Online High-Performance SizeExclusion Chromatography-NuclearMagnetic Resonance for theCharacterization of DissolvedOrganic MatterG W E N C . W O O D S , † M Y R N A J . S I M P S O N , †

B R I A N P . K E L L E H E R , ‡

M A R G A R E T M C C A U L , ‡

W I L L I A M L . K I N G E R Y , § A N DA N D R E J . S I M P S O N * , †

Department of Chemistry, University of Toronto, ScarboroughCampus, Toronto, Ontario, Canada M1C 1A, School ofChemical Sciences, Dublin City University, Dublin 9, Ireland,and Department of Plant and Soil Sciences, Mississippi StateUniversity, Mississippi 39762

Received October 11, 2009. Revised manuscript receivedNovember 25, 2009. Accepted December 5, 2009.

The substantial heterogeneity of dissolved organic matter(DOM) inhibits detailed chromatographic analysis withconventional detectors as little structural information can beobtained in the presence of extensive coelution. Here we examinethe direct hyphenation of high-performance size exclusionchromatography (HPSEC) with nuclear magnetic resonance(NMR)spectroscopytodeterminehowsize-distinguishedfractionsdiffer in composition. The results support the applicability ofusing HPSEC to generate more homogeneous fractions of DOMprior to NMR analysis and demonstrate that structure issignificantly altered with size. The largest fractions are enrichedin carbohydrate- and aromatic-type structures. The midsizedmaterial is substantial and is representative of carboxyl-rich alicyclic molecules (CRAMs). The smallest material hasstrongsignaturesofmaterialderivedfromlinearterpenoids(MDLT).Both CRAMs and MDLT have been recently hypothesized asmajor components of DOM, and detection by HPSEC-NMRconfirms their existence as unique and separable entities. Thispreliminary work focuses on NMR hyphenation to HPSECdue to widespread use of HPSEC to characterize DOM. Onlinehyphenation is useful not only for time-efficient analysis ofDOM but also for that of other highly complex samples suchas those found in many environmental analyses.

IntroductionDissolved organic matter (DOM) constitutes one of the largestreservoirs of actively cycling organic carbon on Earth (1). AsCO2 is a primary product of DOM mineralization, an intimatelink exists between this dissolved pool of carbon and theatmosphere. It has been suggested that enhanced oxidationof marine DOM brought the planet out of a series of severeglaciations and into the Cambrian explosion of life (2).

Understanding DOM mineralization processes is thus criticalto assessing environmental issues such as climate change,but despite considerable research designated to the char-acterization of DOM, the individual molecular structures,and to some extent the major structural components, arestill not well understood. Access to such molecular-levelinformation would provide insight into origins of watermasses, clarity of DOM interactions with environmentalcontaminants, and a better understanding of DOM signifi-cance in the global carbon cycle. Through the use ofmultidimensional NMR, predictions, and simulations, ourresearch group has been able to describe the major categoriesof components present in freshwater DOM (3) but haveconcluded that new analytical techniques will be requiredto elucidate exact structures.

Originating at least in part from the degradation ofbiomolecules, DOM is comprised of a myriad of degradedand reworked products and is subsequently one of the mostcomplex natural mixtures. In laboratory analyses, excessivesignal overlap with most detectors limits the amount ofdetailed information that can be obtained. In attempting todecipher information, researchers frequently employ multipletechniques and instruments, either hyphenated into a singleanalytical run or conducted offline. HPLC is frequentlyhyphenated to mass spectrometry (MS) (4-6). Ultra-high-resolution Fourier transform ion cyclotron resonance MS(FT-ICR-MS) can be used to calculate the molecular formulaof thousands of constituents in DOM and is complementedby offline NMR analyses (7). Spectral information fromUV-vis and fluorescence is frequently used to characterizesources of DOM, and these techniques have been used incombination with high-performance size exclusion chro-matography (HPSEC) (8). Despite such multifaceted ap-proaches, detailed structures remain indiscernible. Evenwhen using advanced multidimensional NMR experimentsthat have successfully unraveled components in soil organicmatter (9-13), the lack of appropriate standards for DOMand extensive spectral overlap result in only basic informationof major structural components (3). Individual structuresthemselves remain elusive and warrant further research.

