High Pressure Size Exclusion Chromatography (HPSEC

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High Pressure Size Exclusion Chromatography (HPSEC) Determination of Dissolved Organic Matter Molecular Weight Revisited: Accounting for Changes in Stationary Phases, Analytical Standards, and Isolation Methods Brandon C. McAdams, George R. Aiken, Diane Marie McKnight, William A. Arnold, and Yu-Ping Chin Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04401 • Publication Date (Web): 29 Nov 2017 Downloaded from http://pubs.acs.org on December 5, 2017

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Environmental Science & Technology

High Pressure Size Exclusion Chromatography (HPSEC) Determination of Dissolved Organic Matter Molecular Weight Revisited: Accounting for Changes in Stationary Phases, Analytical Standards, and Isolation Methods Brandon C. McAdams1, George R. Aiken2, Diane M. McKnight3, William A. Arnold4, Yu-Ping Chin1*

5 6 7 8 9 10 11 12 13 14

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1School

of Earth Sciences, The Ohio State University, 125 S Oval Mall, Columbus, OH 43210, United States, 2U.S. Geological Survey, 3215 Marine Street, Boulder, Colorado 80303, United States, 3Institute of Arctic and Alpine Research (INSTAAR), 4001 Discovery Drive, University of Colorado at Boulder, Boulder, Colorado 80309, United States, 4Department of Civil, Environmental, and Geo-Engineering, University of Minnesota, 500 Pillsbury Drive Southeast, Minneapolis, Minnesota 55455, United States *

Current address: Department of Civil and Environmental Engineering, University of Delaware, Newark, DE 19716, E-mail: [email protected]

Abstract

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We reassessed the molecular weight of dissolved organic matter (DOM)

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determined by high pressure size exclusion chromatography (HPSEC) using

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measurements made with different columns and various generations of polystyrene

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sulfonate (PSS) molecular weight standards. Molecular weight measurements made

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with a newer generation HPSEC column and PSS standards from more recent lots are

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roughly 200 to 400 Da lower than initial measurements made in the early 1990’s These

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updated numbers match DOM molecular weights measured by colligative methods and

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fall within a range of values calculated from hydroxyl radical kinetics. These changes

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suggest improved accuracy of HPSEC molecular weight measurements that we

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attribute to improved accuracy of PSS standards and changes in the column packing.

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We also isolated DOM from wetlands in the Prairie Pothole Region (PPR) using XAD-8,

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a cation exchange resin, and PPL, a styrene-divinylbenzene media, and observed little

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difference in molecular weight and specific UV absorbance at 280 nm (SUVA280)

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between the two solid phase extraction resins, suggesting they capture similar DOM

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moieties. PPR DOM also showed lower SUVA280 at similar molecular weights as DOM

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isolates from a global range of environments, which we attribute to oxidized sulfur in

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PPR DOM that would increase molecular weight without affecting SUVA280.

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Keywords: dissolved organic matter, molecular weight, polydispersity, size exclusion chromatography, prairie pothole region, PPL, XAD-8

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Introduction

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Molecular weight and polydispersity are properties of dissolved organic matter

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(DOM) that play an important role in DOM’s structure, fate, and reactivity from a global

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range of environments. For instance, molecular weight has been correlated to the lability of

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marine and terrestrial organic matter1-4 and, together with polydispersity, has been used to

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determine its sources and understand its fate in aqueous environments.5-8 The

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photochemistry of DOM is impacted by molecular weight and polydispersity, both in terms

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of generating reactive species as well as quenching photoreactions.9-12 In addition,

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molecular weight both influences and is influenced by the formation of metal-DOM

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complexes,13-16 which impact the aggregation and dissolution of natural17 and engineered

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nanoparticles.18, 19 The adsorptive affinity of contaminants to DOM and the adsorption of

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DOM to mineral surfaces has also been linked to molecular weight.20-23 Finally, the

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molecular weight of DOM is tied to the formation of disinfection byproducts (DBPs), filter 2 ACS Paragon Plus Environment

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fouling, and the fate of pollutants during and after water treatment processes.24-26 In short,

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accurate molecular weight measurements of DOM are necessary to understand its role in

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environmental and biogeochemical processes.

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Currently, disparity exists in the literature among molecular weight measurements

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made by high pressure size exclusion chromatography (HPSEC)27-31 and those determined

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using other techniques such as vapor pressure osmometry (VPO),32, 33 high resolution mass

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spectrometry (HRMS),34-39 and more recently using hydroxyl radical (HO●)-DOM oxidation

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kinetics.40 Polystyrene sulfonate (PSS) molecular weight standards are commonly used to

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calibrate HPSEC columns and subtle changes in how they are manufactured has occurred

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since the development of HPSEC as a method for measuring molecular weight. Further,

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changes in column stationary phases may also contribute to the measured values of

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molecular weight. Solid phase extraction (SPE) media, particularly styrene-divinylbenzene

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(PPL) based cartridges, have also gained in popularity for the extraction of DOM. PPL

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generally captures more DOM (~40-60%) than XAD-8 resins (~15-50%) traditionally used

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to isolate the hydrophobic organic acid (HPOA) fraction.41, 42 While some comparison of

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these techniques has been performed,42 differences in molecular weight and polydispersity

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related to differences in the various resins used are absent from the literature.

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Correlations among molecular weight, molar absorptivity or specific UV absorbance

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(SUVA),43 spectral slope, and aromaticity are used by different research groups to infer

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DOM composition.44-49 Chin et al.27, 50 focused on relationships between molecular weight

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and SUVA at 280 nm (SUVA280) for riverine, groundwater, wetland, and lacustrine DOM.

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Unlike DOM in other lakes and wetlands, DOM in the shallow and exposed wetlands of the

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Prairie Pothole Region (PPR) in North America experiences extensive photo-bleaching 3 ACS Paragon Plus Environment


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throughout the summer months, making it unique with respect to natural waters.7, 51, 52 In

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addition, DOM from PPR wetlands and lakes contains high levels of nitrogen and sulfur.53

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Thus, molecular weight estimates from SUVA280 using previously established correlations

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(e.g. as in Chin et al.27) might not apply for these water bodies.

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In this paper, we revisit DOM molecular weight measured by HPSEC, accounting for

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changes in the manufacturing of PSS standards used to calibrate HPSEC molecular weight

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measurements and changes in column packing. Further, we explored the role that the more

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recently developed resins (PPL SPE) might play with respect to capturing the molecular

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weight distribution of DOM relative to previously utilized stationary phases (XAD-8). We

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also examine DOM from PPR wetlands in the context of previous relationships established

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between molecular weight and SUVA280 for rivers, groundwater, and lakes and the effect of

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isolating specific components relative to the whole DOM pool. Lastly, we explore changes in

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molecular weight and polydispersity in PPR wetland DOM from surface to pore waters and

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over an established hydrologic gradient.

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Materials and Methods

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Mobile phase and DOM isolate solutions were made with 18.2 MΩ-cm ultrapure

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water (Milli-Q) (Millipore: Molsheim, France). Methanol (HPLC grade), NaCl, and KH2PO4

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were purchased from Fisher Chemical (Fair Lawn, New Jersey, USA) and used as received.

