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

Abstract

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.

Extreme isomeric complexity of dissolved organic matter found across aquatic environments

“…The fragmentation patterns (the relative intensities of the various fragments) were dominated by neutral losses of CO 2 (244) and H 2 O (218), as has been observed previously (Leenheer et al 2001;Plancque et al 2001;Witt et al 2009;Brown et al 2016;Zark et al 2017) (Fig. 2).…”

“…For example, a loss of vinyl ketene (C 4 H 4 O, 68.026 Da) was observed for some peaks, indicating the presence of cyclic ketone functionality (Harris et al 1967), but this may also be due to loss of carbon suboxide (C 3 O 2 , 67.990 Da) (Huber et al 2007) from the previous precursor peak. These and other more exotic neutral losses may only be investigated with clarity with more advanced techniques that can isolate a single precursor peak (Witt et al 2009;Brown et al 2016). These pre-selection techniques would come with a necessary loss in time resolution, but would greatly improve the scope of the technique that we used.…”

“…These techniques have previously been used separately to confirm that numerous structural isomers contribute to any obtained molecular formula (Leenheer et al 2001;Witt et al 2009;Capley et al 2010;Arakawa and Aluwihare 2015;Cort es-Francisco and Caixach 2015;Brown et al 2016;Zark et al 2017). Chromatographic separation of complex mixtures of DOM has revealed variability in the primary and tertiary structure of their components (Saleh et al 1989;Woelki et al 1997;Reemtsma and These 2003;Namjesnik-Dejanovic and Cabaniss 2004;Woods et al 2011;Arakawa et al 2017), and several recent studies have led to tremendous advances in our understanding of the compositional complexity of terrestrial humic and fulvic acids via chromatographic separation and subsequent high resolution MS analysis (Koch et al 2008;Stenson 2008;Gaspar et al 2010;Brown et al 2016;Sandron et al 2017). Fragmentation has revealed the importance of carboxylic acid and alcohol functionality in fulvic acids (Leenheer et al 2001;Witt et al 2009;Capley et al 2010;Zark et al 2017) and has led to estimates that at least 28 structural isomers are present at any particular molecular mass in seawater (Zark et al 2017).…”

“…Fragmentation has revealed the importance of carboxylic acid and alcohol functionality in fulvic acids (Leenheer et al 2001;Witt et al 2009;Capley et al 2010;Zark et al 2017) and has led to estimates that at least 28 structural isomers are present at any particular molecular mass in seawater (Zark et al 2017). When used in combination with chromatography, fragmentation has allowed the separation and measurement of specific biomarkers, metabolites, or contaminants within a complex mixture (Wu et al 2015;Longnecker and Kujawinski 2017), but has rarely been used to probe the isomeric complexity of the bulk organic mixture (Brown et al 2016).…”

“…Collection of fractions and pre-concentration is a sensible, though time consuming, way of improving sensitivity (Capley et al 2010;Brown et al 2016). However, subtle detail in the elution gradient is lost through combining eluted material into fractions (Brown et al 2016). …”