On-line Coupling of Size Exclusion Chromatography with Electrospray

Feb 13, 2003 - Obviously, a class of well-defined polycarboxylated molecules exist that occurs in all fulvic acid fractions thus far investigated. Wit...
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Anal. Chem. 2003, 75, 1500-1507

On-line Coupling of Size Exclusion Chromatography with Electrospray Ionization-Tandem Mass Spectrometry for the Analysis of Aquatic Fulvic and Humic Acids 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

A method was developed for the analysis of humic and fulvic acids by size-exclusion chromatography-electrospray ionization-tandem mass spectrometry using a completely volatile eluent. Humic and fulvic acids were separted into three peaks. These fractions occupied different mass ranges and showed differences in the fine structure of their mass spectra. The low-molecular-weight (LMW) fraction of fulvic acids is most sensitively determined by ESI-MS, and it appears that previous results obtained by infusion-ESI-MS were primarily determined by this fulvic acid fraction. The average molecular weight of this fractions turned out to be lower than that reported from infusion-ESI-MS measurements. Its scan spectra and the product ion spectra of some of its molecular anions perfectly match those previously obtained from whole fulvic acid mixtures. Obviously, a class of welldefined polycarboxylated molecules exist that occurs in all fulvic acid fractions thus far investigated. With decreasing elution time and increasing molecular weight, detection by ESI-MS loses sensitivity as compared to the parallel UV recording, and the fine structure of the scan spectra becomes increasingly uniform for both fulvic and humic acids. The average molecular weight of the HMW fraction exceeds those values calculated from infusion experiments. Scan spectra and product ion spectra of the high-molecular-weight (HMW) fraction of both the humic and the fulvic acids suggest that the HMW fraction consists of several subunits that originate from the LMW fraction. The invention of liquid chromatography-atmospheric pressure ionization mass spectrometry has drastically changed established methods of water analysis, and it has opened new analytical fields that have previously been inaccessible to mass spectrometry.1 Atmospheric pressure ionization, namely electrospray-ionization (ESI) mass spectrometry, has also provided easy access to the analysis of intact humic and fulvic acids, compound classes that previously had to be chemically destroyed to make them amenable * Corresponding author. Phone: +49-30-31426429. Fax: +49-30-31423850. E-mail: [email protected]. (1) Reemtsma, T. Trends Anal. Chem. 2001, 20, 500-517.

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to mass spectrometry via GC/MS.2 The first paper providing an impression of what ESI-MS can offer was published by McIntyre et al. in 1997.3 In that paper, a triple-quadrupole mass spectrometer was used for analyzing a mixture of humic and fulvic acids from groundwater. Since then, considerable progress has been made concerning resolution and structure analysis. Because of the complexity of fulvic and humic acid mixtures, higher mass resolution than that provided by the most commonly used quadrupole and ion-trap instruments proved helpful. Timeof-flight (TOF) was a step forward,4 but only with Fourier transform ion cyclotron resonance (FTICR)-MS could isobaric mixtures be fully resolved.5,6 Moreover, FTICR-MS allowed the determination of elemental compositions.6-8 But this instrumentation is extremely expensive and scarce and does not seem to be suited for coupling with liquid chromatography. ESI-MS is a soft ionization technique, and the mass spectra obtained for fulvic and humic acids show a quasi-continuous series of m/z signals in either positive or negative mode. Hence, structural information provided by ESI coupled to low resolution MS is very limited. Triple-quadrupole MS3, ion-traps9 and quadrupole-TOF (Q-TOF) MS4 have been used to study fragmentation of fulvic and humic acid anions. Astonishingly, chromatography has only rarely been used to separate and deliver fulvic or humic acids to ESI-MS; instead, samples were introduced by infusion. Given the well-known complexity of fulvic and humic acid mixtures, this is difficult to understand. Size-exclusion chromatography (SEC) is a well(2) Hayes, M. H. B.; MacCarthy, P.; Malcolm, R. L.; Swift, R. S. In Humic Substances II - In Search of Structure; Hayes, M. H. B., MacCarthy, P., Malcolm, R. L., Swift, R. S., Eds.; Wiley: Chicester, 1989, Chapter 24, pp 689-733. (3) McIntyre, C.; Batts, B. D.; Jardine, D. R. J. Mass Spectrom. 1997, 32, 328330. (4) Plancque, G.; Amekraz, B.; Moulin, V.; Toulhoat, P.; Moulin, C. Rapid Commun. Mass Spectrom. 2001, 15, 827-835. (5) Kujawinski, E. B.; Hatcher, P. G.; Freitas, M. A. Anal. Chem. 2002, 74, 413-419. (6) Stenson, A. C.; Landing, W. M.; Marshall, A. G.; Cooper, W. T. Anal. Chem. 2002, 74, 4397-4409. (7) Llewelyn, J. M.; Landing, W. M.; Marshall, A. G.; Cooper, W. T. Anal. Chem. 2002, 74, 600-606. (8) Kujawinski, E. B.; Freitas, M. A.; Zang, X.; Hatcher, P. G.; Green-Church, K. B.; Jones, R. B. Org. Geochem. 2002, 33, 171-180. (9) Leenheer, J. A.; Rostad, C. E.; Gates, P. M.; Furlong, E. T.; Ferrer, I. Anal. Chem. 2001, 73, 1461-1471. 10.1021/ac0261294 CCC: $25.00