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Na2HPO4⦁12H2O was purchased from J.T. Baker (Phillipsburg, New Jersey, USA) and used

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as received. Glassware cleaning reagent (NoChromix™) was purchased from GODAX Labs

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(Cabin John, Maryland, USA) and was prepared as a cleaning solution according to the

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manufacturer’s methods with BDH Aristar 95.0–98.0% H2SO4 purchased from VWR


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International (Radnor, Pennsylvania, USA). A potassium hydrogen phthalate stock solution

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(1001±5 mg C L-1) was purchased from UTRA Scientific (North Kingstown, Rhode Island,

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USA) and diluted with DI and used as dissolved organic carbon (DOC) standards.

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Poly(styrene sulfonate, Na salt) (PSS) standards were purchased from Polysciences,

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Incorporated (Warrington, Pennsylvania, USA) and used as received. Three lots of PSS

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standards manufactured in 1996, 2001, and 2016 were used to analyze the molecular

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weight of DOM isolates in this study. Molecular weight measurements from a previous

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study27 that are also presented herein were made with PSS standards manufactured before

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1992. Information provided by the manufacturer for the 1996, 2001, and 2016 PSS lots

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including nominal molecular weight and molecular weight at peak max as well as

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calibration retention times are provided in Table S1. Isolates analyzed in this study and

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presented in Table 1 are all of the same lot as were analyzed by Chin et al.27 with the

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exception of Suwannee River Fulvic Acid (SRFA) and Lake Fryxell DOM as the original lots

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of those samples had been consumed. The SRFA sample analyzed in this study is from lot

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2S101F from the International Humic Substances Society (IHSS). The Lake Fryxell sample is

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from an isolation performed in 2005.

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Sample Collection and Processing

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Surface and pore water samples were collected in September, 2015 from two PPR

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wetlands (P7 and P8) in the Cottonwoods Lake Study Area (CLSA) located northwest of

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Jamestown, ND, USA (47° 5.878’ N, 99° 6.011’ W). Wetlands in the PPR are categorized into

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three hydrologic groups based on recharge, flow-through, and discharge with increases in

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surface water sulfate along this gradient from recharge to discharge.54 The sediment pore



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waters of these wetlands are highly active redox environments that reflect the evolving

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sulfur chemistry of the groundwater, i.e. higher sulfide concentrations are found in

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discharge compared to recharge and flow-through wetlands.55 The evolving sulfur

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chemistry is also reflected in DOM composition, so that DOM from discharge wetlands has a

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higher sulfur content than DOM from recharge and flow-through wetlands.53 Wetland P7

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borders an agricultural field and chemically represents a flow-through wetland, whereas

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P8 receives no surface runoff from agricultural activity and has higher sulfate

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concentrations characteristic of discharge wetlands in the region.

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Pore waters were extracted from sediment cores by hydraulic extrusion and

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centrifugation then combined into two separate aliquots before filtration and DOM

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isolation. Pore water samples were vacuum filtered through pre-baked (450˚C for four

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hours) 0.7 µm Whatman™ GF/F glass microfiber filters using a glass filter tower, frit, and

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vacuum Erlenmeyer flask, all of which were pre-cleaned with NoChromix™. Surface water

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samples were filtered through AquaPrep™ 0.45 µm high capacity filters (Lot FA1381, Pall

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Corporation, Port Washington, New York, USA) using a peristaltic pump.

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Before isolation, filtered water samples were acidified to pH 2 with 12 N HCl.

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Isolation using Agilent Bond Elut PPL SPE cartridges was performed according to Dittmar

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et al.56 Before use, the cartridges were cleaned with at least 200 mL of HPLC grade

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methanol. The DOM bound to the PPL SPE media was eluted with methanol, which was

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then blown down with argon to obtain a DOM concentrate that was diluted with Milli-Q

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water before freeze-drying. The method of Aiken et al.57 was used for DOM isolation by

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XAD-8 chromatography. Samples were diluted with Milli-Q to a dissolved organic carbon

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(DOC) concentration of approximately 0.4–1.3 mM, acidified to pH 2 with HCl, and loaded 6 ACS Paragon Plus Environment

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onto a pre-cleaned XAD-8 column. Chloride was removed from the HPOA fraction retained

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on the XAD-8 column by rinsing with Milli-Q water until the conductivity of the effluent

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was < 700 µS cm-1. The sample was back eluted from the column with 0.1 M NaOH and run

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through a proton-saturated cation exchange resin (AG-50W, Bio-Rad Laboratories) to

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remove sodium ions. The pH of de-salted HPOA samples was approximately 3.3.

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DOM Analyses

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HPSEC was performed using a Waters Protein-Pak™ 7.8×300mm column (Part

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number WAT084601) on a Waters 1515 isocratic HPLC pump.27 A mobile phase solution of

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0.1 M NaCl solution buffered with 1 mM M Na2HPO4 and 1 mM M KH2PO4 was pumped at

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1.0 mL min-1 over an elution window of 15 minutes. PSS standards, acetone, and DOM

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isolates were prepared in the same solution as the mobile phase. Filtered whole water PPR

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wetland samples were diluted by half into a twice-concentrated mobile phase solution so

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that the final ionic strength and pH of the samples matched the mobile phase composition.

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PSS standards with nominal molecular weights of 18000, 8000, 4600, and 1800 Da were

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used for calibration, with the exception of the 2016 standards where the ‘1800 Da’

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standard is actually 1000 Da. Replicate HPSEC measurements showed excellent agreement

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between chromatograms (Figures S1 and S3). Peaks in standards and experimental

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solutions were detected using a Waters 2487 dual λ absorbance detector at 224 nm except

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for acetone, which was detected at 280 nm. Number (Mn) and weight (Mw) average

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molecular weights were calculated using the following equations detailed elsewhere:27, 58, 59

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= ℎ ℎ ⁄ (1)

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and

= ℎ ℎ (2)

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where hi is the height of the sample SEC curve eluted at volume “i” and Mi is the molecular

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weight at eluted volume “i” as calculated by the calibration curve. Polydispersity is defined

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as Mw/Mn and reflects the mass distribution of components in the DOM measured (e.g. DOM

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composed of components with the same molecular weight will have unity

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polydispersity).27, 58, 59

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Light absorbance of DOM isolates and filtered PPR wetland waters was measured

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on a Shimadzu UV-1800 or a Cary 60 spectrophotometer. The HPSEC mobile phase was

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used as the blank. Repeat absorbance measurements were within 1%. Dissolved organic

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carbon of DOM isolate solutions and PPR wetland samples was determined by Pt-catalyst

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combustion and non-dispersive infrared absorbance of the evolved CO2 using a Shimadzu

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TOC-V. Accuracy of DOC analyses determined using check standards diluted from a third-

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party stock was within 6%, and precision of replicate DOC measurements was within 1%.

