Isolation and molecular characterization of dissolved organic phosphorus using electrodialysis‐reverse osmosis and solution ³¹P‐NMR

Abstract

The composition of dissolved organic phosphorus (DOP), which directly impacts its biological and chemical reactivity, remains poorly constrained due to methodological and operational challenges. A series of compound‐specific isolation experiments and bulk estuarine seawater isolations were conducted to assess the ability of coupled electrodialysis (ED) and reverse osmosis (RO) to isolate DOP from seawater for molecular characterization using solution 31P‐NMR analysis. These isolations were carried out using a recently developed small‐volume (24 L) ED/RO system. Compound‐specific isolation experiments (n = 7) averaged 64.1% ± 9.2% for final DOP recovery, while recoveries for estuarine seawater (n = 7) were on average 83.8% ± 10.7%. Nearly complete mass balance of DOP was observed for the compound‐specific isolations (88.1% ± 11.2%) with ED responsible for the majority of DOP loss (16% ± 5%). Isolation recovery was impacted by compound molecular weight rather than charge‐to‐size ratio. Isolation bias between bulk dissolved organic carbon and DOP was negligible. Solution 31P‐NMR confirmed that ED/RO isolation does not impact molecular integrity or DOP composition. In addition, solution 31P‐NMR accurately estimated several components (phosphonomethyl‐glycine, D‐glucose‐6‐phosphate, L‐α‐phosphatidylcholine) of an artificial complex DOP matrix. However, we suggest further refinements for ED/RO operation and significant caveats remain for 31P‐NMR sample preparation and the interpretation of 31P‐NMR spectra. Hydrolysis from NaOH dissolution was found to impact the ability to accurately resolve diester composition (ca. 40% underestimation) and discrimination between terminal and central phosphoanhydride atoms. Nonetheless, our results describe a viable option for the isolation and molecular characterization of marine DOP.

Resolving marine dissolved organic phosphorus (DOP) composition in a coastal estuary

“…One caveat is that monoP may not be as efficiently recovered using the ED/RO technique relative to diP. While our isolation results indicated almost complete recovery and characterization of the entire DOP pool (Table 1), Bell et al (2017) found a significant relationship between isolation recovery and MW. Smaller compounds, such as phosphomethyl-glycine (MW = 169), were recovered with 50% efficiency while larger compounds, such as RNA (assumed MW = 3360), were recovered near 80%.…”

“…Similar to low MW monoP compounds, pyroP may also have been under-recovered. Yet, it is also possible that alkaline dissolution impacted our results by degrading polyP (Bell et al 2017), although Turner et al (2003) found their polyP standard to be stable for 9 d in NaOH-EDTA. Terminal P from di-and triphosphate mononucleotides could also be included in the pyroP signal, although this would result in an ester bonded P peak (i.e., nucleoP α ) between −9 and −11 ppm (Figs.…”

“…A detailed description of the ED/RO isolation process, including instrument operation and an assessment of P recovery, mass balance, and isolation biases is found in Bell et al (2017). Briefly, prior to isolation, the ED/RO system undergoes an acidic and alkaline cleaning treatment.…”

“…The integrity of the isolate was thus tracked through P measurements in the final ED/RO isolate phase (Iso), after lyophilization (Lyo), upon immediate re-dissolution prior to 31 P-NMR analysis (Diss) and using the 31 P-NMR spectra (NMR). Solution 31 P-NMR experiments were performed as described by Bell et al (2017) on a Bruker Avance III-HD 400 MHz using a 5-mm broadband Prodigy cryoprobe. The T 1 relaxation times (0.9-2.9 s) were determined for a series of DOP compound standards.…”

Characterization of phosphorus in algae from a eutrophic lake by solution 31P nuclear magnetic resonance spectroscopy

“…Therefore, the forms and concentrations of P in algae shall be evaluated while algae debris is decomposing. However, until now, studies of the species, concentrations, and effects of P o in algae of eutrophic lakes have been limited because of the complexity and limitations of analytical methods (Turner et al 2005;Bell et al 2017).…”