© 2003 American Chemical Society Published on Web 02/13/2003

established separation method for these analytes,10 and it has been extensively used to characterize humic substances, traditionally by using UV detection,11 and by an on-line detection of dissolved organic carbon (DOC).12-14 SEC is also an important technique for the off-line fractionation of humic and fulvic acids, for example, for NMR analyses.15 Coupling of SEC with ESI-MS was expected to offer several advantages: (a) A separation of analytes according to their hydrodynamic volume adds an additional dimension to the mass spectrometric analysis and increases the amount of information provided by the analysis. (b) The chromatographic separation reduces the competition for the electrical charges during the formation of gas-phase ions in the electrospray interface and improves the ionization of analytes. This can be helpful for those fractions that are more difficult to ionize, as it was expected for the high molecular weight fraction of fulvic and humic acids.6 (c) A separation of inorganic sample constituents from the organic compounds should again increase the ionization efficacy. Moreover, it reduces the risk of adduct formation and, thus, decreases the complexity of the spectra obtained. Adduct formation is not always easily recognized. Despite these expected benefits, SEC has rarely been coupled to ESI-MS for analyzing fulvic and humic acids and, indeed, only little progress has been achieved as compared to the infusion approach.16,17 Nevertheless, we decided to improve SEC coupling to MS and to use the SEC-MS to obtain insight into the complexity and into the structure of aquatic humic and fulvic acids. Scan analyses and fragmentation of molecular anions in the MS/MS mode should provide novel insight into the structural information on building blocks of these oligomeric materials that help to elucidate the structure of these mysterious materials. MATERIALS AND METHODS Reference Materials. All studies were performed with Suwannee River fulvic acid (SRFA) and humic acid (SRHA) standards from the International Humic Substances Society (IHSS). Standard solutions of 1 g/L (scan analyses) or 2 g/L (MS/MS experiments) were freshly prepared in ultrapure water before use. Polystyrene sulfonate standards (sodium salts) were from Polymer Standards Service (Mainz, Germany). Instrumentation and Mass Spectrometry. The SEC was performed on a HP 1100 (Hewlett-Packard) liquid chromatography system with a diode array detector that was coupled to a Quattro LC triple-quadrupole mass spectrometer (Micromass, Manchester, (10) Miles, C. J.; Brezonik, P. L. J. Chromatogr. 1983, 259, 499-503. (11) Chin, Y.-P.; Aiken, G.; O’Loughlin, E. Environ. Sci. Technol. 1994, 26, 18531858. (12) Gloor, R.; Leidner, H.; Wuhrmann, W.; Fleischmann, T. Water Res. 1981, 15, 457-462. (13) Huber, S. A.; Frimmel, F. H. Environ. Sci. Technol. 1994, 28, 1194-1197. (14) Her, N.; Amy, G.; Foss, D.; Cho, J.; Yoon, Y.; Kosenka, P. Environ. Sci. Technol. 2002, 36, 1069-1076. (15) Piccolo, A.; Conte, P.; Trivellone, E.; van Lagen, B.; Buurman, P. Environ. Sci. Technol. 2002, 36, 76-84. (16) Persson, L.; Alsberg, T.; Kiss, G.; Odham, G. Rapid Commun. Mass Spectrom. 2000, 14, 286-292. (17) Pfeifer, T.; Klaus, U.; Hoffmann, R.; Spiteller, M. J. Chromatogr., A 2001, 926, 151-159.

