PH effects on solid phase extractable dissolved organic matter: Expanding the analytical window

Marine dissolved organic matter (DOM) is the largest exchangeable organic carbon pool, holding comparable amounts of carbon as CO2 in the atmosphere and exceeding all biomass. DOM constituents are made up of thousands of unique organic compounds with astounding molecular diversity, featuring a wide range of hydrophobicity, size, and acidic or basic properties. Modern high resolution, high accuracy, and fast cycle time mass spectrometers can provide deep molecular insights into complex mixtures but require compatible samples. The inorganic matrix and low DOM concentrations have required organic biogeochemists to rely on Agilent Bond Elut Priority PoLutant (PPL) Solid Phase Extraction (SPE) to isolate and concentrate DOM through hydrophobic interactions. Currently, the standard SPE method has been optimized to maximize recovery? of dissolved organic carbon (DOC) through sample acidification and methanol elution. However, there is a lack of full understanding of the effect of adjusting the sample pH on the extraction efficiency of different DOM compounds. This study investigated the effects of pH modification on the SPE recoveries and the effects of various procedures on the isolated DOM. This study collected water samples from three sites to represent different marine systems (Lavaca River, Baffin Bay and Gulf of Mexico) with unique sources and signatures of DOM. Samples were acidified to pH 2, kept at natural pH, basified to pH 10 or run sequentially where the permeate was further isolated. Various modified methanol elution solvents were tested, comparing methanol, acidic methanol, basic methanol, and combinations of both. The isolated DOM was chemically characterized in positive mode separated with reverse-phase high performance liquid chromatography (RP-HPLC) and in negative mode with Anion Exchange Ion Chromatography (AEX-IC) on an Orbitrap Fusion Tribrid mass spectrometer (OT-FTMS) using data dependent acquisition (DDA). The standard SPE procedure with acidification yielded the highest recoveries but showed bias against dissolved organic nitrogen (DON). The samples that were not acidified, including natural pH or pH 10, yielded a different fraction of enigmatic DOM with a higher nitrogen percentage than the DOM isolates using the standard procedure. We found that through isolating DOM from a water sample at natural pH and pH 2, two fractions of DOM can be isolated, including hydrophobic DOM, acidic DOM, and basic DOM. These results suggest in order to better represent DOM from marine systems, collecting both fractions and analyzing in both positive and negative modes provide a more comprehensive and representative isolate of DOM.