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Results and Discussion

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Changes in Molecular Weight Standards 8 ACS Paragon Plus Environment

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Number and weight average molecular weights of DOM isolates measured using the

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more recent and more accurate PSS standards from 1996, 2001, and 2016 were roughly

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200-400 Da less than measurements made with older PSS standards used in a previous

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study27 (Table 1). Standards manufactured in 1996 and 2001 produced similar results for

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both Mn and Mw across the DOM isolates analyzed. Standards manufactured in 2016

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produced DOM Mn similar to those determined using the 1996 and 2001 standards but had

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consistently higher Mw values. Differences between DOM molecular weights measured by

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HPSEC in this study and values from past studies are attributed to improvements in the

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precision and accuracy of PSS manufacturing. Such improvements are also supported by

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changes in the information provided by the PSS manufacturer PolySciences, Inc., wherein

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they now provide specific molecular weights at the peak maximum rather than nominal

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values (e.g. the 2001 ‘18000 Da’ PSS standard has a peak maximum at 15800 Da, Table S1).

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The differences between DOM Mw measured using the 2016 standards and values

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measured with the 1996 and 2001 standards are also associated with this change in

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information provided by the manufacturer, suggesting that standards from 1996 and 2001

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may be more accurate than 2016 standards, especially for Mw measurements. For example,

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the 2016 ‘18000 Da’ PSS standard no longer provides information about peak maximum

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molecular weight, which, as stated above, was less than 18000 Da for the previous

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generations of PSS standards. This change in information provided by the manufacturer

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effectively biases the calibration curve at the upper end, thereby leading to higher

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measured Mw when using the 2016 PSS standards compared to the 1996 and 2001

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standards. In addition, the low 2016 PSS standard is 1000 Da rather than 1800 Da (as it

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was for the 1996 and 2001 PSS), but had a similar retention time at the highest peak signal. 9 ACS Paragon Plus Environment


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This similarity in retention time may bias the calibration curve at the lower molecular

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weight range, accounting for the similar Mn measured using the 1996 and 2001 standards.

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A definitive molecular weight for the lowest molecular weight PSS standard is difficult to

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constrain because of the high polydispersity of this polymer compared to higher molecular

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weight standards (Figure S2). We found that choosing the highest peak signal, however,

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and not necessarily the first peak signal, provided the best fit for the calibration curve.

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Molecular weight normalized chromatograms (where retention time is converted to

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log molecular weight based upon a specific calibration curve) of Suwannee River Fulvic

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Acid (SRFA) measured with the 2001 PSS standards relative to those reported by Chin et

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al.27 reveal an apparent bias toward lower molecular weight values when measured with

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the newer standard (Figure 1). Therefore, even though the two peaks show a similar peak

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max between 103 and 103.5 Da, Mw and Mn measured with 2001 standards are lower than

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those previously reported.27 Peak broadening, however, cannot be attributed to changes in

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the calibration curve and is a function of differences in the stationary phase composition. A

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simple displacement of the chromatograms to lower molecular weight would indicate a

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change caused solely by the changes in PSS standard manufacturing. The column used in

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this study was purchased in 2011, roughly 20 years after the column used in the initial

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study. Because the column used in this study is the same model and diol modified silica

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stationary phase as used in the 1994 study,27 we suggest that subtle differences in the

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nature of the column packing (possibly morphology and packing configuration) are

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responsible for the observed broadening of the peak and not changes in the chemical

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properties of the column. Thus, we attribute the lower reported molecular weight (Table 1)

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to both changes in the stationary phase morphology and composition of the molecular 10 ACS Paragon Plus Environment

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weight standards. The lower Mn values match those obtained by vapor pressure osmometry (VPO)32, 33

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and are within ranges calculated from HO● reactions with DOM40 (Figure 2). Further, these

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values are closer to, but still larger than molecular weights reported using high-resolution

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mass spectrometry (HRMS).34-39 A colligative technique, VPO relies on a decrease in the

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vapor pressure of a solute as dictated by the molality of DOM in solution for a known mass

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of DOM in solution. Accuracy of VPO measurements is well constrained by choosing the

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simplest solution phase (i.e. H2O as in Pavlik and Perdue33). Organic solvents that show

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greater change in vapor pressure with DOM molality (e.g. tetrahydrofuran) increase the

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sensitivity of measurements, but these approaches must account for residual water in

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solution by treating H2O as an inorganic molecule and correcting for the relative humidity

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in the droplet being measured.32 Additional corrections for dissociated protons and other

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inorganic ions must also be made regardless of solvent composition, such that decreases in

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solution vapor pressure are attributable to DOM only.60, 61 Thus, by measuring changes in

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solution vapor pressure—a physical property of a solution phase that will change based on

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the number of molecules available for nucleation—the molality of DOM in solution is

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obtained for a known mass of DOM, which results in its number average molecular weight

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(i.e., Mn). Relative concentrations of individual components, however, cannot be

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determined, and so VPO cannot provide information about the mass distribution of

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molecules present in the DOM (i.e., polydispersity) nor its weight average molecular

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weight.

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High resolution mass spectrometry (HRMS) provides detailed information about the

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chemical composition and structure of DOM and has contributed significantly to knowledge 11 ACS Paragon Plus Environment


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of DOM reactivity, transport, fate, and origin.62 Nonetheless, HRMS is limited in

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determining molecular weight as it has been shown to be biased towards lower molecular

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weight measurements. Some have observed that larger DOM molecules are not preserved

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in the ionization process, leading to apparently smaller molecular weight DOM than may

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have initially existed in solution (i.e., as measured by VPO).36, 63 In fact, These and

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Reemtsma36 utilized size exclusion chromatography in-line with ESI-HRMS to elucidate the

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fate of large molecular weight components in the ionization process. They found high

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molecular weight moieties to be poorly ionized by settings used to measure low molecular

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weight components and that the increased voltage necessary to obtain a strong signal with

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the high molecular weight fraction resulted in fragmentation to smaller constituents.

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Others have also shown little difference in the HRMS analyzed molecular weight of DOM

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fractions that have been separated either by dialysis39 or ultrafiltration,12 further

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supporting the limitations of HRMS in providing accurate molecular weight measurements.

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Therefore, we suggest that the actual (i.e., as would occur in nature) molecular weights of

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isolates measured in this study may be somewhere between HPSEC and VPO

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measurements and the lower value HRMS measurements.

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More recently, the upper and lower limits for DOM molecular weight have been

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determined using HO●-DOM oxidation kinetics.40 This model does not distinguish between

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aggregates and the smaller molecules that compose those aggregates and so is most

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comparable to VPO and HPSEC measurements. A molar representative rate constant (krep,

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molDOM-1 L s-1) determined as the mode of published rate constants for a range of organic

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molecules (composed only of C, H, N, and O) with known molecular weights was used to

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approximate the composition of DOM.40 This representative rate constant was then divided 12 ACS Paragon Plus Environment

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by published rate constants (kDOM, converted to gDOM-1 L s-1) for DOM isolates with known

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carbon compositions to calculate a Mn in gramsDOM molDOM-1. A precise Mn is not obtained

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due to the wide range of reported rate constants even for the most extensively studied

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isolates (e.g., SRFA). Using reasonable adjustments to krep and accounting for all reported

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kDOM, a range of Mn is determined that represents upper and lower bounds of possible Mn

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for the isolates measured. Our HPSEC measured Mn now fall within this range (Figure 2).

