Environ. Sci. Technol. 2005, 39, 3507-3512
Comparative Investigation of Low-Molecular-Weight Fulvic Acids of Different Origin by SEC-Q-TOF-MS: New Insights into Structure and Formation THORSTEN REEMTSMA* AND ANJA THESE Department of Water Quality Control, Technical University of Berlin, Sekr KF 4, Strasse des 17 Juni 135, 10623 Berlin, Germany
Size exclusion chromatography (SEC) coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (ESIQ-TOF-MS) was used to analyze the elemental composition and structure of low-molecular-weight fulvic acid molecules. It is shown that the set of hundreds of individual molecules form a homogeneous and structurally unique class of compounds that can be clearly differentiated from any other class of biogenic matter investigated to date. The molecular composition of low-molecular-weight fulvic acids in isolates of very different origin (surface water, groundwater, peat) is virtually indistinguishable. Significant and characteristic differences are, however, recognized when qualitative information and quantitative information provided by ESI-Q-TOF-MS are linked to each other. The relative frequency of the various molecules in each mixture can differ significantly, with the peat showing higher intensity of the aromatic and less carboxylated molecules of this set, whereas the aquatic fulvic acids show a strong contribution of the molecules with less aromaticity and a higher carboxylate content. The identity of fulvic acid molecules in isolates of different origin implies that no specific source material is required for fulvic acid formation but that they may be formed from different sources by different oxidative processes.
Introduction Fulvic acids, an operationally defined fraction of humic substances, comprise up to 30-50% (1) or even more of total organic material in natural waters. As a highly water-soluble fraction of natural organic matter, fulvic acids take part in transport, transformation, and burial of organic matter and, thus, participate in global carbon cycling. Sources, formation processes, composition, and properties of humic substances have been investigated for almost two centuries (2). Despite considerable progress, namely, in spectroscopic methods, a major limitation remained, as chromatography is not suitable for isolating a single fulvic acid molecule from these very complex natural mixtures. Thus, only average properties of fractions of humic substances and similarities between such fractions could be investigated. Individual and intact humic molecules remained hidden in these mixtures and their properties invisible and unknown. * Corresponding author fax:
[email protected]. 10.1021/es0480466 CCC: $30.25 Published on Web 04/13/2005
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As far as fulvic acids are concerned, a fundamental change was initiated recently by the application of electrospray ionization mass spectrometry (ESI-MS) (3), a technique that is ideally suited for the mass spectrometric analysis of ionic and polar organic compounds from aqueous solution. Highresolution mass spectrometry was recently proven to allow separation of individual molecular species from the complex fulvic acid mixtures (4, 5), and it was used to determined molecular formulas from natural organic matter (6, 7). Moreover, molecular formulas of several thousands of highmolecular-weight (8) and about two hundred low-molecularweight fulvic acids (9) have been determined. These findings outlined that fulvic acids, though being highly complex mixtures, exhibit certain regularities in their structure and their composition. But molecular formulas series of fulvic acids published so far (8, 9) came from only one reference material, the Suwannee River fulvic acids (SRFA) (10). A deepened knowledge on the molecules that form fulvic acid mixtures and on differences and similarities of fulvic acid molecules in isolates of different origin would improve our understanding of the sources and of the ways of formation of this material. This may also become a basis for a rationale chemical definition of this yet only operationally defined class of organic matter. In this study, fulvic acid isolates of diverse origin were investigated with respect to their molecular composition and molecular structures using size exclusion chromatography quadrupole time-of-flight mass spectometry (SEC-Q-TOFMS). SEC allows a coarse separation of fulvic acid mixtures according to the molecular size into three fractions (11), and Q-TOF-MS provides sufficient mass resolution to allow calculation of molecular formulas of either the molecular ions of low-molecular-weight fulvic acids or of their fragment ions (9). Moreover, product ion spectra generated after collison-induced dissociation (CID) of the molecular ions are indicative of functional groups and properties of the carbon skeleton of the individual molecules. One of the advantages of SEC-MS over infusion-MS is the potential to distinguish between molecular ions of low mass and lowmass fragments of molecular ions of higher molecular weight (12). This work focuses on low-molecular-weight fulvic acids, because these compounds are detected more sensitively by ESI-MS as high-molecular-weight fulvic molecules (12) and the mass resolution of a TOF-MS would not have be sufficient to derive molecular formulas for fulvic acids of higher molecular weight. It has been repeatedly suggested that fulvic acids of higher molecular weight may have a polymeric character, formed from low-molecular-weight fulvic acids (12, 13). Therefore, knowledge obtained for low-molecularweight fulvic acid molecules may also be of relevance for fulvic acids of higher molecular weight.