NMR is a powerful detector for unknown constituents inmixtures, but hyphenated NMR is a fitting solution whenfaced with the unprecedented complexity of DOM. If suitableseparation can be obtained with an instrument online withNMR, more detailed information can be obtained than withoffline analyses. Online HPLC-NMR finds application in suchareas as the analysis of pharmaceuticals, food products, andcontaminants, with the intent of resolving mixtures ofmetabolites and degradation products (14-17). Samplemixtures are simplified chromatographically and directlydetected by NMR without extensive laboratory procedures.The greatest advantage is simply that samples are analyzedquickly and frequent data acquisition is possible (i.e., “slices”can be taken more often than in comparable offlinetechniques, translating into less coelution per fraction). Thedrawbacks to the online system include sample “dilution”during HPLC separation, leading to sensitivity issues withNMR detection as well as problems with solvent signalsuppression of nondeuterated HPLC solvents. The conse-quence of these drawbacks is that compromises must bemade when designing experimental parameters, but the endresult is unparalleled detection capabilities with only a singlechromatographic run.

Online HPLC-NMR has found widespread use in phar-maceutical screening but rarely in environmental applica-tions (17), with only a preliminary online analysis of DOM

* Corresponding author phone: (416) 287-7547; fax: (416) 287-7279; e-mail: andre.simpson@utoronto.ca.

† University of Toronto.‡ Dublin City University.§ Mississippi State University.

Environ. Sci. Technol. 2010, 44, 624–630

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to date (18). The development of HPLC-NMR techniques issignificant not only for DOM elucidation but also for analysisof other complex environmental samples such as organicmatter in soils, sediments, and the atmosphere. Here weexamine the structural components of DOM with NMRhyphenated to HPSEC (a commonly used chromatographictechnique with DOM). As such this work describes thesuccessful hyphenation of HPSEC with NMR to examine thesize-distinguished structural variability in DOM.

Materials and MethodsSample Collection and Preparation. Three DOM sampleswere used for this study. Nordic Reservoir natural organicmatter (NRNOM) and Suwannee River natural organic matter(SRNOM) were purchased from IHSS. Both samples wereisolated via reverse osmosis following 0.4 µm filtration (detailson the IHSS Web site, http://ihss.gatech.edu/ihss2). The thirdsample was collected from a wetland in the Lynde ShoresConservation Area, Whitby, Ontario, in August of 2007. TheLynde Shores sample (LSDOM) was collected via pressurefiltration with 0.45 µm PVDF membranes and isolated onDEAE cellulose. Samples were extracted under N2 with 0.1M NaOH, ion exchanged to remove Na+, and lyophilized(18, 19). All samples were prepared in the HPSEC mobilephase, adjusted to pH 12 with NaOH, and syringe filtered(0.45 µm).

HPSEC Separation. HPSEC separation and HPSEC-NMRanalyses were conducted on an Agilent HP1100 HPLC system,equipped with a column heater, diode array detector (DAD),and fraction collector (Foxy Jr., ISCO) and controlled withHystar software (Bruker), version 3.0. Readers should notethat the DAD signal was in most cases swamped due to thelarge and concentrated injections required for NMR spec-troscopy. As such the DAD data are not considered here.Future studies designed to use both the DAD and NMR asparallel detectors would need a software-controlled splitterand dilutor prior to DAD. Two columns, Ultrahydrogel 250and 120 (Waters, rated pH 2-12), were used in series. Columnperformance was assessed daily using a mixture of poly-(styrenesulfonic acid) standards, and no qualitative orquantitative changes were noted to occur over the durationof this study. Additionally, quantitative recovery of DOM wasassessed by collecting all eluate after injections both withand without the columns, and 100.1 ( 2.3% recovery wasnoted on the basis of multiple injections. Although thecolumn was calibrated with PSS standards, molecular weightestimates of DOM are not presented. The hydrodynamic radiias well as interactions with the stationary phase will differbetween common commercial standards and humic sub-stances as debated heavily in the literature (20). Appropriatecalibration standards for DOM are difficult to assess pendingbetter insight into molecular structures and aggregation ofthis complex material (21). HPSEC is used here as a meansto size-separate DOM, and molecular weights are notpresented due to concerns as to the accuracy of such data.

An isocratic solvent system at 40 °C was used for HPSECseparations with an aqueous buffer comprised of 0.1 M NaCland 0.03 M NH4Cl, adjusted to pH 11 with NH4OH. A similarbuffer has been cited elsewhere (22), but a lower NaClconcentration was used here for purposes of NMR compat-ibility (which is particularly crucial for fraction collectionwhere salts are concentrated during drying). Separation wasnot significantly affected by this reduction in salts. For directlycoupled HPLC-NMR experiments, a 90:10 H2O/D2O versionof the buffer was used.