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Despite shifts to lower molecular weights than those measured by Chin et al.,27 the

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correlation between Mw and SUVA280 was still observed (Figure 3a), but the slopes and y-

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intercepts differ. Because Mw measured with 2016 standards are most similar to those

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measured by Chin et al.,27 the slope of the correlation (presented as ± 95% confidence

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intervals) from the 2016 PSS measured Mw values vs. SUVA280 (3.93±0.66, r2 = 0.86) is

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statistically similar to the slope from the Chin et al.27 data (4.49±0.72, r2 = 0.89). For clarity,

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we removed the Mw value reported for Aldrich humic acid from our earlier paper, which

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biased the regression line previously.27 In contrast, Mw values measured with 1996 and

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2001 PSS are lower than those measured with 2016 PSS. Further, the slopes of the

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correlations for Mw measured using 1996 PSS (3.26±0.67, r2 = 0.80) and 2001 PSS

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(3.31±0.58, r2 = 0.84) are statistically smaller than those from the 2016 PSS measured Mw

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and the Chin et al.27 data. Regardless, for a broad range of DOM isolates included in these

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correlations (i.e. derived from rivers, groundwater, and lakes) higher molecular weight

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DOM still strongly correlates with SUVA280, and could be used to make rough quantitative

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estimates of molecular weight from light absorbance.

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Positive correlations between Mn and SUVA280 were also observed (Figure 3b), and we propose using this relationship (Mn = 1.43(±0.32)×SUVA280 + 151(±112), r2 = 0.77, pslope 13 ACS Paragon Plus Environment


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= 0.0044 for 2016 PSS standards) to roughly estimate the number average molecular

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weight of DOM because Mn represents a more practical unit (as opposed to weight-average

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molecular weight) for the reasons discussed above. While this equation is useful for a

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variety of DOM isolates as presented here, it cannot be assumed that this relationship will

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apply to all samples. For example, nitrate and iron64 in DOM samples can affect SUVA

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measurements without affecting molecular weight. In addition, as discussed below, DOM

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from aquatic environments that differ widely from the fulvic acids used in this study (e.g.

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high sulfur environments) may not follow the same trend. Thus, those wishing to utilize the

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above relationship to estimate molecular weight (both Mn and Mw) from SUVA280 should

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note these concerns.

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Effect of Isolation Methods on molecular weight using Prairie Pothole Region DOM

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All data generated or analyzed during this study by the U.S. Geological Survey are

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included in the main text of this publication. Remarkably, PPR surface water DOM isolated

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by PPL SPE showed little difference in SUVA280 and molecular weight relative to XAD-8,

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with the most marked changes occurring for P7 surface SUVA280 (174 L mole-OC-1 cm-1 by

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PPL SPE compared to 203 L mole-OC-1 cm-1 by XAD-8) and P8 surface molecular weight

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(Mw of 1339 Da by PPL SPE compared to Mw of 1500 Da by XAD-8). PPL SPE was able to

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extract roughly 13% more DOM as measured by DOC than XAD-8 for P8 surface waters, but

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was statistically identical for P7 surface waters, further supporting that little difference

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exists between PPL SPE and XAD-8 chromatography when isolating PPR surface water

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DOM.

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The relationship between Mn and SUVA280 (we chose to use Mn for the reasons


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stated previously) for PPR DOM isolates exhibits significantly higher Mn values for the same

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extinction coefficient value relative to the relationships observed for DOM isolates derived

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from rivers, lakes, and groundwater (Figure 4). For this aspect of the study, we compared

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molecular weights calibrated with the 2016 PSS standards. Thus, estimates of PPR DOM

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molecular weights using Mn-SUVA280 correlations derived from riverine, groundwater, and

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lake DOM would underestimate the Mn of PPR isolates by 100 to 300 Da. While a “universal”

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correlation between Mn and SUVA280 is useful for DOM derived from diverse water bodies,

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it does not appear to be as applicable for PPR and other similar wetland derived DOM.50

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PPR wetland DOM has been shown to have significant amounts of high O/C ratio CHOS

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components with low double bond equivalency,53 which could increase molecular weight

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without contributing to light absorbance. In addition, PPR DOM isolates have a similar

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slope (1.43±0.45, r2 = 0.72) in their Mn-SUVA280 relationship to the river, lake, and

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groundwater DOM isolates (1.43±0.32, r2 = 0.77) (Figure 4). Therefore, the chromophoric

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nature of the PPR DOM isolates is likely similar in size to the other isolates studied, but

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their association with oxidized sulfur groups adds molecular mass that shifts the y-

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intercept of the Mn-SUVA280 relationship by roughly 150 Da. For example, the addition of

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two –SO3 functional groups (–SO3 has a molecular mass of 80 da) could explain this offset.

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Future attempts at creating models to estimate DOM properties from readily available

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parameters should consider PPR wetland DOM and other high sulfur content DOM as

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inherently different than DOM from other environments.

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Isolation of PPR DOM by PPL SPE56 and XAD-857 reveals an opposite trend observed

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for DOM derived from other surface waters. We observed a shift to lower molecular

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weights and higher SUVA280 relative to the filtered whole waters (Table 2, Figure S3). These 15 ACS Paragon Plus Environment


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changes in DOM properties suggest high molecular weight fractions of PPR DOM that

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preferentially absorb at the lower wavelength used in the HPSEC detector (224 nm), but

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not at 280 nm, are poorly retained by both PPL SPE and XAD-8 isolation. While inorganic

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constituents such as bisulfide65 and nitrate absorb light at 224 nm, they would elute last as

340

sharp peaks on the HPSEC chromatogram and bias measurements toward lower molecular

341

weights, leading to lower measured molecular weight for whole waters compared to

342

isolates. However, low molecular weight peaks are not seen in chromatograms of PPR

343

whole water (Figure S3), suggesting that the separation mechanism in the PPL SPE reflects

344

intrinsic properties of PPR DOM. Because the waters were collected in September, these

345

high molecular weight fractions that absorb at 224 nm (but not at 280 nm) may be a

346

product of DOM photo-bleaching or primary production (throughout the summer) of DOM

347

that does not absorb at higher wavelengths. Others have observed patterns of photo-

348

bleaching throughout a summer in PPR DOM that decrease chromophores with little

349

change in molecular weight.7, 51 Also, autochthonous DOM resulting from algal blooms may

350

result in the formation of high molecular weight, low chromophore, more hydrophilic

351

polysaccharides.66-68

352

Increases in molecular weight and DOC concentration were also observed from

353

surface water to pore water. Extraction efficiencies by PPL SPE for pore waters were

354

roughly twice those for surface waters, implying a higher percentage of more hydrophobic

355

organic matter in PPR wetland pore waters. These patterns could be tied to condensation

356

of recalcitrant hydrophobic components69 coupled with reductive dissolution of metal

357

oxide minerals that would result in the release of previously adsorbed large hydrophobic

358

fractions of DOM, as has been suggested for other pore water environments.50, 70 16 ACS Paragon Plus Environment

Page 16 of 33

Page 17 of 33


359

The fractionation that occurred during the isolation of PPR DOM illustrates that

360

Mn-SUVA280 relationships of DOM isolates (as illustrated in Figures 3 and 4) may not be

361

reliable for whole water samples (Figure S4), especially high molecular weight, low

362

chromophoric DOM samples. Thus, to calculate molecular weight from light absorbance

363

properties for whole water samples, it would be best to establish a unique correlation (e.g.