U.K.) with a Z-Spray electrospray ionization source. The drying gas (N2) flow was 850 L/h, and the nebulizer gas (N2) flow rate was 90 L/h. The probe temperature was held at 250 °C, and the ion source temperature, at 120 °C. A capillary voltage of 2.85 kV was used, and the cone voltage was set to 25 V unless stated otherwise. The MS/MS experiments were performed with argon as collision gas at a pressure of 1.3 × 10-3 bar and a collision energy of 20 eV. The mass spectrometer was operated in the negative ion mode with a data collection rate of 16 points/Da. Centroid data were routinely stored, and the m/z- range 150750 scanned in 2.3 s. For the analysis of the high molecular weight (HMW) fractions, three separate analyses were performed with the ranges m/z 100-1100, 1000-2000, and 2000-3000 scanned in 2.3 s. Continuum data were collected from m/z 200 to 300 in 2.75 s. All product ion spectra were recorded in continuum mode. Size-Exclusion Chromatography. A PL Aquagel-OH 30 SEC column from Polymer Laboratories (Shropshire, U.K) of 250 × 4.6-mm i.d. and a particular size of 8 µm was used. The column had a nominal molecular weight range of 100-30 000 U. Separation was performed at a flow rate of 0.3 mL/min with an eluent consisting of 80/20 (v/v) water/methanol mixture with 10 mM NH4HCO3 at a column temperature of 40 °C. Data Treatment. The mass spectra used for interpretation and for the calculations were summed over a retention time window of ∼0.5 min. For molecular weight calculations, these mass spectral data were transferred from the mass spectrometer software Masslynx 3.5 (Micromass) to a spreadsheet program. The background noise was determined from a baseline spectrum over the same mass range for a time period of 0.5 min. The average background intensity over a m/z range of 100 was determined and subtracted from the intensity data of the analyte spectrum. This was repeated for the whole analyte spectrum. Weight-average and number-average molecular weights were then calculated from the background-subtracted intensity data according to the established formulas (e.g., ref 18). RESULTS AND DISCUSSION Eluent Composition. For coupling of SEC to ESI-MS, eluents have to be completely volatile, including the inorganic additives that are added to reduce electrostatic interactions between analytes and the polymeric phase. Phosphate buffers that are routinely used in SEC of dissolved organic matter cannot be used. Furthermore, columns of a smaller volume are preferable, since flow rates directed to the electrospray interface are usually lower (e.g., 0.3 mL/min) than those used in conventional SEC with UV or DOC detection.13,14 Different volatile eluents were tested with SR fulvic acid, and eluent composition turned out to influence both the SEC separation and the ESI-MS detection (Figure 1). Concerning chromatographic separation, pure water is insufficient (Figure 1a). Adding formiate as an inorganic additive to reduce ionic interactions with the column material and some methanol to reduce hydrophobic interactions improves the separation (Figure 1b), but better results were achieved with bicarbonate as the electrolyte (Figure 1c). If no methanol was used as organic modifier, elution times increased, which may indicate retardation due to hydrophobic interactions with the column material (Figure 1d). (18) Lesec, J. J. Liquid Chromatogr. 1985, 8, 875-923.

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Figure 2. Semilog plot of the molecular weights of polystyrene sulfonate standards versus their retention time in SEC. The numbers denote the average molecular weights (Mw) of some of the standards. (SEC eluent, 10 mM ammonium bicarbonate in water/methanol (80/20)).

