Environ. Sci. Technol. 2005, 39, 8382-8387
Structure-Dependent Reactivity of Low Molecular Weight Fulvic Acid Molecules during Ozonation ANJA THESE AND THORSTEN REEMTSMA* Department of Water Quality Control, Technical University of Berlin, Sekr KF 4, Strasse des 17 Juni 135, 10623 Berlin, Germany
Size-exclusion chromatography coupled to quadrupole time-of-flight mass spectrometry (SEC-Q-TOF-MS) was used to study changes in the molecular composition of a Suwannee River fulvic acid isolate by ozonation. The composition of all three SEC fractions showed strong changes and a relative increase of the low molecular weight anions. Further mass spectrometric investigations focused on the low molecular weight fulvic acid molecules, where a preferential removal of fulvic acid molecules with a low oxidation state (low O/C ratio) and a high degree of unsaturation (low H/C ratio) was observed. Besides their elemental composition, also the structure of the fulvic acid molecules influenced their reactivity toward ozone. The data suggest that molecules with a more extended carbon skeleton and less carboxylate substituents showed higher reactivity whereas some highly unsaturated molecules did not show measurable removal up to a specific ozone dose of 2.5 mg/mg of DOC due to sterical shielding of the reactive structures. Newly formed molecules were determined by SEC-Q-TOF-MS, which were characterized by a very high number of carboxylate groups (high O/C ratio) and a highly saturated carbon skeleton (high H/C ratio). These investigations explain on a molecular level many observations previously made with whole mixtures or fractions of natural organic matter.
Introduction Electrospray-ionization high-resolution mass spectrometry (ESI-MS) allows detection and investigation of single fulvic acid molecules out of the complex isolates (1-6). Today the elemental composition of hundreds to thousands of fulvic acid molecules is known (3-5), and information on the structures of fulvic acid molecules has been obtained by MS/ MS measurements (3, 5-9). These various investigations revealed a high degree of regularity in fulvic acid mixtures, from intensity variations over elemental compositions to their molecular structure. Recently, size-exclusion chromatography-quadrupole time-of-flight mass spectrometry (SECQ-TOF-MS) has been used to compare fulvic acid isolates of different origins, and a very high degree of coincidence was recognized (6). With ESI high-resolution mass spectrometry, which makes individual molecular species amenable to analysis, it should also be possible to study the chemical reactivity of fulvic acid molecules. This could substantially deepen our understanding of the reactivity of these molecules and the reaction * Corresponding author phone: +49-30-31426429; fax: +49-3031423850; e-mail:
[email protected]. 8382
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mechanisms and processes and could help to explain the products eventually being generated. Ozonation of natural organic matter (NOM), the majority of which consists of fulvic acids, is an important process in drinking water preparation (10). Ozonation has been shown to improve the flocculation of NOM and coagulation of particles (11) as well as NOM removal in biofilters and to reduce NOM’s potential to form disinfection byproducts in chlorination (12) even without a significant decrease of the organic carbon content. By using characterization methods, it was shown that ozonation of natural organic matter leads to decoloration as well as to decreases of the UV absorbance and of the average molecular weight, while polarity and acidity increase (ref 12 and references therein). Thus, fulvic acids are subjected to marked chemical alterations by ozonation. These changes have never been visible on a molecular level, and despite intensive research, most disinfection byproducts generated by ozonation are yet unidentified (13). In this study SEC-Q-TOF-MS is used to study on a molecular level the alteration of a fulvic acid mixture during ozonation. This approach differs from previous attempts to use ESI-MS to follow transformation of NOM in oxidative processes such as UV irradiation (14) and chlorination (15) in three respects: (a) SEC is used here to separate the whole mixture according to the hydrodynamic volume, thereby reducing the polydispersity of the mixture entering the mass spectrometer at a given time (9), (b) high-resolution (TOF) MS allows separation of molecular species with the same integer mass in the low molecular weight range and determination of their molecular formulas (5), and (c) MS/MS coupling (Q-TOF) provides information on the structure of the various molecules (5). By this approach the different reactivities of individual fulvic acid molecules toward ozone become visible, enabling us to link the reactivity with their elemental composition and molecular structure. Moreover, the ability to resolve isobaric molecules, i.e., ions with the same integer mass but different exact masses (molecular formula), allows original fulvic acid molecules and molecules that are newly formed during the ozonation process of fulvic acids to be distinguished.