Fraction Collection. HPSEC-eluted fractions were ac-quired for comparison to online techniques. Fractions of theDOM were taken in 2 min intervals over the duration thatDOM eluted from the HPSEC column. This process wasrepeated 30 times, and combined fractions were lyophilized

and stored for later analysis. Lyophilized samples werereconstituted with D2O and analyzed via 1H NMR.

Solution 1H NMR. HPSEC-NMR analyses were achievedon a Bruker Avance 500 MHz spectrometer at 298 K. A dual-tuned 1H-19F flow probe (120 µL) fitted with an activelyshielded z-gradient was used for NMR detection. Forcontinuous-flow analyses run at 0.5 mL/min, 16 scans wereacquired, while at 0.05 mL/min 88 scans were acquired.Stopped-flow experiments were performed using 96 scans.Fraction-collected and whole DOM samples were furtheranalyzed in D2O (with 10 µL of NaOD added to whole samplesto ensure solubility) with a 5 mm, triple-resonance broad-banded inverse (TBI) probe using 128 scans. For all NMRexperiments 16 384 time domain points were used with arecycle delay of 2 s. NOESY presaturation (pulse programNOESYPR1D) was used with a 400 µs mixing time to suppressthe signal from the mobile phase (water at 4.7 ppm).

During Fourier transform the residual water signal wasreduced using a Gaussian function centered at the waterfrequency (∼4.7 ppm) and corresponding to a bandwidth inthe transform spectrum of 0.7 ppm (23). Reduced contribu-tions from water allow the profiles and projections from theDOM signal to be more reliably discerned. The disadvantageis that the region around the water appears artificially“smoothed” and is seen most clearly in samples with lowS/N ratio (e.g., Figure 2, row C). Readers should recognizethis as an artifact from the processing employed.

Spectra were apodized through multiplication with anexponential decay corresponding to 1 Hz and processed usinga zero filling factor of 2. All 1D spectra generated from onlineexperiments were compiled into pseudo-2D NMR chro-matograms (1D 1H NMR spectra along the x-axis and HPSECelution volume on the y-axis) using Bruker Topspin (Bruker),version 2.1.

Results and DiscussionOnline Techniques. Hyphenated HPLC-NMR may be ac-complished in either continuous-flow (also called on-flow)or stopped-flow mode, with advantages and disadvantagesto both. Both methods include directly flowing eluate fromthe HPLC to the NMR flow cell. Continuous-flow is ac-complished with the HPLC pump and NMR running simul-taneously, whereas stopped-flow involves stopping the pumpin intervals during which time NMR experiments are run onthe static sample (controlled here via Hystar software andstopped for ∼10 min per fraction). Continuous-flow is limitedin the number of scans per spectrum (translating into reducedsensitivity) due to limited time in the NMR cell. The processof continuously flowing also causes inhomogeneities withinthe magnetic field which can lead to reduced spectralresolution. In continuous-flow, however, chromatographicseparation is not interrupted and the influence of diffusionis much less compared to that in stopped-flow. Thus, despitedrawbacks, continuous-flow is useful for fast screening ofsamples and/or comparison to stopped-flow. In contrast,stopped-flow allows the sample to be shimmed, generatinga more homogeneous field inside the NMR cell, as well asthe possibility to collect more scans per slice, hence improvingdetection limits (15). Due to the potential for complementaryinformation and/or for comparison, we examined onlineanalyses in both stopped- and continuous-flow. Stopped-flow experiments were run in time-slice mode, whichessentially stops flow when the detection cell is refreshedwith new material, ensuring that all material is detected.With continuous-flow, a limited number of scans can becollected, and thus, we examined the effect of a reducedflow by dropping the rate by 10-fold (i.e., run at both 0.5 and0.05 mL/min). Readers should keep in mind throughout theremainder of the text that compromised chromatography isa necessity of successful online HPLC-NMR. The reduced

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sensitivity of NMR compared to traditional HPLC detectorsrequires that conditions such as large sample load and slowerflow are used to achieve sufficient material within the NMRflow cell. HPLC is essentially a means of separation prior toNMR analysis, and optimal separation, although desirable,is not necessary to obtain structural information from NMR(17).