Materials and Methods Materials. Fulvic Acids. The following fulvic acid reference materials were purchased from the International Humic Substance Society (IHSS): Suwannee River (SRFA) from Georgia, USA, Waskish Peat (WPFA) from Minnesota, and Nordic aquatic (NAFA) originating from Hellrudmyra pond, Norway. A groundwater fulvic acid (GOFA) was provided by M. Wolf (GSF-Institute for Groundwater Ecology, Neuherberg) and originated from a groundwater aquifer located in a depth of 134-137 m in Lower Saxony, Germany (14). The groundwater had a dark brown color and an elevated DOC concentration of 97 mg/L. Isolation of fulvic acids was VOL. 39, NO. 10, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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performed by reverse osmosis, sorption on a XAD-8 column, and cation exchange (15). Instrumentation. Size Exclusion Chromatography. SEC was performed using a Waters Alliance 2795 HPLC system and a PL Aquagel column from Polymer Laboratories (Shropshire, UK) with 250 × 4.6 mm ID and a particle size of 8 µm. The column had a molecular weight range from 100 to 30000 Da. A sample volume of 30 µL was injected and separated with an eluent of 80:20 (v/v) water/methanol + 10 mM NH4HCO3 at a flow rate of 0.3 mL/min at room temperature. The column effluent was split by a T-piece with about 0.1 mL/min directed toward the mass spectrometer. Details of the SEC separation have been given elsewhere (11). Mass Spectrometry. The mass spectrometric data were obtained by a Q-TOF Ultima mass spectrometer (Micromass, Manchester, UK) equipped with an electrospray source (Zspray) operated in negative ion mode. The capillary voltage was set to -2500 V. Nitrogen was used as the source cone gas (50 L/h), as nebulizer gas (90 L/h), and for desolvation (500 L/h). In all experiments the cone voltage was set to 35 V and the RF Lens 1 to 35 V. Spectra were recorded over a range of m/z 150-1000 with a scan time of 1.0 s. All data were recorded in the continuum mode. The source was equipped with a lock spray, and cluster ions of 0.1% H3PO4 were used as lock masses. It was possible to switch between the V-mode with a mass resolution (m/∆m at fwhm) of 9000 and the W-mode which was adjusted in our experiments to a mass resolution of 16 000 (leucine enkephalin, m/z 556). For MS/MS experiments the collision energy was set to 18 eV and a mass range of m/z 30-500 was scanned in 1.0 s. All MS data presented here originate from the last SEC peak that was attributed to low-molecular-weight fulvic acids (11). Data Treatment. All MS data were analyzed with Masslynx 4.0 software. Spectra were generated by summing as many spectra as possible in a given chromatogram. For the data treatment process “mass measuring” neither background subtraction nor any smoothing function was used. TOF mass correction was done using two phospate ions (m/z 195, 293) from the lock-mass spray, and the signal was centered to the peak top. For the 3D van Krevelen plots the signal intensity of all 45 fulvic acid molecules in the range m/z 151-179 was determined from a summed mass spectrum. Spectrum summation was performed over the same number of scans in the same retention time window (0.67 min width, third signal) of the SEC-chromatogram of the four reference materials. H/C, O/C, and intensity data for each molecular formula in each sample were compiled, and the intensity data of each sample were normalized to the largest signal in that mass range. A 3D contour plot was created by Origin 6.0 (Microcal Software Inc, Northampton, MA) from these normalized data.