Materials and Methods Materials. All experiments were performed with Suwannee River fulvic acid (SRFA) that was purchased from the International Humic Substance Society (IHSS). Ozonation. Pure oxygen was supplied to a high-voltage discharge ozone generator (Sorbios, GSG 1.2) continuously at a flow rate of 35 L/h. The ozone inlet gas concentration was 25 g/m3. Ozone concentrations in the inlet and outlet gas were detected by UV analyzers (BMT, Berlin, Germany) and electronically recorded every 5 s. Ozonation was performed stepwise in a gas wash bottle with 30 mL of an aqueous fulvic acid solution of 2.0 g/L (pH 9 adjusted with 1 M NaOH). Ozonation was followed by monitoring the increase in the outlet gas ozone concentration, and each ozonation step was stopped when it had reached almost the inlet value (∼20 g/m3). An aliquot of 1.5 mL of the solution was withdrawn for investigation, the pH value readjusted, and the next ozonation step performed. This sequence was continued until the outlet concentration almost instantaneously increased to about 20 g/m3 and, thus, no ozone consumption was measurable. Typically four ozonation steps were performed. The ozone consumption (mg) was calculated from the difference between the outlet and inlet ozone 10.1021/es050941h CCC: $30.25
2005 American Chemical Society Published on Web 09/28/2005
FIGURE 1. Characteristics of a fulvic acid isolate before (top) and after (bottom) ozonation with 2.5 mg of O3/mg of DOC. (a) SEC chromatograms with UV (220-550 nm, gray) and MS-TIC (m/z 150 to 750, black). The absolute intensity of each trace ()100%) is provided in the upper right corner of the graphs. MS spectra of the first (b), second (c), and third (d) SEC peaks. Spectra (b) are combined from two measurements (m/z 150-1000 and m/z 1000-2000). concentrations for each 5 s interval (at constant flow) and summed over the time of ozonation. The dissolved organic carbon (DOC) content and the UV absorbance at 254 nm were measured by standard procedures, and ESI-MS analyses were performed within a few days after storage in a refrigerator. SEC-MS Analyses. Fulvic acid solutions before and after ozonation were separated by SEC using a 250 × 4.6 mm i.d. hydrophilic polymer column (particle size 8 µm, PL Aquagel, Polymer Laboratories, Shropshire, U.K.) and an eluent of 80:20 (v/v) water/methanol + 10 mM NH4HCO3. This SEC system was coupled to a diode array detector and to one of two mass spectrometers, depending on the mass resolution required (9). For low-resolution MS analyses SEC was coupled to a triple-quadrupole mass spectrometer (Quattro LC, Micromass, Manchester, U.K.) with electrospray ionization in the negative ion mode, using a mass resolution of 800-1000 (m/∆m at fwhm) (9, 16). For high mass resolution the SEC was coupled to a Q-TOF Ultima mass spectrometer (Micromass) operated in either the V-mode (m/∆m 8000) or the W-mode (m/∆m 12000, m/z 556). Spectra were recorded over a range of m/z 150-1000 with a scan time of 1.0 s in the continuum mode. For MS/MS experiments, a collision energy of 18 eV was used and a mass range of m/z 30-500 was scanned in 1.0 s (5, 6). All high-resolution MS data presented here originate from the last SEC peak that was attributed to low molecular weight fulvic acids (9). Data processing and generation of 3D van Krevelen diagrams have been described recently (6).
Results and Discussion Ozonation of Fulvic Acid. All ozonations of fulvic acids were performed in the laboratory at a concentration of 2 g/L (DOC about 1 g/L). This high concentration was necessary to allow for a direct SEC-MS analysis of the ozonated solutions without any preconcentration. In this way selective losses of polar ozonation products could be avoided. The bulk parameters of these experiments suggest that ozonation and organic matter transformation proceeded as known for very diluted fulvic acid solution as the UV absorbance decreased very rapidly, whereas a decrease in DOC content occurred at ozone dosages of more than 2.5 mg/mg of DOC only (17).