Figure 1. Total ion chromatogram (TIC, range m/z 150-750) of Suwannee River fulvic acid obtained by SEC-ESI-MS in the negative mode with different eluents: (a) pure water, (b) ammonium formiate (10 mM) in water/methanol (80/20), (c) ammonium bicarbonate (10 mM) in water/methanol (80/20), and (d) ammonium bicarbonate (10 mM) in water.

Bicarbonate was also advantageous with respect to the MS detection, since the sensitivity was 5-10-fold higher than with formiate additive. A basic pH, as with the bicarbonate eluent (pH 8.2), is preferable for the detection of anions in the negative ion mode, whereas an acidic pH (pH 6.5 for the formiate eluent) would be favored for the detection of cations in the positive ion mode. Most organic matter dissolved in water is acidic and occurs in anionic form, namely fulvic and humic acids. The separation by SEC was evaluated by analyzing a series of polystyrene sulfonate standards (Figure 2). A fairly linear calibration curve for the log Mw versus the retention time (Rt) was obtained from 1200 to 15 000 amu. Contrary to many SEC applications in which SEC retention times are used to calculate molecular weights, a linear interrelationship between log MW and Rt is not essential here, because SEC is used for separation purposes only, and detection is performed by mass spectrometry. Rather, the nonlinear range from 1000 down to 200 amu is especially useful, because it provides a high chromatographic resolution in this molecular weight range. SEC Separation and MS Detection. The TIC chromatogram of the fulvic acids shows three distinct peaks with maximums at 7.7, 8.1, and 8.9 min (Figure 3b). Unfortunately, this separation cannot be compared with previous work on SEC-MS, because SRFA have not been analyzed by these authors.16,17 The chromatograms of fulvic acid obtained by SEC with either UV or MS detection differ strongly. In comparison to UV254 detection, ESIMS favors the late eluting compounds. This effect is even more 1502 Analytical Chemistry, Vol. 75, No. 6, March 15, 2003

pronounced for the humic acids analyzed under the same conditions (Figure 3c,d). Two effects may have contributed to this significant difference between the UV and the MS signals: (i) Not all components of the two reference materials show the same molar absorptivity, but it is likely to change over the chromatographic run. For example, the early eluting material (Rt 7.7 min) exhibited light absorbance up to 580 nm, whereas the latest peak (Rt 8.9 min) absorbed up to 270 nm only. It has previously been shown that HMW compounds tend to be more aromatic and to exhibit a higher molar absorptivity.11 (ii) More importantly, recent results concerning the analysis of fulvic acids by ESI-MS suggest that ESI shows a bias in favor of low-molecular-weight (LMW) compounds.6 Moreover, the mass specrometer used in this study had a nominal mass range of 4000 amu, and it is well-known that the sensitivity of quadrupole systems decreases in the high-mass region. Since HMW compounds elute before LMW compounds in SEC, SEC-ESI-MS is expected to preferentially detect late eluting substances. Doubling the cone voltage from 20 to 40 V had no significant effect on the TIC in single-MS mode. This may be due to some counterbalancing effects, because a higher CV favors the acceleration of heavier ions toward the sample cone and their introduction into the mass spectrometer, while it also induces “in-source” fragmentation of HMW, leading to a decreasing signal intensity for this group. This is hardly visible in the scan spectra of the HMW fraction, but the effect of CV becomes more obvious when more selective MS/MS experiments are performed (see below). Mass Spectra Obtained by SEC-ESI-MS. The average scan spectra of the three peaks in the TIC of the fulvic and humic acids are given in Figure 4. They outline that SEC clearly separated the fulvic acid mixture into signals with different mass spectra. According to the separation mechanism of SEC, the first eluting peaks were formed by compounds of higher molecular weight (Figure 4a,d). The late eluting peaks were due to LMW compounds with exponentially decreasing signal intensities with increasing m/z ratios in the respective mass spectra (Figure 4c,f). A striking feature of the HMW spectra of both the fulvic and the humic acids (Figure 4a,d) was the wavy pattern of the signal intensity, with maximums in the range of m/z 200, 550, and 950. The humic acids exhibited a fourth maximum around m/z 1500 (Figure 4d). Many ESI-MS spectra of fulvic acids found in the

Figure 3. SEC chromatograms obtained with UV detection at 254 nm (a,c) and ESI-MS detection from m/z 150 to m/z 750 (b,d): (left) fulvic acid and (right) humic acid (SEC eluent, 10 mM ammonium bicarbonate in water/methanol (80/20)).