Results and Discussion Elemental Composition. We have recently published molecular formulas of 220 low-molecular-weight SRFA fulvic acid molecules in the mass range 190-340 Da (9). These formulas can be graphically arranged in a van Krevelen diagram (6, 16) that displays the H/C ratio versus the O/C ratio of each molecule (Figure 1). This plot clearly defines a region of H/C- and O/C-values in which low-molecularweight SRFA molecules occur. The molecular formulas in fulvic acid mixtures can be arranged into three homologue series (insert in Figure 1): (a) a series of hydrogen homologues with a decreasing number of C-C double bonds per molecule (+2.015 Da); (b) alkyl homologues with an increasing number of aliphatic carbons (+ 14.015 Da); (c) and from our data (9) we conclude that a third homology is generated by the repeated sequential 3508
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FIGURE 1. Van Krevelen diagram of 220 molecular formulas of lowmolecular-weight fulvic acids in SRFA (mass range m/z 198-344). The insert shows how hydrogen homologues, alkyl homologues, and molecules with increasing degree of oxidation are arranged in this diagram. oxidation of methyl groups via alcohols to carboxylic acids. The superimposition of alkyl homologues (b) and oxidation homologues (c) results in the formation of isobaric molecules with a mass difference of 36 mDa, which have been previously recognized using high-resolution mass spectrometry (5, 8, 9). These homologies are, however, formal and need not necessarily indicate real structure homologues. A helpful parameter to sort fulvic acid molecules according to their elemental composition is the so-called nominal mass series z* (eq 1) (8), a parameter that was originally developed for complex hydrocarbon mixtures (17).
(
z* ) mod
- 14 [NM 14 ])
(1)
NM denotes the nominal mass of the respective molecule. In this notation, molecules with a distance of 14 Da share the same z*-value. A series of hydrogen homologues is characterized by a stepwise increasing z*-value. In the van Krevelen diagram of low-molecular-weight SRFA, all molecules with z* ) -14 are located right in the middle of the set, whereas molecules with a nominal mass value z* ) -6 are situated at the outer boundaries (Figure 1). According to the wavy intensity distribution in the mass spectra of fulvic acids, molecules with z* ) -14 (or -12) occur with high intensity and those with z* ) -6 with lowest intensity in this mass range (Figure 2, (9)). Thus, those homologues occurring in high intensity are located toward the center of that region in the van Krevelen diagram that is occupied by fulvic acids. If a series of homologues is formed under thermodynamic control, one would expect that those compounds occurring in the middle of such series should prevail, whereas the members showing extreme compositions should be least frequent. It is noticeable that the nominal mass series z* ) -6 falls apart into two groups, one with high H/C- and high O/Cvalues adjacent to z* ) -8, the other one with low H/C- and low O/C-values adjacent to the z* ) -4 series (Figure 1). Thus, z* ) -6 comprises the end of one and the beginning of another series of homologues in SRFA. It is reasonable that such end-members are least frequent and that their intensity in the mass spectrum is low. In this sense, the molecular composition of fulvic acids with z* ) -6 defines the boarders of existence of low-molecular-weight fulvic acids in terms of their H/C- and O/C-values. Those with low H/Cand O/C-values (lower left region, Figure 1) are characterized
FIGURE 3. Van Krevelen diagram (H/C vs O/C) of the individual formulas of fulvic acid molecules in the mass range m/z 275-303 of the SRFA O, WPFA 0, GOFA 2, and NAFA o isolates, determined in the V-mode of the Q-TOF-MS.
FIGURE 2. Left: Enlarged sections (m/z 275-315) of Q-TOF-MS scan spectra of the low-molecular-weight fraction of fulvic acids of different origin (a) SFRA, (b) WPFA, (c) GOFA, (d) NAFA. Right: Product ion spectra of m/z 295 of (e) SRFA, (f) GOFA. Note that each product ion spectrum is the superimposition of products from all isobaric anions. Fragments are related to carboxylate, [, and to hydroxy, O. by a high aromatic and a low carboxylate portion in the molecule. A further decrease in the H/C- and the O/C-ratio is chemically feasible, but it would result in pure aromatic compounds that cannot be attributed to the fulvic acid fraction. At the other end, the z* ) -6 series with high H/Cand O/C-values (top right, Figure 1) consists of mostly aliphatic polycarboxylated compounds. If more oxygen was introduced into these molecules, carbon-carbon bonds have to be split, leading to the destruction of the molecule. Here the molecular formula information provided by highresolution mass spectra and the intensity information in these spectra match perfectly, showing that it is most useful to link qualitative and quantitative information on individual fulvic acid molecules obtained hy high-resolution MS. Fulvic Acids of Different Origin. We were interested to know whether the composition of low-molecular-weight aquatic fulvic acids isolated from a freshwater swamp in Florida (SRFA) (18) was specific for only