364

between SUVA280 and Mn) for whole water samples from the environment to be studied. In

365

addition, for the reasons stated above, seasonal changes in DOM properties (e.g. via

366

primary productivity and photobleaching)7 coupled with non-DOM light absorbing

367

substances may impact the accuracy of such correlations.

368

Acknowledgements

369

We thank two anonymous reviewers for their helpful comments and the following

370

individuals. Rob Lindberg, Dan Dunlap, and Brent Curtiss from Ohio State's IT department

371

helped recover data generated with MS-DOS compatible software (Figure 1) for this paper.

372

Jill Kerrigan ran many of the absorbance measurements reported in this paper. Dave

373

Mushet and Matthew Solensky of the USGS NPWRC along with Paula Dalcin-Martins and

374

Garret Smith provided vital help with sample collection from PPR wetlands. Sara

375

Breitmeyer and Brett Poulin (USGS) performed DOM isolation by XAD-8. This work was

376

supported by NSF grants EAR 1246594 and EAR 1245135.

377

Supporting Information Available.

378

These include chromatograms, DOM light absorbance and molecular weights, standards

379

data, and SUVA-molecular weight correlations.



380

References

381

(1) Amon, R. M. W.; Benner, R. Rapid-Cycling of High-Molecular-Weight Dissolved Organic-Matter in the Ocean. Nature. 1994, 369 (6481), 549-552, DOI: 10.1038/369549a0.

382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408

(2) Amon, R. M. W.; Benner, R. Bacterial utilization of different size classes of dissolved organic matter. Limnol. Oceanogr. 1996, 41 (1), 41-51, DOI: 10.4319/lo.1996.41.1.0041. (3) Marschner, B.; Kalbitz, K. Controls of bioavailability and biodegradability of dissolved organic matter in soils. Geoderma. 2003, 113 (3-4), 211-235, DOI: 10.1016/S00167061(02)00362-2. (4) van Hees, P. A. W.; Jones, D. L.; Finlay, R.; Godbold, D. L.; Lundström, U. S. The carbon we do not see—the impact of low molecular weight compounds on carbon dynamics and respiration in forest soils: a review. Soil Biol. Biochem. 2005, 37 (1), 1-13, DOI: 10.1016/j.soilbio.2004.06.010. (5) Nguyen, H. V.; Hur, J. Tracing the sources of refractory dissolved organic matter in a large artificial lake using multiple analytical tools. Chemosphere. 2011, 85 (5), 782789, DOI: 10.1016/j.chemosphere.2011.06.068. (6) Seders Dietrich, L. A.; McInnis, D. P.; Bolster, D.; Maurice, P. A. Effect of polydispersity on natural organic matter transport. Water Res. 2013, 47 (7), 2231-2240, DOI: 10.1016/j.watres.2013.01.053. (7) Ziegelgruber, K. L.; Zeng, T.; Arnold, W. A.; Chin, Y. P. Sources and composition of sediment pore-water dissolved organic matter in prairie pothole lakes. Limnol. Oceanogr. 2013, 58 (3), 1136-1146, DOI: 10.4319/lo.2013.58.3.1136. (8) Cuss, C. W.; Gueguen, C. Relationships between molecular weight and fluorescence properties for size-fractionated dissolved organic matter from fresh and aged sources. Water Res. 2015, 68, 487-497, DOI: 10.1016/j.watres.2014.10.013. (9) Wenk, J.; Eustis, S. N.; McNeill, K.; Canonica, S. Quenching of excited triplet states by dissolved natural organic matter. Environ. Sci. Technol. 2013, 47 (22), 12802-12810, DOI: 10.1021/es402668h.


Page 18 of 33

Page 19 of 33

409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438


(10) McKay, G.; Couch, K. D.; Mezyk, S. P.; Rosario-Ortiz, F. L. Investigation of the Coupled Effects of Molecular Weight and Charge-Transfer Interactions on the Optical and Photochemical Properties of Dissolved Organic Matter. Environ. Sci. Technol. 2016, 50 (15), 8093-8102, DOI: 10.1021/acs.est.6b02109. (11) McKay, G.; Roasio-Ortiz, F., Photochemical Reactivity of Organic Matter and its Size Fractions. In Surface Water Photochemistry, Calza, P.; Vione, D., Eds. Royal Society of Chemistry: Cambridge, 2016; pp 77-95. (12) Maizel, A. C.; Remucal, C. K. Molecular Composition and Photochemical Reactivity of Size-Fractionated Dissolved Organic Matter. Environ. Sci. Technol. 2017, 51 (4), 21132123, DOI: 10.1021/acs.est.6b05140. (13) Wrobel, K.; Sadi, B. B. M.; Wrobel, K.; Castillo, J. R.; Caruso, J. A. Effect of metal ions on the molecular weight distribution of humic substances derived from municipal compost: Ultrafiltration and size exclusion chromatography with spectrophotometric and inductively coupled plasma-MS detection. Anal. Chem. 2003, 75 (4), 761-767, DOI: 10.1021/ac0261193. (14) Gao, Y.; Korshin, G. Effects of NOM properties on copper release from model solid phases. Water Res. 2013, 47 (14), 4843-4852, DOI: 10.1016/j.watres.2013.04.055. (15) Beckler, J. S.; Jones, M. E.; Taillefert, M. The origin, composition, and reactivity of dissolved iron(III) complexes in coastal organic- and iron-rich sediments. Geochim. Cosmochim. Ac. 2015, 152, 72-88, DOI: 10.1016/j.gca.2014.12.017. (16) Kuhn, K. M.; Neubauer, E.; Hofmann, T.; von der Kammer, F.; Aiken, G. R.; Maurice, P. A. Concentrations and Distributions of Metals Associated with Dissolved Organic Matter from the Suwannee River (GA, USA). Environ. Eng. Sci. 2015, 32 (1), 54-65, DOI: 10.1089/ees.2014.0298. (17) Deonarine, A.; Lau, B. L.; Aiken, G. R.; Ryan, J. N.; Hsu-Kim, H. Effects of humic substances on precipitation and aggregation of zinc sulfide nanoparticles. Environ. Sci. Technol. 2011, 45 (8), 3217-3223, DOI: 10.1021/es1029798. (18) Louie, S. M.; Tilton, R. D.; Lowry, G. V. Effects of molecular weight distribution and chemical properties of natural organic matter on gold nanoparticle aggregation. Environ. Sci. Technol. 2013, 47 (9), 4245-4254, DOI: 10.1021/es400137x.