Figure 4. ESI-mass spectra obtained for fulvic acid (a-c) and humic acid (d-f) of all three chromatographic signals: (a,d) first SEC peak, (b,e) second SEC peak, (c,f) third SEC peak. Spectra a and d were combined from two scan analyses each (m/z 100-1100 and 10002000).

literature do not show a similar pattern, but a monomodal distribution of ion intensities. Some recently published spectra

for fulvic acids of other origin by Q-TOF in the negative ion mode4 and of SRFA with FTICR in the positive ion mode6 show a bimodal intensity pattern. Owing to the SEC separation, one can assume that the signals in the low m/z range of the HMW spectra (Figure 4c,f) are due to fragment ions generated by in-source fragmentation of the HMW compounds. And the nonstatistical frequency distribution (neither Poisson nor Gaussian) in these spectra indicates that the fragmentation of HMW compounds occurs at preferential sites of the HMW molecules. A comparison of the spectra series from the LMW to the HMW fraction of SRFA (Figure 4a-c) and SRHA (Figure 4d-f) further suggests that the larger molecules fragment along the LMW subunits of which they were possibly formed. For two reasons, these wavy spectra of the HMW fraction have not been determined before: (i) Using infusion-ESI-MS, HMW compounds were analyzed together with LMW compounds, and the ions in the lower m/z range of the HMW spectra were superimposed by the more prominent molecular anions of the LMW compounds, and (ii) the parallel introduction of HMW and LMW compounds into the electrospray interface likely suppressed the ionization of the HMW compounds. Thus, Figure 4 clearly demonstrates the benefit of using SEC-MS fur humic acid analysis. Not only the coarse but also the fine structure of the mass spectra changed from peak to peak (Figure 5). The latest chromatographic peak of the fulvic acids (Figure 5a) exhibits the pattern previously described for fulvic acids as a whole: each nominal mass is present, with a strong prevalence of the odd anion masses (M - H-) that belong to molecules with an even molecular weight. Using infusion FTICR mass spectrometry with an ultrahigh resolving power, it has been shown that each of these unit mass signals is formed by a considerable number of compounds with different exact masses.5,6,8 With a SEC separation preceding the MS detection, the signals in the SEC-MS spectra can be expected to be considerably less complex. The occurrence of a 2-amu spacing has been ascribed to varying degrees of unsaturation in the respective molecules,3 and exact mass determinations supported this interpretation.6,19 Though each nominal mass is present, intensity maximums occur at a typical distance of 14 amu3. This can be attributed to a homologous series differing in their number of methylene groups, as confirmed by FTICR-MS.6 (19) Brown, T. L.; Rice, J. A. Anal. Chem. 2000, 72, 384-390.

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Figure 5. Extended ESI-MS spectra of the three chromatographic signals of fulvic acid (left) and humic acid (right): (a,d) last SEC peak, (b,e) second SEC peak, and (c,f) first SEC peak.