this material or whether it was a general feature of fulvic acids and valid also for those of other origin. Therefore, fulvic acid isolates from a Minnesota peat (WPFA) (10), an inland tarn in Norway (NAFA) (19), and a deep groundwater from Germany (GOFA), whose FA fraction is believed to have been formed by microbially mediated oxidation from sedimentary organic matter (20), were analyzed by SEC-Q-TOF-MS. All four fulvic acid isolates show the same regularities in the scan spectra with a pronouced 2-Da spacing and the wavy signal intensity with a 14-Da period (Figure 2a-d). Intensity maxima occur at a z*-value of -12 (m/z 295, 309) and minima at z* ) -6 (m/z 287, 301) in all isolates. All molecular formulas detected in the other three isolates were compared with those found in SRFA for the mass ranges m/z 150-200 and m/z 275-303. A total of 98% of the exact
masses in the range m/z 150-200 agreed with those found in SRFA; for the range m/z 275-303, about 85% agreement with SRFA were found (Figure 3). The lower agreement in this higher-mass range is due to analytical restrictions, as the relative resolution of a mass spectrometer decreases with increasing molecular mass. Under these conditions the signal intensities of some molecular anions located at the boundaries of the H/C- and O/C-area of fulvic acids were too weak to allow exact mass determination. Even though some of the molecules were not found in SRFA, they all fit into the homologue series and, thus, in the regularities defined above. Product ion spectra recorded from several, but not all, molecular anions of the four FA isolates in this mass range are virtually indistinguishable (Figure 2e,f). We conclude that low-molecular-weight fulvic acids from different sources not only have some structural elements in common or exhibit similar regularities in their mass spectra but also that they consist of an identical set of several hundreds of molecules. Despite the large number of individual molecules, lowmolecular-weight fulvic acids are a very homogeneous class of compounds as fragmentations observed in the product ion spectra of all members yet investigated are extremely similar. This supports and extends previous findings obtained by low-resolution mass spectrometry (11, 13, 21, 22). It may be argued that larger compositional differences can occur only in fractions of higher molecular weight as only those allow for a wider structural diversity. However, there is no report yet available that molecules of higher molecular mass show fragmentations other than those discussed here for the low-molecular-weight fraction. Differences between the four isolates can, however, be found in the relative intensity of the 3-5 isobaric anions that occupy one nominal mass, with a mass difference of 0.36 mDa (Figure 4). In the aquatic fulvic acid (SRFA, Figure 4a), the intensity distribution is of an almost Gaussian shape with the most intense signal located toward the middle of each group of isobaric mass signals. Contrary to that, the peat fulvic acids (WPFA, Figure 4b) show a Poisson-like intensity distribution in which the maximum is shifted toward the very left signal of this group, with the highest O/C- and lowest H/C-ratio of these isobaric molecules. Remarkably, no additional molecular signal with a higher O/C-ratio occurs in this group of isobaric ions, suggesting that the very left molecule exhibits the highest degree of oxidation that is possible in fulvic acid mixtures. Any further oxidation would probably result in a thermodynamically unstable molecule, thus leading to its oxidative degradation. VOL. 39, NO. 10, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Relative Frequency (%) of Structural Elements in Fulvic Acid Isolates Determined by 13C NMR Spectroscopya WPFA SRFA NAFA a
FIGURE 4. Further enlarged sections (m/z 164.8-165.4) of the TOF mass spectra showing the exact masses of the isobaric anions ([M-H]-) of (a) SRFA and (b) WPFA.
FIGURE 5. 3D van Krevelen diagram of low-molecular-weight fulvic acids. (a) WPFA, (b) SRFA, (c) GOFA, and (d) NAFA (each plot calculated from the same 45 molecular formulas in the mass range m/z 151-179 and normalized to the signal of highest intensity in that mass range). A combination of these molecular intensity data with the H/C- versus O/C-ratios of each molecule results in a 3D van Krevelen diagram (6). Figure 5 displays the contour plots generated from the intensity data of all detected signals in the mass range m/z 151-179 for each of the four fulvic acids mixtures. Each plot was normalized to the molecular signal of highest intensity in this mass range. According to the differences directly visible in the spectra (Figure 4), WPFA and SRFA also exhibit quite different 3D plots (Figure 5a,b). One intensity maximum of the peat sample (WPFA, Figure 5a) is located at low O/C (0.2)- and low H/C (0.8)-values, indicating aromatic compounds with a relatively low carboxylate content. This agrees with the high aromaticity in WPFA determined by 13C NMR spectroscopy (Table 1, (10)). This high-aromaticity/low-oxygen maximum is not found 3510
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aromatic
aliphatic
carboxylic
36 24 31
20 33 18
19 20 24
Ref 10.