SEC-UV analyses before and after ozonation (2.5 mg of O3/mg of DOC) show a clear shift toward longer retention times, corresponding to a decrease in the average molecular mass of the fulvic acid mixture upon ozonation (Figure 1a). The corresponding MS traces (Figure 1a) do not show the same clear shift, which is likely due to the lower sensitivity of ESI-MS toward fulvic acids of higher molecular weight (16). Clear quality changes due to ozonation are already visible in low-resolution mass spectra recorded by a quadrupole MS instrument (Figure 1b-d). A clear shift toward ions of lower m/z values occurs in all three SEC fractions, and the two early eluting fulvic acid fractions of higher molecular weight (Figure 1b,c) appear to lose their oligomeric character that was reflected in their tri- and bimodal intensity distribution (9, 16). The SEC-UV chromatograms and, more clearly, these scan spectra obtained from the SEC peaks confirm the well-known trend that the average molecular weight of fulvic acid isolates decreases upon ozonation (12, 18, 19). The further study focused on the low molecular weight fulvic acids that elute in the third chromatographic peak (Figure 1d, Rt ≈ 7.5 min) because this fraction is detected most sensitively by ESI-MS (16). A Q-TOF-MS instrument with a mass resolution of about ∼10000 was used as it resolved most molecular ions with the same integral mass. Due to the suggested oligomeric character of fulvic acids, compositional changes observed in the low molecular weight fraction are expected to be of importance also for the fractions of higher molecular weight. Fulvic Acid Composition and Structure. ESI highresolution mass spectrometry has previously shown that fulvic acid isolates are very complex mixtures of a large number of individual molecules, but with a high degree of regularity at different levels, from elemental composition to molecular structure (5, 6). The z* value has been shown to be a useful parameter for structuring ESI-MS data of fulvic acids (3). It divides a spectrum into sections of 14 mass units and helps to reveal regularities in the elemental composition of fulvic acids (3, 5, 6). The molecular composition of a fulvic acid isolate can be visualized in a van Krevelen diagram (20) in which each single dot displays the O/C and H/C ratios of the molecular formula of one of its molecules (Figure 2a). Of the ∼300 fulvic acid VOL. 39, NO. 21, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. (a) van Krevelen diagram of the molecular formulas of ∼300 low molecular weight fulvic acids in the mass range m/z 150-344 determined by SEC-TOF-MS. (b) Putative structures of four representative fulvic acid molecules. O/C and H/C ratios of each of the molecules are close to the averages of the respective quadrant.
FIGURE 3. TOF-MS scan spectra of low molecular weight fulvic acids before (top) and after (bottom) ozonation with 2.5 mg of O3/mg of DOC. (a, b) Spectra of the range m/z 264-306. Numbers in italics denote the z* values. (c, d) Extended section for the mass range m/z 295.0-295.2. molecules in the mass range m/z 150-344, those with a common z* value (that is every 14th nominal mass) occupy a certain section of the diagram. Molecules with z* ) -12 or -14 fall in the middle of the set, and they also occur in highest intensity in the scan spectra of low molecular weight fulvic acids (Figure 3a), whereas low-intensity ions exhibit z* values of -6 and are located at the outer boundaries of the van Krevelen diagram (Figure 2a) (6). 8384
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Product ion spectra obtained by MS/MS analyses with the SEC-Q-TOF-MS provide information on structural moieties of fulvic acid molecules (5, 6). Such product ion spectra have been shown to be dominated by successive losses of CO2 (-43.990 amu) with a parallel loss of H2O (-18.011 amu), indicating that almost all oxygen is bound in carboxylate groups and a maximum of two hydroxy moieties are found in low molecular weight fulvic acids (5). Only weak signals of decarbonylation (-27.995 amu) occurred, which may indicate a minor contribution of quinoid structures in these