Benefits of multidimensional fractionation for the study and characterization of natural organic matter

• Published in: Journal of Chromatography A Vol. 1470; pp. 84 - 96
• Main Authors: Brown, Taylor A., Jackson, Benjamin A., Bythell, Benjamin J., Stenson, Alexandra C.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 28-10-2016
• Subjects: Benzopyrans - analysis; Chemical Fractionation; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; FT-ICR MS of humic substances; Humic Substances - analysis; Molecular Weight; MS/MS of humic substances; Multidimensional HPLC; Radicals in humic substances; Separation of humic substances; Tandem Mass Spectrometry; Separation of humic substances; FT-ICR MS of humic substances; Multidimensional HPLC; MS/MS of humic substances; Radicals in humic substances
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2016.10.005

•A robust 2D-HPLC method with high orthogonality between dimensions was developed.•LCMS/MS provides an evaluation of decreases in isomeric complexity in the 2nd D.•NOM radicals were detected in the CAS- like region of van Krevelen plots.•Support for fulvic acids as a unique compound class was discovered.
The belief that chromatographic separation of complex environmental mixtures or natural organic matter (NOM) produces featureless humps from which little, if anything, can be learned is still pervasive. Meanwhile improvements in chromatography and the use of information-rich detection methods have led to meaningful fractionation and revealed consistent data. Here, we build on this work and developed a robust, facile two-dimensional separation with high orthogonality between dimensions. We illustrate that re-injections of fractions (both in the first and in the second dimension) leads to individual peaks at the expected retention times and use information-rich detection to investigate the basis on which NOM is fractionated. We demonstrate unprecedentedly feature-rich chromatograms are observed even with standard UV detection for polar NOM fractions. The second stage of fractionation is demonstrated to separate isomers, providing a direct look at isomeric complexity in NOM as well as a tool to reduce it. Consistent with expectation, but confirmed for the first time through mass spectral data, radicals were detected for NOM components that were generally nonpolar and grouped in the condensed aromatic structure – like region of van Krevelen plots. High-resolution tandem mass spectral data, furthermore, suggests that many higher-MW components of fulvic acids (especially the highly oxidized ones) have formulas that do not match any known compounds in the literature, supporting the hypothesis that fulvic acids are a unique compound-class. Combined, the data illustrate that meaningful reduction in complexity reveals new compositional and structural detail and avails new avenues of investigation.