in any of the other mixtures (5b-d). Aromatic compounds with a low degree of carboxylation have a comparatively low water solubility and high sorption tendency, and it is therefore reasonable that they occur in much lower relative concentration in aqueous samples than in a peat elutriate. Molecules with similar elemental characteristics but of higher molecular weight have recently been suggested to indicate oxygenated black carbon (7). The second maximum in WPFA (O/C: 0.35, H/C: 0.9; Figure 5a) reflects more typical fulvic acid molecules with less aromaticity and a higher carboxylate content. The SRFA reference material shows a more uniform distribution with only one maximum (O/C: 0.2, H/C: 1.3; Figure 5b), indicating that more aliphatic compounds predominate in this mixture. This, again agrees with results provided 13C NMR spectroscopy for the whole SRFA mixture (Table 1). The two other aquatic fulvic acids (GOFA and NAFA) show one intensity maximum at the same value as SRFA, but they are both dominated by a second maximum at higher O/Cand lower H/C-ratios (Figure 5c, d). A higher contribution of carboxylated fulvic acid molecules in the NAFA isolate, as previously recognized by 13C NMR data (Table 1), is well reflected in the second maximum of its 3D van Krevelen diagram (O/C: 0.55, H/C: 0.9; Figure 5d). The groundwater fulvic acids (GOFA, Figure 5c) were assumed to be derived from sedimentary organic matter (20), and one could expect some similarities with the peat isolate (WPFA, Figure 5a). However, since GOFA was isolated from groundwater and not from soil, it lacks the high-aromaticity/low-oxygen maximum of WPFA. Both samples do, however, almost agree in the second intensity maximum (O/C: 0.37, H/C: 1.0; Figure 5a,c). It is worth remembering that differences between these four isolates as visible from Figure 5 are not due to the occurrence of different molecules but only due to different intensity distributions of the identical set of low-molecularweight fulvic acid molecules. This interpretation of the 3D van Krevelen diagrams of fulvic acids is only preliminary, as these are the first data on low-molecular-weight fulvic acid molecules obtained in this way. The mass range covered here is limited (m/z 150-180), and the appearance of the contour plot may change when a larger mass range is included. Moreover, it cannot be excluded that some decarboxylation of fulvic acid molecules has occurred. This fragmentation process would significantly decrease the O/C- and increase the H/C-values as compared to the parent molecule. However, as all analyses in this study were performed under the same analytical conditions, one can assume that the extent of fragmentation was the same for all four samples and the results were, thus, still comparable. Anyhow, these graphs already show that it is inevitable to combine qualitative information (elemental composition) and quantitative data (relative frequency) of each molecule in these complex mixtures to determine differences between fulvic acid isolates of different origin. This combination allows us to link high-resolution MS data of individual molecules of fulvic acid mixtures with spectroscopic data of the whole mixture. Structure Information. The Q-TOF instrument provides access to exact mass data of the fragment ions generated by CID from all isobaric fulvic acid molecules. In this way,
FIGURE 6. (a) Potential structures of the molecule C10H12O5 (212.068 Da). (b) H/C versus O/C plot of the eight alkyl homologues and the four hydrogen homologues as well as the three additional degrees of oxidation of C10H12O5 that were determined in SRFA. structural information can be obtained for each molecular formula (9). These product ion spectra (Figure 2e,f) are dominated by series of decarboxylations with weaker signals from parallel losses of water (8, 9, 13, 23) and sporadic signs of decarbonylation (8). Thus, carboxylate groups and aliphatic hydroxy groups and a very few carbonyl and aromatic hydroxy moieties are the only functional groups visible from the product ion spectra. This fragmentation pattern proves that fulvic acids are a unique class of compounds, as similar spectra have never been reported yet for any other biogenic material, including fulvic acid model compounds (21). Due to this scarcity of fragments many functional groups can be excluded, namely, ethers and esters along with other oxygenous structures such as flavonoid skeletons. Hence, structures of fulvic acid molecules appear to be largely independent from any source material. Moreover, the simple product ion mass spectra of lowmolecular-weight fulvic acids also reflect properties of the carbon skeleton of these compounds. Complex extended carbon skeletons are unlikely to occur, as such skeletons would be prone to fragmentation. Additionally, the skeleton must be branched to accommodate several