439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469

(19) Jiang, C.; Aiken, G. R.; Hsu-Kim, H. Effects of Natural Organic Matter Properties on the Dissolution Kinetics of Zinc Oxide Nanoparticles. Environ. Sci. Technol. 2015, 49 (19), 11476-11484, DOI: 10.1021/acs.est.5b02406. (20) Davis, J. A.; Gloor, R. Adsorption of dissolved organics in lake water by aluminum oxide. Effect of molecular weight. Environ. Sci. Technol. 1981, 15 (10), 1223-1229, DOI: 10.1021/es00092a012. (21) Chin, Y. P.; Aiken, G. R.; Danielsen, K. M. Binding of pyrene to aquatic and commercial humic substances: The role of molecular weight and aromaticity. Environ. Sci. Technol. 1997, 31 (6), 1630-1635, DOI: DOI 10.1021/es960404k. (22) Meier, M.; Namjesnik-Dejanovic, K.; Maurice, P. A.; Chin, Y. P.; Aiken, G. R. Fractionation of aquatic natural organic matter upon sorption to goethite and kaolinite. Chem. Geol. 1999, 157 (3-4), 275-284, DOI: 10.1016/S0009-2541(99)00006-6. (23) Sun, H.; Song, Q.; Luo, P.; Wu, P.; Wu, J. Sorption of phenanthrene on single-walled carbon nanotubes modified by DOM: effects of DOM molecular weight and contact time. Environ. Sci.: Proc. Imp. 2013, 15 (1), 307-314, DOI: 10.1039/c2em30569k. (24) Neale, P. A.; Antony, A.; Gernjak, W.; Leslie, G.; Escher, B. I. Natural versus wastewater derived dissolved organic carbon: implications for the environmental fate of organic micropollutants. Water Res. 2011, 45 (14), 4227-4237, DOI: 10.1016/j.watres.2011.05.038. (25) Wang, C.; Zhang, X.; Wang, J.; Liu, S.; Chen, C.; Xie, Y. Effects of organic fractions on the formation and control of N-nitrosamine precursors during conventional drinking water treatment processes. Sci. Total Environ. 2013, 449, 295-301, DOI: 10.1016/j.scitotenv.2013.01.080. (26) Aschermann, G.; Jeihanipour, A.; Shen, J.; Mkongo, G.; Dramas, L.; Croue, J. P.; Schafer, A. Seasonal variation of organic matter concentration and characteristics in the Maji ya Chai River (Tanzania): Impact on treatability by ultrafiltration. Water Res. 2016, 101, 370-381, DOI: 10.1016/j.watres.2016.05.022. (27) Chin, Y. P.; Aiken, G.; O'Loughlin, E. Molecular weight, polydispersity, and spectroscopic properties of aquatic humic substances. Environ. Sci. Technol. 1994, 28 (11), 1853-1858, DOI: 10.1021/es00060a015.


Page 20 of 33

Page 21 of 33

470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500


(28) Zhou, Q. H.; Cabaniss, S. E.; Maurice, P. A. Considerations in the use of high-pressure size exclusion chromatography (HPSEC) for determining molecular weights of aquatic humic substances. Water Res. 2000, 34 (14), 3505-3514, DOI: 10.1016/S0043-1354(00)00115-9. (29) Her, N.; Amy, G.; Foss, D.; Cho, J.; Yoon, Y.; Kosenka, P. Optimization of method for detecting and characterizing NOM by HPLC-size exclusion chromatography with UV and on-line DOC detection. Environ. Sci. Technol. 2002, 36 (5), 1069-1076, DOI: 10.1021/es015505j. (30) Perminova, I. V.; Frimmel, F. H.; Kudryavtsev, A. V.; Kulikova, N. A.; Abbt-Braun, G.; Hesse, S.; Petrosyan, V. S. Molecular weight characteristics of humic substances from different environments as determined by size exclusion chromatography and their statistical evaluation. Environ. Sci. Technol. 2003, 37 (11), 2477-2485, DOI: 10.1021/es0258069. (31) Schwede-Thomas, S. B.; Chin, Y. P.; Dria, K. J.; Hatcher, P.; Kaiser, E.; Sulzberger, B. Characterizing the properties of dissolved organic matter isolated by XAD and C-18 solid phase extraction and ultrafiltration. Aquat. Sci. 2005, 67 (1), 61-71, DOI: 10.1007/s00027-004-0735-4. (32) Aiken, G. R.; Malcolm, R. L. Molecular-Weight of Aquatic Fulvic-Acids by VaporPressure Osmometry. Geochim. Cosmochim. Acta 1987, 51 (8), 2177-2184, DOI: 10.1016/0016-7037(87)90267-5. (33) Pavlik, J. W.; Perdue, E. M. Number-Average Molecular Weights of Natural Organic Matter, Hydrophobic Acids, and Transphilic Acids from the Suwannee River, Georgia, as Determined Using Vapor Pressure Osmometry. Environ. Eng. Sci. 2015, 32 (1), 2330, DOI: 10.1089/ees.2014.0269. (34) Kujawinski, E. B.; Hatcher, P. G.; Freitas, M. A. High-resolution Fourier transform ion cyclotron resonance mass spectrometry of humic and fulvic acids: Improvements and comparisons. Anal. Chem. 2002, 74 (2), 413-419, DOI: 10.1021/ac0108313. (35) Stenson, A. C.; Marshall, A. G.; Cooper, W. T. Exact masses and chemical formulas of individual Suwannee River fulvic acids from ultrahigh resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectra. Anal. Chem. 2003, 75 (6), 1275-1284, DOI: 10.1021/ac026106p.



501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531

(36) These, A.; Reemtsma, T. Limitations of electrospray ionization of fulvic and humic acids as visible from size exclusion chromatography with organic carbon and mass spectrometric detection. Anal. Chem. 2003, 75 (22), 6275-6281, DOI: 10.1021/ac034399w. (37) Rostad, C. E.; Leenheer, J. A. Factors that affect molecular weight distribution of Suwannee river fulvic acid as determined by electrospray ionization/mass spectrometry. Anal. Chim. Acta 2004, 523 (2), 269-278, DOI: 10.1016/j.aca.2004.06.065. (38) Mawhinney, D. B.; Rosario-Ortiz, F. L.; Baik, S.; Vanderford, B. J.; Snyder, S. A. Characterization of fulvic acids by liquid chromatography-quadrupole time-of-flight mass spectrometry. J. Chromatogr. A. 2009, 1216 (9), 1319-1324, DOI: 10.1016/j.chroma.2008.12.068. (39) Remucal, C. K.; Cory, R. M.; Sander, M.; McNeill, K. Low molecular weight components in an aquatic humic substance as characterized by membrane dialysis and orbitrap mass spectrometry. Environ. Sci. Technol. 2012, 46 (17), 9350-9359, DOI: 10.1021/es302468q. (40) Appiani, E.; Page, S. E.; McNeill, K. On the use of hydroxyl radical kinetics to assess the number-average molecular weight of dissolved organic matter. Environ. Sci. Technol. 2014, 48 (20), 11794-11802, DOI: 10.1021/es5021873. (41) Her, N.; Amy, G.; McKnight, D.; Sohn, J.; Yoon, Y. M. Characterization of DOM as a function of MW by fluorescence EEM and HPLC-SEC using UVA, DOC, and fluorescence detection. Water Res. 2003, 37 (17), 4295-4303, DOI: 10.1016/S0043-1354(03)00317-8. (42) Green, N. W.; Perdue, E. M.; Aiken, G. R.; Butler, K. D.; Chen, H. M.; Dittmar, T.; Niggemann, J.; Stubbins, A. An intercomparison of three methods for the large-scale isolation of oceanic dissolved organic matter. Mar. Chem. 2014, 161, 14-19, DOI: 10.1016/j.marchem.2014.01.012. (43) Weishaar, J. L.; Aiken, G. R.; Bergamaschi, B. A.; Fram, M. S.; Fujii, R.; Mopper, K. Evaluation of specific ultraviolet absorbance as an indicator of the chemical composition and reactivity of dissolved organic carbon. Environ. Sci. Technol. 2003, 37 (20), 4702-4708, DOI: 10.1021/es030360x.