Figure 1 illustrates the chromatographic variability ofconducting the above-mentioned modes on SRNOM. RowsA, B, and C depict stopped-flow, slow continuous-flow, andfast continuous-flow, respectively. For these rows, columnI illustrates 2D NMR chromatograms. The y-axes of these 2Dchromatograms contain profiles of SRNOM elution withmobile-phase volume and are constructed from the sum ofNMR signals (excluding solvent signal from water). Exami-nation of these y-axes illustrates that continuous-flow elutedmaterial over larger volumes than stopped-flow trials andthat continuous-flow resulted in later apex elution (see alsoTable 1). Theoretically it would be expected that peaks instopped-flow would be broader due to the substantialdiffusion that is permitted to occur from both motionlessconditions and considerably longer run times (e.g., here ∼17h for stopped-flow vs ∼4 h for slow and ∼1 h for fastcontinuous-flow). Diffusion is also likely to promote DOMdisaggregation, and thus, stopped-flow should result insmaller material at larger retention volumes. Stopped-flowinstead generated a sharper peak and earlier apex thancontinuous-flow, contrary to what was expected.

A plausible explanation for these elution differences isthe effects of sample viscosity. As noted to occur in preparativeHPLC, “viscous fingering” occurs with large sample injections,

causes a delay in retention volume, and is known to generatefalse banding after apex material has passed (24, 25). In simpleterms, viscous fingering occurs when a large plug of sampleis injected into a less viscous mobile phase and is essentially“run over” by the mobile phase, resulting in poor separation,broadened peaks, and false “bands” at the tail (which wasnoted to occur with samples run in continuous-flow mode).Stopped-flow, permitting abundant diffusion, would beexpected to generate ample time for the sample edges to mixwith the mobile phase and therefore generate fewer viscousfingering effects. The early elution and tighter sample bandsproduced by stopped-flow indeed suggest that viscousfingering did not occur nearly as much as with continuous-flow conditions. To further test the role viscosity might play,much less concentrated/viscous samples were also run andfound to elute earlier than more concentrated samples (Table

FIGURE 1. Comparison of HPSEC-NMR techniques (SRNOM, 100 mg/mL, 100 µL injection). Column I (rows A-C): pseudo-2Dchromatograms from online analyses, elution profiles along the y-axes. Column I (row D): sum profiles from selected NMR regionsfrom column I, row A (indicated by dashed lines and colored regions): red, aromatics, 6.5-7.8 ppm; green, carbohydrates, 3.2-4.5ppm; blue, carboxyl-rich alicyclic molecules (CRAMs), 1.6-3.2 ppm; purple, material derived from linear terpenoids (MDLT), 0.6-1.6ppm (as highlighted in column I, row A). These profiles are discussed later in the paper but included here so that the reader canvisualize how the sum profiles are created from 2D HPLC-NMR data sets. Rows A, B, and C: NMR spectra from stopped-flow, slowcontinuous-flow, and fast continuous-flow, respectively. Row D (columns II-IV): NMR spectra from offline fraction collection.Columns II, III, and IV: NMR spectra of material eluted at 50%, 75%, and 95% of the total sample elution volume. Readers should beaware that under continuous-flow conditions (rows B and C) the sample is only in the NMR cell for a finite amount of time; hence,the number of scans cannot be increased. However, in the case of stopped-flow (row A) and fraction collection (row D) the numberof scans can be increased substantially, permitting the detection of components at much lower concentrations. The asteriskindicates carbohydrates; methoxyl from lignin also contributes to this region.

TABLE 1. Parameters and Apex Retention Volume forHPSEC-NMR Analyses

methodflow rate(mL/min) sample

concn(mg/mL)

apex retentionvol (mL)

stopped-flow 0.5 SRNOM 100 14.25continuous-flow 0.05 SRNOM 100 16.83continuous-flow 0.5 SRNOM 100 16.08fraction collection 0.5 SRNOM 100 16.14stopped-flow 0.5 SRNOM 20 13.88stopped-flow 0.5 SRNOM 5 13.63stopped-flow 0.5 NRNOM 100 14.25stopped-flow 0.5 LSNOM 100 14.63

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1). This finding further suggests that viscosity acts to delayand broaden eluting material in the more concentratedsamples. An alternative theory might be that late elution isthe result of a reduction in conformational size withincreasing concentration, but NMR research has shown thatnot to be the case with SRNOM (21), and thus, it is not likelyto be the case here. Further comparison of the two continu-ous-flow experiments reveals that slow flow resulted inbroader and later elution than fast flow trials (Figure 1(column I, row C, and column I, row B); see also Table 1),suggesting that both diffusion and viscosity affect samplesrun under slow continuous-flow conditions.