carboxylate groups and to provide resistance to fast aerobic microbial degradation, and it must allow the introduction of conjugated double bonds. An aromatic ring may occur in some of the low-molecular-weight fulvic acids but not in all of them, as only 50% of the molecular formulas provide 4 or more carbon-carbon double-bond equivalents (DBE) that are required for an aromatic system (9), while only 30% accommodate 5 or more carbon-carbon double bonds. Assuming that the primary source of fulvic acids was fresh biogenic matter that contained oxygen in many different forms, these mass spectral data require that during its transformation into fulvic acids extensive hydrolysis and oxidation reactions occur that simplify the molecular structures of the parent compounds and reduce the diversity of functional groups. As a result of the oxidation processes, most oxygen, then, occurs in carboxylate groups at primary carbons. Aliphatic and aromatic hydroxy groups and carbonyl moieties may indicate incomplete oxidation or the inclusion of oxygen at tertiary and secondary carbons. Two structure proposals for one low-molecular-weight fulvic acid species (C10H12O5) that fulfill all these criteria are given in Figure 6a. Of these two, the left variant may be more
likely, because one could expect the conjugated aliphatic dicarboxylic acid (right structure) to be more reactive and less stable. It is not possible to explain the whole range of O/C- and H/C-ratios covered by the formal homologues of this molecule with variation in the extremeties of one carbon skeleton only (Figure 6b). Such homologue series as shown here for C10H12O5 can be found for any member of the fulvic acid mixtures, and they are the basis for the large complexity of these mixtures and the occurrence of isobaric ions with 36-mDa mass difference. Two structure proposals for a fulvic acid molecule based on low-resolution ESI-MS data have been published before. These proposals largely agree with the criteria developed above as they consist of polycarboxylated side chains that are bound to a cyclic core (13, 21, 23). Additionally, a molecular formula that would correspond to one of these proposals was later determined in SRFA (9). A structure proposal based on exact mass data was given by Cooper and co-workers for a molecule of higher molecular weight (8) putatively derived from lignin material. Considering its structural diversity with ether and ester bonds and several hydroxy groups, this proposal is structurally much more complex than the low-molecular-weight fulvic acids determined here. However, compounds of this structure may well serve as precursor material. Implications Concerning Formation, Sources, and Definition. The identity of hundreds of low-molecular-weight fulvic acids in isolates of very different origin can be achieved only by a very intensive oxidative reworking of different biogenic precursor material. This reworking transforms the yet unknown source compounds so effectively that they lose their previous molecular identity to a large extent and become members of the new class, the fulvic acids. Therefore, it can hardly be the source material that determines the elemental composition and the structures of low-molecular-weight fulvic acids. Rather it is suggestable that the thermodynamic stability inherent to these products may be the force that drives their formation and that leads to the evolution of the characteristic molecular structures and the systematics between the hundreds of molecules that are found in all these mixtures. It is, then, no longer astonishing that large identical sets of molecular homologues in isolates from very different aquatic compartments were found. Further research is needed to elucidate whether similar structural regularities and similarities can be found for fulvic acids of higher molecular weight and to understand the relation between fulvic acids of higher and low molecular weight on a molecular level. The results of recent mass spectrometric investigations of fulvic acids using electrospray ionization improve our understanding and change our perception of the chemical nature of fulvic acids. This development may finally allow replacement of the yet purely operational definition of fulvic acids by a very clear and verifiable chemical definition in terms of elemental composition, molecular structures, and homologies.
Acknowledgments The authors thank M. Wolf (GSF-Institute for Groundwater Ecology, Neuherberg) for providing the GOFA material and C. Thomas (Waters, Eschborn) for access and assistance at the Q-TOF mass spectrometer. This work was supported by grants from the German Research Council (DFG, Bonn; RE 1290/4-2, 4-3).
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Received for review December 10, 2004. Revised manuscript received March 11, 2005. Accepted March 17, 2005. ES0480466