Page 22 of 33

Page 23 of 33

532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561


(44) Helms, J. R.; Stubbins, A.; Ritchie, J. D.; Minor, E. C.; Kieber, D. J.; Mopper, K. Absorption spectral slopes and slope ratios as indicators of molecular weight, source, and photobleaching of chromophoric dissolved organic matter. Limnol. Oceanogr. 2008, 53 (3), 955-969, DOI: 10.4319/lo.2008.53.3.0955. (45) He, X. S.; Xi, B. D.; Wei, Z. M.; Jiang, Y. H.; Geng, C. M.; Yang, Y.; Yuan, Y.; Liu, H. L. Physicochemical and spectroscopic characteristics of dissolved organic matter extracted from municipal solid waste (MSW) and their influence on the landfill biological stability. Bioresour. Technol. 2011, 102 (3), 2322-2327, DOI: 10.1016/j.biortech.2010.10.085. (46) Stedmon, C. A.; Amon, R. M. W.; Rinehart, A. J.; Walker, S. A. The supply and characteristics of colored dissolved organic matter (CDOM) in the Arctic Ocean: Pan Arctic trends and differences. Mar. Chem. 2011, 124 (1-4), 108-118, DOI: 10.1016/j.marchem.2010.12.007. (47) Porcal, P.; Dillon, P. J.; Molot, L. A. Photochemical production and decomposition of particulate organic carbon in a freshwater stream. Aquat. Sci. 2013, 75 (4), 469-482, DOI: 10.1007/s00027-013-0293-8. (48) Wang, H.; Holden, J.; Zhang, Z.; Li, M.; Li, X. Concentration dynamics and biodegradability of dissolved organic matter in wetland soils subjected to experimental warming. Sci. Total Environ. 2014, 470-471, 907-916, DOI: 10.1016/j.scitotenv.2013.10.049. (49) Wünsch, U. J.; Stedmon, C. A.; Tranvik, L. J.; Guillemette, F. Unraveling the sizedependent optical properties of dissolved organic matter. Limnol. Oceanogr. 2017, DOI: 10.1002/lno.10651. (50) Chin, Y. P.; Traina, S. J.; Swank, C. R.; Backhus, D. Abundance and properties of dissolved organic matter in pore waters of a freshwater wetland. Limnol. Oceanogr. 1998, 43 (6), 1287-1296, DOI: 10.4319/lo.1998.43.6.1287. (51) Waiser, M. J.; Robarts, R. D. Photodegradation of DOC in a shallow prairie wetland: evidence from seasonal changes in DOC optical properties and chemical characteristics. Biogeochemistry. 2004, 69 (2), 263-284, DOI: 10.1023/B:BIOG.0000031048.20050.4e.



562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593

(52) McCabe, A. J.; Arnold, W. A. Seasonal and spatial variabilities in the water chemistry of prairie pothole wetlands influence the photoproduction of reactive intermediates. Chemosphere. 2016, 155, 640-647, DOI: 10.1016/j.chemosphere.2016.04.078. (53) Sleighter, R. L.; Chin, Y. P.; Arnold, W. A.; Hatcher, P. G.; McCabe, A. J.; McAdams, B. C.; Wallace, G. C. Evidence of Incorporation of Abiotic S and N into Prairie Wetland Dissolved Organic Matter. Environ. Sci. Technol. Lett. 2014, 1 (9), 345-350, DOI: 10.1021/ez500229b. (54) Goldhaber, M. B.; Mills, C. T.; Morrison, J. M.; Stricker, C. A.; Mushet, D. M.; LaBaugh, J. W. Hydrogeochemistry of prairie pothole region wetlands: Role of long-term critical zone processes. Chem. Geol. 2014, 387, 170-183, DOI: 10.1016/j.chemgeo.2014.08.023. (55) McAdams, B. C.; Adams, R. M.; Arnold, W. A.; Chin, Y. P. Novel Insights into the Distribution of Reduced Sulfur Species in Prairie Pothole Wetland Pore Waters Provided by Bismuth Film Electrodes. Environ. Sci. Technol. Lett. 2016, 3 (3), 104-109, DOI: 10.1021/acs.estlett.6b00020. (56) Dittmar, T.; Koch, B.; Hertkorn, N.; Kattner, G. A simple and efficient method for the solid-phase extraction of dissolved organic matter (SPE-DOM) from seawater. Limnol. Oceanogr.: Meth. 2008, 6 (6), 230-235, DOI: 10.4319/lom.2008.6.230. (57) Aiken, G. R.; Mcknight, D. M.; Thorn, K. A.; Thurman, E. M. Isolation of Hydrophilic Organic-Acids from Water Using Nonionic Macroporous Resins. Org. Geochem. 1992, 18 (4), 567-573, DOI: 10.1016/0146-6380(92)90119-I. (58) Yau, W. W.; Kirkland, J. J.; Bly, D. D. Modern Size Exclusion Chromatography. WileyInterscience: New York, 1979. (59) Chin, Y. P.; Gschwend, P. M. The Abundance, Distribution, and Configuration of Porewater Organic Colloids in Recent Sediments. Geochim. Cosmochim. Ac. 1991, 55 (5), 1309-1317, DOI: 10.1016/0016-7037(91)90309-S. (60) Reuter, J. H.; Perdue, E. M. Calculation of molecular weights of humic substances from colligative data: Application to aquatic humus and its molecular size fractions. Geochim. Cosmochim. Ac. 1981, 45 (11), 2017-2022, DOI: 10.1016/0016-7037(81)90056-9. (61) Aiken, G. R.; Gillam, A. H., Determination of molecular weights of humic substances by colligative property measurements. In Humic Substances II, Hayes, M. H. B.; 24 ACS Paragon Plus Environment