Along with differences in chromatographic profiles, theNMR data provide information as to the separation ofstructural entities in the fast, slow, and stopped-flow experi-ments. In all cases the NMR spectrum for the major fractioneluting at the apex is dominated by carboxyl-rich alicyclicmolecules (CRAMs), signified by the dominant signal from∼1.6 to 3.2 ppm (Figure 1, column II, rows A-C). By thelatest fractions (95% elution), however, CRAMs are depletedin the stopped-flow and fast continuous-flow experimentsbut are still prominent in the slow continuous-flow. Thereduced variability apparent in the slow trial compared tothe others suggests that slow continuous-flow provides theleast effective separation.

Offline fraction collection of SRNOM is illustrated in Figure1 (row D, columns II-IV). These spectra are similar to theonline spectra, but differences exist that are likely due to the8× greater sample volume collected for the offline fractions(1.00 mL vs 0.125 mL elution volume). These offline spectrarepresent 240 times more material (8× larger fractions and30 HPLC runs), 240 times higher salt content, longer NMRexperiments, and material freeze-dried following fractioncollection. Excess repetitions of HPLC runs were conductedto acquire more sample material from the most dilute frontand tail fractions in hopes of obtaining information unob-tainable from online analyses. No structural information wasgained that was not discernible in online experiments.Fraction collection is, however, more readily available toresearchers, and depending on the type of separation orresearch goals, fraction collection may in many instances bemore practical than online HPLC-NMR. The goal of researchpresented here is to develop online techniques that are inmany ways complementary to offline techniques as bothhave advantages and disadvantages. For example, the onlineexperiments result in a degree of carryover within the NMRflow cell (dependent upon the volume and design of the flowcell and connections). Fraction collection, in turn, is limitedin the sense that increasing the numbers of fractions becomesan issue of resources and labor. From our research experience,100 fractions could be accomplished within∼1 h (continuous-flow) and ∼17 h (stopped-flow) while online. The comparableoffline technique took weeks to obtain and run 25 fractions.Improved HPLC separations in the future (with potentiallyhundreds to thousands of peaks) would make fractioncollection extremely laborious to achieve the sort of resolution(i.e., minute fractions) capable with online HPLC-NMR.Additionally, salts from buffers are concentrated whilefractions are collected, which can make accurate matchingand tuning difficult for NMR.

Of the online methods compared in this study, stopped-flow provided the best separation (due to reduced viscousfingering) and has the potential for enhanced sensitivity overother methods (as one can increase the number of scans).Though diffusion could be problematic, evidence suggeststhat more time for equilibration between the sample andsolvent system in stopped-flow reduces undesirable viscosityeffects which at least in this study outweigh negative effectsfrom diffusion. Environmental samples are frequently veryheterogeneous and due to the vast numbers of constituents

present in very small quantities require large HPLC injections.The effects of viscosity are therefore an important considerationof online HPLC-NMR with environmental applications.

Effects of Concentration. Concentrations as low as 5 mg/mL are known to result in the aggregation of DOM intoconformationally larger material (21). Thus, it is importantto investigate whether increased concentration leads toreduced separation, either from sample aggregation or fromcolumn overload, which in turn leads to a loss of informationvia NMR detection. Figure 2 illustrates strong similarities inthe eluting material between the three tested concentrations(5, 20, and 100 mg/mL). At 5 mg/mL (Figure 2, row C), theearly- and late-eluting material produced spectra with lowS/N ratio and sample peaks are overshadowed by largeartifacts resulting from water suppression during dataprocessing (see the Materials and Methods). At such lowconcentrations, distortions from the Gaussian filter used toreduce the water signal are emphasized and weak samplesignals are difficult to discern. This is best exemplified by theregion at ∼3.7 ppm adjacent to the water and highlightedwith an arrow in Figure 2 (column I, row A). This signal arisesfrom a combination of lignin methoxyl and carbohydrates(21), is clearly seen in the more concentrated sample, is likelypresent in the 20 mg/mL separation, and is not discerniblein the 5 mg/mL separation. Figure 2 demonstrates that elutingmaterial appears similar at all tested concentrations but alsoillustrates the importance of using large sample quantitiesto obtain sufficient NMR signal for more detailed and robustidentifications.

