Size exclusion chromatography with online ICP-MS enables molecular weight fractionation of dissolved phosphorus species in water samples
Graphical abstract
Introduction
Phosphorus (P) is an important and often limiting element in terrestrial and aquatic ecosystem and is at the center of important environmental challenges from a perspective of excess and scarcity. Phosphorus is essential because of its biochemical role in RNA, DNA and cellular processes (Gifford et al., 2015). Modern agriculture heavily relies on (mineral) phosphate fertilizers to achieve high yields and feed an ever-growing population (Rittmann et al., 2011, Venkatesan et al., 2016). Excess organic and inorganic phosphate, mainly from plant and animal agricultural waste streams, enters waterways and leads to algae blooms in fresh and seawater that threaten ecosystems. While the most advanced wastewater treatment systems remove (and in some cases attempt to capture and reuse) inorganic P reasonably well, they still discharge organic P (OP) into waterways (Mayer et al., 2016). Research has been performed on eutrophication and P cycling in estuaries, wetlands, lakes etc. over the last decade and identified the bioavailability of OP as a major and underestimated issue (Björkman and Karl, 2003, Ni et al., 2016, Pant et al., 2002). OP is known to be a complex mixture of many different species including DNA and RNA fragments, phospholipids, phosphoproteins, and phosphate esters (Baldwin, 2013, Worsfold et al., 2008). In addition to the unknown fractions of OP, total P (TP) in environmental systems is also comprised of condensed inorganic phosphates (polyphosphates) and colloidal P (Moorleghem et al., 2011). Studies have addressed the mineralization of these unknown forms, but all from a rather coarse perspective as chemically the OP and colloidal P remains poorly characterized. The complexity and lack of chemical information on TP (organic and some inorganic) in the environment, hampers our understanding of the mineralization of the material, its dynamic partitioning in water/sediment/particle systems and eventually its lifetime. The lack of understanding of the P structures (molecular weight distribution, functional group composition), also limits the design of P mitigation and recovery approaches.
The various fractions of P in the environment are often determined via indirect methods. For example, dissolved organic phosphorus (DOP) measurement in the environment is calculated either as the difference between total dissolved P (TDP) and dissolved reactive P (DRP), or measured in terms of DRP after pretreatment or digestion of samples (Worsfold et al., 2008). Such measurements, however, fail to provide fundamental chemical data on the diverse nature of TP (e.g. size distributions, functional groups, etc.). The most common tool used in the study of the speciation of P in terrestrial systems is 31P NMR spectroscopy. The method is well established and showed that typically a large part of P is under the form of orthophosphate and polyphosphate (Cade-Menun, 2005). NMR also allowed demonstrating the presence of phosphate esters, DNA, RNA, phospholipids and glycophosphates, all derived from biological processes (Bell et al., 2017). Advanced (high resolution) mass spectrometry was only attempted in a few studies but allowed for the discovery of select biomarker species. Most of the characterization work focused on soils and sediments, and less research is available for 31P NMR and MS on organic matter extracted from water, in part because isolation and concentration techniques have not been developed to target P like they have been for bulk carbon or nitrogen (Aiken et al., 1992, Chin et al., 1994, Herckes et al., 2007, Leenheer et al., 2007).
Ion/liquid chromatography coupled to inductively coupled plasma mass spectrometry (ICP-MS) have been successfully applied to determine the speciation of P in soils and foodstuffs (Koplík et al., 2002, Persson et al., 2009, Shah and Caruso, 2005). The advantage of using ICP-MS is that it detects TDP (both organic and inorganic) in samples unlike commonly used spectrophotometric and colorimetric P methods that detect only orthophosphates. Chromatographic separation prior to TDP analysis by ICP-MS could serve as a powerful and robust tool to conveniently analyze various fractions of P in the environment. Two studies have successfully applied size exclusion chromatography (SEC)-ICP-MS technique to investigate the soluble P species and other elements in soybean flour, white bean seeds, and barley grain tissues (Koplík et al., 2002, Persson et al., 2009). By using SEC-ICP-MS, the authors were able to determine the molecular weight (MW) distribution of P in food samples in a single run without the need for complex and successive ultrafiltration techniques. Though SEC coupled to an organic carbon detector (SEC-DOC) is a commonly used technique in determining the MW distribution of DOC in waters (Her et al., 2003, Nam et al., 2008, Wang et al., 2013), to the best of our knowledge, a similar approach has not been applied for the determination of size distribution of P in environmental waters.
The goal of this study was to develop a robust SEC-ICP-MS method to conveniently determine the MW distribution of P in environmental matrices, specifically in waters. The developed method was tested on surface waters, primary and secondary wastewater effluents, aerosols, and wetland samples to identify the abundant MW fraction of P in these environmental matrices. The performance of the SEC-ICP-MS method was compared against a typically used ultrafiltration technique for MW fractionation. SEC-DOC was additionally applied to aqueous samples in order to qualitatively determine the fraction of P associated with organic matter. Though this method does not directly identify OP in samples, it provides critical molecular information and improved understanding of the dynamic nature of P in the environment.
Section snippets
Chemical reagents
All chemicals used were ACS grade and purchased from Sigma-Aldrich (MO, USA) unless specified otherwise. Trace metal grade hydrochloric acid (33–36%) was purchased from J.T. Baker (Ultrex II, JT Baker Inc., NJ, USA). Deionized water (>18.3 MΩ cm, NANOpure Infinity, LA, USA) was used throughout the experiment.
SEC-ICP-MS
Chromatographic separation was performed using Toyopearl HW-50S resin (20–40 μm size exclusion resin; hydroxylated methacrylate matrix). The resin was gravity packed in a stainless-steel
Mobile phase, calibration, and method optimization of P detection in SEC-ICP-MS
Various mobile phase buffers and eluent pH were tested on the column to optimize for retention time and peak shape of P. Supplemental Fig. S1 shows the peak shapes and retention time of orthophosphate injected into the SEC-ICP-MS system. Similar to the observation made for the SEC-DOC system (Her et al., 2003), increasing the ionic strength of the mobile phase increased the retention of P (orthophosphate) in the column and also improved the peak shape (DI water vs. 20 mM NaCl - conductivity of
Conclusions
The present study shows that a combination of SEC and ICP-MS can serve as a powerful and robust tool to characterize unknown fractions of P in the environment to further our understanding of the occurrence and cycling of P in natural and built systems. The study results point out that the abundant fraction of P in the environment is < 600 g/mol in most cases. The average fraction of orthophosphate in various environmental matrices ranged from 21 to 98% of TDP, with the lowest value observed for
Acknowledgment
This material is based upon work supported by the National Science Foundation under grant number DEB-1637590, Central Arizona-Phoenix Long-Term Ecological Research (CAP LTER). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the NSF.
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