Page 24 of 33

Page 25 of 33

594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625


MacCarthy, P.; Malcolm, R. L.; Swift, R. S., Eds. John Wiley and Sons Ltd: Chichester, 1989; pp 515-544. (62) Sleighter, R. L.; Hatcher, P. G. The application of electrospray ionization coupled to ultrahigh resolution mass spectrometry for the molecular characterization of natural organic matter. J. Mass Spectrom. 2007, 42 (5), 559-574, DOI: 10.1002/jms.1221. (63) Kujawinski, E. B.; Freitas, M. A.; Zang, X.; Hatcher, P. G.; Green-Church, K. B.; Jones, R. B. The application of electrospray ionization mass spectrometry (ESI MS) to the structural characterization of natural organic matter. Org. Geochem. 2002, 33 (3), 171-180, DOI: 10.1016/S0146-6380(01)00149-8. (64) Poulin, B. A.; Ryan, J. N.; Aiken, G. R. Effects of iron on optical properties of dissolved organic matter. Environ. Sci. Technol. 2014, 48 (17), 10098-10106, DOI: 10.1021/es502670r. (65) Guenther, E. A.; Johnson, K. S.; Coale, K. H. Direct ultraviolet spectrophotometric determination of total sulfide and iodide in natural waters. Anal. Chem. 2001, 73 (14), 3481-3487. (66) Jiao, N.; Herndl, G. J.; Hansell, D. A.; Benner, R.; Kattner, G.; Wilhelm, S. W.; Kirchman, D. L.; Weinbauer, M. G.; Luo, T.; Chen, F.; Azam, F. Microbial production of recalcitrant dissolved organic matter: long-term carbon storage in the global ocean. Nat. Rev. Microbiol. 2010, 8 (8), 593-599, DOI: 10.1038/nrmicro2386. (67) Biddanda, B.; Benner, R. Carbon, nitrogen, and carbohydrate fluxes during the production of particulate and dissolved organic matter by marine phytoplankton. Limnol. Oceanogr. 1997, 42 (3), 506-518, DOI: 10.4319/lo.1997.42.3.0506. (68) Biersmith, A.; Benner, R. Carbohydrates in phytoplankton and freshly produced dissolved organic matter. Mar. Chem. 1998, 63 (1-2), 131-144, DOI: 10.1016/S0304-4203(98)00057-7. (69) Reemtsma, T.; These, A.; Springer, A.; Linscheid, M. Differences in the molecular composition of fulvic acid size fractions detected by size-exclusion chromatographyon line Fourier transform ion cyclotron resonance (FTICR-) mass spectrometry. Water Res. 2008, 42 (1-2), 63-72, DOI: 10.1016/j.watres.2007.06.063. (70) O'Loughlin, E. J.; Chin, Y. P. Quantification and characterization of dissolved organic carbon and iron in sedimentary porewater from Green Bay, WI, USA. Biogeochemistry. 2004, 71 (3), 371-386, DOI: 10.1007/s10533-004-0373-x. 25 ACS Paragon Plus Environment


Page 26 of 33

626

Table 1. Weight average molecular weight (MW), number average molecular weight) (Mn),

627

and polydispersity (PD) of dissolved organic matter isolates from Chin et al.26 and using

628

2001 and 2016 polystyrene sulfonate (PSS) standards in this study. Chin et al.26 Samplea

MW (Da)

2001 PSS

Mn (Da)

PD

MW (Da)

Mn (Da)

2016 PSS PD

MW (Da)

Mn (Da)

PD

Coal Creek FA

2230

1180

1.9

1693

752

2.3

1838

782

2.4

Groundwater FA

1000

639

1.6

810

456

1.8

780

417

1.9

Suwannee River FA

2310

1360

1.7

1987

909

2.2

2230

968

2.3

Lake Fryxell

1080

713

1.5

856

416

2.1

918

436

2.1

Missouri River FA

1460

839

1.7

1207

602

2.0

1310

644

2.0

Ohio River FA

1330

705

1.9

1127

546

2.1

1243

574

2.2

Yakima River FA

1560

800

1.9

1320

614

2.2

1427

633

2.3

1264

527

2.4

1404

559

2.5

Pony Lake FA aFA

= Fulvic Acid


Page 27 of 33


629

630

Figure 1. Suwannee River Fulvic Acid chromatograms from Chin et al.27 (dashed) and this

631

study of lot 2S101F (solid) normalized to max peak height and over log molecular weight.

632

Log molecular weight was calculated from retention times (in minutes) using calibration

633

curves wherein log(molecular weight) = calibration slope × retention time. The calibration

634

slope is the slope of a linear regression between the log molecular weight of the PSS

635

standards and the retention time at which the PSS standards elute.



636 637

Figure 2. Number average molecular weight (Mn) measured via high pressure size

638

exclusion chromatography (HPSEC) using 2016 PSS standards compared to Mn previously

639

measured by vapor pressure osmometry (VPO) compiled in Chin et al.27 (blue circles) and a

640

range of Mn values calculated by Appiani et al.40 using hydroxyl radical oxidation kinetics

641

(represented by the light blue lines). Dashed line represents a 1:1 fit where HPSEC and VPO

642

measurements are in unity.


Page 28 of 33

Page 29 of 33


643

644 645

Figure 3. a) Weight average (Mw) and b) number average (Mn) molecular weights over

646

molar absorptivity (SUVA280) for DOM isolates measured by Chin et al.27 () using PSS

647

standards manufactured before 1994 and DOM isolates measured in this study using PSS



648

standards manufactured in 1996 ( ), 2001 ( ), and 2016 ( ). Linear regressions show

649

shallower slopes and lower y-intercepts for Mw,Mn-SUVA280 relationships with molecular

650

weights measured using newer PSS standards, reflecting a more accurate molecular weight

651

measurement that matches values obtained by VPO.


Page 30 of 33

Page 31 of 33


652

653

Figure 4. Number-average molecular weights (Mn) compared to molar absorptivity

654

(SUVA280) for PPR DOM isolates including regression lines for river, lake, and groundwater

655

DOM isolates presented in Figure 3. Circles () indicate PPR DOM isolated by XAD-8 and

656

squares () indicate PPR DOM isolated by PPL. PPR DOM isolates show a similar slope to

657

the other isolates studied but a positive shift in the y-intercept reflecting contributions to

658

Mn from oxidized sulfur components in PPR DOM that do not contribute to SUVA280.



Page 32 of 33

659

Table 2. Weight average (MW) and number average (Mn) molecular weights, polydispersity

660

(PD), molar absorptivity (SUVA280), DOC concentrations, extraction efficiencies (PPL SPE),

661

and DOM fractionations (XAD) for DOM isolates and whole water DOC of PPR wetland

662

surface and pore waters.

Sample

MWa

MNa

(Da)

(Da)

PD

SUVA280

DOC

(L mole OC-1 cm-1)

(mM)

Extraction Efficiencyb (%)

P8 Pore 1

1950

974

2.0

215

3.71

P8 Pore 2

2175

1073

2.0

237

4.62

P8 Surface

1646

842

2.0

184

2.31

P8 Pore (PPL SPE)

1486

681

2.2

266

80.1

P8 Surface (PPL SPE)

1339

627

2.1

263

43.5

P8 Surface (XAD)

1500

771

1.9

276

P7 Pore 1

1975

939

2.1

141

3.36

P7 Pore 2

1310

729

1.8

159

3.10

P7 Surface

1605

811

2.0

135

2.37

P7 Pore (PPL SPE)

1170

572

2.0

169

70.3

P7 Surface (PPL SPE)

1110

538

2.1

174

36.1

P7 Surface (XAD)

1190

630

1.9

203

aMW

HPOA, HPI, TPIA fractionc (%)

30, 32, 26

35, 28, 23

and MN measured using PSS standards manufactured in 2016.

bReported

as the quotient of the DOC of the PPL SPE effluent divided by the DOC of the whole water.

cHydrophobic

organic acid (HPOA), low molecular weight hydrophilic (HPI), and transphilic organic acid (TPIA) fractions of PPR wetland dissolved organic matter as percentages, respectively.

663 664


Page 33 of 33


TOC Art picture used was taken by BC McAdams. 84x47mm (300 x 300 DPI)

ACS Paragon Plus Environment

High Pressure Size Exclusion Chromatography (HPSEC

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