molecules (3, 5). No other fragmentation was observed, so ester or ether linkages cannot be prominent. Finally, the considerable stability of fulvic acids in the natural environment calls for branchings in the aliphatic structures to improve resistance against microbial degradation. On the basis of these mass spectrometric results, we have recently proposed structures of low molecular weight fulvic acids (6). However, all these structure proposals are based solely on the mass spectrometric information outlined above. Therefore, substitutional isomers of these structures are very possible, while the elemental composition, the character and number of the functional groups, and the number of carboncarbon double bonds are not in doubt. To relate the elemental information provided in a van Krevelen diagram with molecular structure, Figure 2b shows putative structures of four fulvic acid molecules, each representative of one of the quadrants of the van Krevelen diagram. On the left side less oxygenated structures are found. On the lower left side molecules exhibit a high number of carbon-carbon double bonds; these structures resemble molecules previously identified in NOM isolates that have been ascribed to black carbon sources (21). On the upper left side less aromatic and more aliphatic compounds are located. Despite their relatively high H/C ratio, a considerable number of carbon-carbon double bonds can be found, since the number of carboxylate groups and, thus, of carbon-oxygen double bonds is low. Contrary to that, the structures found on the upper right side tend to be saturated aliphatic and highly carboxylated. Fulvic acid molecules on the lower right side are also highly carboxylated. Therefore, less double bonds are located in the carbon skeleton, rendering them less aromatic as compared to their neighbor on the lower left side. Reactivity of Fulvic Acid Molecules. Intensities in Scan Spectra. As recently shown the comparison of the relative signal intensities of molecules in ESI-MS spectra of different samples analyzed under the same instrumental conditions provides insight into compositional differences at the molecular level (6). Such relative signal intensities of the individual molecular ions may also be useful to detect compositional changes in one fulvic acid mixture induced by ozonation. Despite the drastic changes induced by ozonation (Figure 1), the wavy intensity distribution with a periodicity of 14 amu is maintained (Figure 3a,b), but the intensity maxima are shifted from z* ) -12 before ozonation (Figure 3a) to z* ) -8/-10 after ozonation (Figure 3b). According to the van Krevelen diagram (Figure 2a), such a shift in the z* values indicates a relative increase of molecules with higher H/C and O/C values, i.e., of more saturated and more oxidized molecules. Obviously, fulvic acid molecules with a low degree of oxidation and a high degree of unsaturation are preferentially oxidized, leaving the more saturated and less reactive molecular species with higher H/C values behind. These first observations of fulvic acid transformations by ozonation at the molecular level are in line with quality changes observed for whole fulvic acid mixtures, such as a decrease in color and UV absorbance (12) and a relative depletion of aromatic against aliphatic structural elements as determined by NMR spectroscopy (see, e.g., ref 17). The
TABLE 1. Characteristics of Five Isobaric Fulvic Acid Molecules Determined as Molecular Anions by SEC-Q-TOF-MS Analysis at m/z 295 (z* ) -12)a sum formula C12H8O9 C13H12O8 C14H16O7 C15H20O6 C16H24O5
DBEb COOHc 9 8 7 6 5
4 4 3 3 2
O/C
H/C
ox. stated
0.75 0.62 0.50 0.40 0.31
0.67 0.92 1.14 1.33 1.50
+0.833 +0.308 -0.143 -0.533 -0.875
C-C skeletone DBEf C8H4O C9 H12 C11H13O C12H20 C14H24O
5 4 4 3 3
a For structure proposals refer to Figure 4. b Double bond equivalents (for CxHyOz, DBE ) (2x + 2 - y)/2. c Maximum number of carboxylate groups (derived from MS/MS experiments). d Average oxidation state of the carbon. e Formula of the carbon skeleton (after removal of all carboxylate groups). f Carbon-carbon double bond equivalents () DBE - number of carboxylate groups).