Structural Information. Recent work on the structuralidentification of DOM has identified material thought to bederived from cyclic terpenoids known as CRAMs (3, 26) andmaterial derived from linear terpenoids (MDLT) (3) as majorcomponents of DOM. Recent 1H and 13C NMR research onDOM and humic acids provides evidence of aromatics andcarbohydrates as major constituents of conformationallylarger DOM, while CRAMs and MDLT generally constitutesmaller material (21, 27, 28). For the analyses of spectradescribed in this study, constituents present in 1D 1H NMRspectra were assigned the following chemical shifts: aromatics(6.5-7.8 ppm), carbohydrates (3.2-4.5 ppm), CRAMs (1.6-3.2ppm), and MDLT (0.6-1.6 ppm).

The 1D slices from all experiments reveal that the bulkof carbohydrate- and aromatic-type material eluted in theearly slices (Figures 2, column I, and 3, column II). The apexmaterial was in turn characterized by a strong CRAMsignature (Figures 2, column II, and 3, column III), and thelate-eluting material was characterized by strong signals fromthe MDLT region as well as sharp peaks in the carbohydrateregion suggestive of small sugars (Figure 1, column IV). Toanalyze the elution order of major components present ineach sample, 1D profiles were generated from selectedregions of the pseudo-2D NMR chromatograms usingAdvanced Chemistry Development (ACD laboratories) Spec-trum Manager (version 11.0). As indicated by the coloredregions in Figure 1 (column I, row A), profiles of the aromatics,carbohydrates, CRAMs, and MDLT were generated bycompiling the sums of these regions. An example of theresulting profiles is illustrated in the bottom left corner ofFigure 1 (column I, row D). The insets in Figure 3 illustratethe component order of elution for three DOM samples(SRNOM, NRNOM, and LSNOM) and are discussed in detailbelow. The maxima of the four material types elutedindependently and with the general elution volumes ofaromatics < carbohydrates < CRAMs < MDLT. This trend wastypical for all experiments and samples with the exceptionof LSNOM. LSNOM did not separate well as indicated by anelution order of aromatics)carbohydrates)CRAMs < MDLT(further discussion below).

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Variability of Environmental Samples. To assess envi-ronmental variability, DOM samples isolated from three verydifferent bodies of water were compared. Figure 3 illustrateswhole DOM samples (column I) and HPSEC-eluted materialat 25%, 50%, and 75% of the total sample elution volume(columns II-IV). The whole sample NMR spectra of all threesamples consist of similar broad NMR profiles (characteristicof DOM). The profiles of size-distinguished fractions, how-

ever, reveal clear molecular differences between samples.This variability is key to elucidating and understanding theorigins of the organic material as well as the physical andbiological processes that act to degrade and mineralize DOM.The fractions displayed in Figure 3 are merely representativeof the types of material eluted prior to, during, and after thechromatographic apex of each sample; these fractions donot add up to make the whole of the bulk samples but do

FIGURE 2. Comparison at varying concentrations (stopped-flow, SRNOM, 100 µL injection). Rows A, B, and C: NMR spectra of 100,20, and 5 mg/mL, respectively. Columns I, II, and III: material eluted at 25%, 50%, and 75% of the total sample elution volume. Thearrow is discussed in the text.

FIGURE 3. Comparison of DOM samples (stopped-flow, 100 mg/mL, 100 µL injection). Rows A, B, and C: SRNOM, NRNOM, andLSNOM, respectively. Column I: whole samples run in a traditional 5 mm NMR tube. Columns II, III, and IV: material eluted at 25%,50%, and 75% of the total sample elution volume (100 mg/mL, stopped-flow). The insets in column I (rows A-C) illustrate elutionprofiles from HPSEC-NMR; the axes are the retention volume (mL). (Refer to the Figure 1 caption for color and chemical shiftassignments.) The asterisk indicates carbohydrates; methoxyl from lignin also contributes to this region.

