. Unlike the complex mixtures we encounter in living things, humic substances are not constrained to a relatively short list of building blocks (e.g., amino and nucleic acids). Therefore, the problem of structural characterization is far more complex. Furthermore, most techniques that provide structural information can do so only for pure compounds. When mixtures are analyzed, such techniques provide a composite average for all components. Using only such techniques to elucidate the structure of humic substances is tantamount to using national averages of personal traits to identify a single individual. High resolution mass spectrometry is a technique that does provide compositional information on individual analytes within complex mixtures, so long as those analytes have different molecular masses. To have different molecular masses, analytes must be comprised of different ratios of elements, i.e., they must have different molecular formulas. However, for any given elemental composition, a number of different structures are possible. The larger the molecule, the more structures are possible. Smaller molecules may have tens to hundreds of possible structures; larger molecules may have thousands to millions. Molecules that have the same formula but different structures are referred to as isomers. Humic substances are presumed to consist of large numbers of isomers for each molecular composition. Because high resolution mass spectrometry cannot distinguish between isomers, it is essential that humic substances be separated before mass spectrometry is used to characterize them. In this project, we developed separation methods that begin to reduce the structural complexity of humic materials. We used Suwannee River Fulvic Acid, a common standard for humic materials. First, we separated it into approximately one hundred different fraction based mainly on the polarity of its constituent components. High resolution mass spectrometry was then used to characterize a number of the fractions collected. Through the combination of information derived from the order in which the analytes were separated (i.e., elution order), how readily the analytes could complex metals of different size and charge, how readily the analytes could exchange hydrogen for deuterium atoms, and what type of elemental compositions resulted when the analytes were broken into smaller pieces, we learned not only that the separation had proceeded as intended but also gained more insight into the structural make-up of the analytes. Because most molecules in humic substances are large enough that between hundreds and millions of different structures are possible, we took three fractions from the first separation and separated them again. The second separation was designed to be as different from the first as possible. Both separations were tested on mixtures of known compounds until the second separation resulted in a different order or notably longer separation times. Mass spectral data indicate that this separation, too, was successful. Because the first separation resulted in one hundred fractions, the structural complexity in each fraction was approximately one hundredth of the original. The second separation reduced that complexity by another factor of approximately one hundred for a combined reduction of approximately one ten-thousandth. Through this work we have shown that meaningful reduction in structural complexity of humic materials is possible and have illustrated how mass spectral data can be maximized to reveal structural detail for these ubiquitous and critical analytes.