higher reactivity of less saturated fulvic acid molecules is also in agreement with the known electrophilic nature of ozone, which requires centers of high electron density such as carbon-carbon double bonds to react with (18, 22). However, from the experimental conditions it cannot be excluded that some oxidation by OH radicals occurred, a process which is generally considered less selective (10). Isobaric Ions. The signals shown in Figure 3a,b for each integer m/z value (e.g., m/z 295) are the superimposition of the intensities of a number of isobaric ions which share the same integer mass but differ in their exact mass. For the mass range considered in this study a Q-TOF-MS instrument is suitable to resolve these isobaric anions and to determine their molecular formulas (5). The exact masses of a set of fulvic acid anions with the same integer mass (Figure 3c) usually differ by 36 mDa, corresponding to the formal replacement of one oxygen by a carbon and four hydrogen atoms (2, 3, 5) (Table 1). This difference originates from the superimposition of two homologue series, a series of hydrogen homologues and a series of oxidation homologues (6). According to the different elemental compositions of these isobaric anions (Table 1), one would expect that these molecules are not equally reactive toward ozone. A comparison of the signal intensity of a set of isobaric ions before and after ozonation (Figure 3c,d) shows which fulvic acid molecules react faster than others, which are completely removed and which remain.
Time-resolved data for the m/z 295 set exemplarily show the influence of the molecular composition on the reactivity toward ozone (Figure 4a). Generally, the reactivity decreased with increasing degree of oxidation from C15H20O6 to C12H8O9. Correspondingly, the concentration of the most oxidized molecule (C12H8O9) remained constant even after 2.5 mg of O3/mg of DOC (Figure 4a), whereas the least oxidized molecule (C16H24O5) was quantitatively removed by an ozone dose of 2.2 mg/mg of DOC already. These findings correspond to expectation as far as thermodynamics are considered, because oxidation of the least oxidized molecule provides the largest gain in energy. However, as previously hypothesized (17, 22), the reactivity toward ozone is a matter not only of the elemental composition of a molecule but also of its structure. The number of carbon-carbon double bonds decreases from C12H8O9 to C16H24O5 (Table 1), whereas the reactivity toward ozone increases (Figure 4a). This seems to contradict the expectation that more unsaturated molecules are more reactive. An explanation can be deduced from the potential structures of these five isobaric molecules (Figure 4b): the more oxidized molecules with a larger number of carboxylate groups (Figure 4b, top) have less carbon atoms available to form the carbon skeleton (8 out of 12 for C12H8O9), and this skeleton must accommodate four carboxylate groups. Therefore, in C12H8O9 the carbon-carbon double bonds are all conjugated with and sterically shielded by the carboxylate groups. Especially the latter may hamper the electrophilic attack by ozone. Contrary to that, the least oxidized molecule of this group of isobaric ions (C16H24O5) has a quite extended carbon skeleton (Figure 4b, bottom) that accommodates only two carboxylate groups. This results in a more accessible molecular structure that eases attack by ozone and renders this compound more reactive despite its lower number of carbon-carbon double bonds. Thus, within a group of isobaric molecules those with a higher H/C ratio appear to be more reactive toward ozone, leading to a decreasing H/C ratio in such a group upon ozonation. Formation of New Molecules. The Q-TOF-MS spectra also allow determination of ozonation products that occur at exact masses not detected before ozonation. Clear signs of ozonation products were obtained in those sections of the mass spectra that correspond to z* values of -4 and -2 (Figure 3a). The fulvic acid molecules detected at these z* values before ozonation were located at the lower left side in the van Krevelen diagram, exhibiting relatively low O/C and H/C
FIGURE 4. (a) Change in relative signal intensities (I/I0) for five isobaric fulvic acid anions of m/z 295 (z* ) -12) determined by SECTOF-MS. The noisy line for C12H8O9 is due to a low absolute signal intensity. (b) Structure proposals for these molecules (for elemental details refer to Table 1). VOL. 39, NO. 21, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 5. Change in relative signal intensities for isobaric anions of (a) m/z 303 (z* ) -4) and (b) m/z 305 (z* ) -2) determined by SEC-TOF-MS. (c) Structure proposals for the ozonation products of (b). values (Figure 2a). These molecules with a low oxidation state and a high number of carbon-carbon double bonds (characteristic example shown in Figure 2b) are highly reactive toward ozone and were quantitatively removed already by a specific ozone dose of 0.9 mg/mg of DOC, whereas highly oxidized ozonation products occurred at these z* values of -4 and -2. This is exemplarily shown for two sets of isobaric ions (Figure 5). The original fulvic acid molecule C15H12O7 (m/z 303.0505 for [M - H]-) has low O/C (0.47) and H/C (0.8) ratios and a z* value of -4. It is completely removed by an ozone dosage of