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illustrate the variability in composition between samples.With the largest material, the NRNOM sample has thestrongest carbohydrate contribution (methoxyl from ligninmay also resonate here) compared to the other samples. Withthe midsized fraction, NRNOM has the weakest contributionof CRAMs, while the smallest fraction has the strongest MDLTcomponent. The SRNOM and LSNOM samples appear mostsimilar to each other by HPSEC-NMR, but differences notapparent from the total NMR spectra are apparent. Forexample, the SRNOM contains more MDLT in the smallerfraction than the LSNOM, despite the fact that LSNOMcontains more MDLT as a whole. Further analysis of apexretention volumes (Table 1) reveals that the LSNOM elutedlater than the other samples, indicative of smaller material(or greater viscosity). The SRNOM and NRNOM samples, inturn, eluted earlier than the LSNOM sample but with retentionvolumes equal to one another. Finally, the profiles ofaromatics, carbohydrates, CRAMs, and MDLT for all threesamples are illustrated in the insets of Figure 3, column I.From these insets it is apparent that the LSNOM sampleseparated the least, with nearly all component maximacoeluting while the SRNOM and NRNOM samples haddistinct elution volumes for the four major groups. The insetin column I (row B) further illustrates that the NRNOM samplehad the most successful chromatographic separation with asecond major peak after apex elution. This second peak isrich in MDLT-type material and is structurally very distinctfrom the earlier material (hence the higher contributionsfrom the purple trace in the second peak).

The variation in HPSEC elution could be the result of anumber of factors. The samples originated from diverse watersources and were collected in different years with varyingmethods of isolation. The SRNOM and NRNOM samplesoriginated from a blackwater river and drinking reservoir,respectively, and were both concentrated via reverse osmosis(in 1999 and 1997). The LSNOM sample was collected froma productive marshland in 2007 and concentrated on DEAEcellulose followed by alkaline extraction. All samples werestored in an identical fashion (freeze-dried, sealed, and inthe dark) but factors such as origin, parent vegetation,microbial inputs, degradation processes, mentioned isolationtechniques, and age could contribute to the variability inHPSEC separations. The LSNOM sample was notably themost difficult to separate into chemically distinct fractions,while the NRNOM sample was the easiest and even produceda second major chromatographic peak (refer to the inset inFigure 3, column I, row B). More research is necessary todetermine the reasons for elution variability between samples.These differences do, however, demonstrate that while allsamples are similar in terms of their 1D NMR (column I),HPSEC-NMR provides additional information on the sub-components within DOM which may prove useful inunderstanding how components vary temporally and spa-tially and their role in the formation and persistence of DOMin the environment.

Despite the compromises necessary for online HPSEC-NMR, analyses of slices eluted at regular intervals indicatevariations in material from the largest to the smallest sizedfractions, and material generally eluted in the order of (1)aromatics, (2) carbohydrates, (3) CRAMs, and (4) MDLT. Anoteworthy phenomenon is the strong NMR signal of CRAM-type material during apex elution (i.e., the biggest fractionof the sample). This supports the existence of CRAMs as aseparable and unique entity in DOM that has only recentlybeen proposed in the literature (26). Similarly, MDLTdominates the smaller size fraction, supporting the hypothesisthat MDLT also contributes significantly to DOM and is aunique and separable entity. Comparison of whole samplespectra to size-separated fractions suggests that DOMseparates into structurally different material that varies

between samples. It has been previously proposed that whilebiomolecules undergo similar biogeochemical processes, itseems likely that there are similarities between samples thathave undergone significant degradation (26). Whether thedifferences found in the separations here are caused more bysource material or degradation warrants further research.

With this initial HPSEC-NMR hyphenation, we haveshown that online HPLC-NMR can be used for the structuralanalyses of DOM. This technique is useful and perhapsadvantageous over other analytical techniques in that samplescan be analyzed very quickly and that large quantities ofdata and detailed information are made available in a shortperiod of time. Of particular interest for further research isto examine stationary phases better suited for the separationof DOM constituents. Improved chromatography online withNMR has the potential to provide unsurpassed structuralcharacterization of DOM and is a technique applicable toanalyses of other complex natural samples.

AcknowledgmentsWe thank the Natural Science and Engineering ResearchCouncil of Canada (Discovery Grant, A.J.S.), the InternationalPolar Year (IPY), the Science Foundation of Ireland (GrantGEOF509), and the Irish Environmental Protection Agency(STRIVE program) for providing funding. We also thank thegovernment of Ontario for providing an Early ResearcherAward (A.J.S.).

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