In the mass range selected in Figure 5 (m/z 234-272) the spectra of the earlier eluting fractions (Figure 5b, c) are less well structured. For the HMW fraction of fulvic acids, the intensity of the even masses comes close to the odd anion masses (Figure 5c). A similar trend is observed for the humic acids (Figure 5df). This points at the fact that these spectra show fragment ion distributions rather than molecular ion distributions, as in Figure 5a and d. Hydrogen shifts can occur during fragmentation, and this weakens the odd-to-even prevalence. However, the similarity of the fragment ion distributions (Figures 5c,f) with the LMW molecular anion distribution (Figure 5a,d) suggests that the HMW compounds are built from these LMW precursors. No indication for a significant portion of multiple charges was found in any of the spectra (Figure 5). Multiple charging of fulvic acids has previously been complained of (refs 9, 16, 17) and suspected as the reason for erroneous molecular weight determinations.9 More recent studies confirmed that multiple charging need not be significant.6 The ammonium ions in the SEC eluent used here may further reduce the formation of multiply charged anions, since ammonium can serve as a proton donor. On one hand, the absence of multiple charging is highly desirable, because this reduces the complexity of the spectra and eases their interpretation. On the other hand, this limits the molecular mass range of dissolved organics accessible to the ESI-MS analysis to the instrumentally provided m/z-range of the respective mass spectrometer. Calculation of Mn and Mw from Mass Spectra. Electrospray ionization mass spectrometry was initially assumed to provide direct and, thus, unequivocal information on the molecular weight distribution of fulvic and even humic acids. Indeed, ESI-MS can be used to calculate average molecular weights of polymer mixtures, for example, of poly(methyl methacrylate), provided that no fragmentation occurs in the interface and that the response factor of all oligomers is known or identical.20 As discussed above, the first prerequisite is, however, not met in the case of fulvic (20) Simonsick, C. N.; McEwen, W. J.; Larsen, B. S.; Ute, K.; Hatada, K. J. Am Soc. Mass Spectrom. 1995, 6, 906-911.

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Table 1. Number Average (Mn) and Weight Average (Mw) Molecular Weight and Polydispersity (Mw/Mn) of the Three Fractions of Fulvic and Humic Acids as Determined by SEC-ESI-MS peak no. 1 2 3 1 2 3

RT window

Mw

Mw/Mn

SR Fulvic Acid 6.80-7.25 720 7.25-7.70 480 7.70-8.15 320 8.50-8.95 250

1150 770 440 280

1.6 1.6 1.4 1.1

SR Humic Acid 6.75-7.20 930 7.20-7.70 830 7.70-8.30 270 8.75-9.20 180

1680 1100 350 200

1.8 1.3 1.3 1.1

Mn

and humic acid analyses by ESI-MS, and in addition, the second one cannot be fulfilled because pure standards are impossible to obtain. Nevertheless, spectra generated by infusion of fulvic acids into ESI-MS have been repeatedly used for the calculation of number average (Mn) and weight average (Mw) molecular weights.9,17,19 Despite the existing doubts on the validity of such calculations, the same approach was followed here with the intention to compare the results obtained by SEC-ESI-MS with those of infusion-ESI-MS. When calculating average molecular weights from averaged mass spectra generated by SEC-MS (Figure 4), it became clear that the way electronic and chemical noise in the mass spectra was treated substantially influenced the average molecular mass calculation. Differences of 20% between the lowest and the highest calculated value can be easily obtained from the same spectrum. Apparently, this aspect was not adequately addressed in previous Mn and Mw calculations. For the molecular weight calculations, the chromatograms (Figure 3) were separated into four sections (Table 1). The first SEC peak (Figure 1) was split into two sections of 0.5 min to keep a uniform width of data accumulation. Mn and Mw values decreased and converged with increasing elution time for both the fulvic

and the humic acids (Table 1), leading to a decreasing polydispersity with decreasing molecular weight. The results compiled in Table 1 outline that the material underlying the first SEC-MS signal is not uniform, but shows a significant decrease in its average molecular weight with increasing retention time. In general, the decrease in the calculated average molecular weight with increasing retention time confirms that a separation according to the molecular weight of the SRFA and SRHA was obtained by the SEC separation preceding the MS analysis. As a result of the chromatographic separation and the distinction and elimination of noise, these data are unique and cannot be compared directly with previous data obtained by infusionESI-MS in the negative mode. As one could expect, the LMW fraction provided lower and the more abundant HMW fraction higher average molecular weights than were obtained by infusionESI-MS analyses, n which Mn values of 591 and 699 and Mw values of 914 and 714 have been calculated for SRFA.9,19 For the SRHA, no ESI-MS data have previously been reported. For the reasons given above, molecular weight data obtained by ESI-MS have to be used with great care because they underestimate HMW compounds. Previous discrepancies of infusion-ESI-MS data with other detection methods have been attributed to calibration problems in SEC or aggregate formation in vapor pressure osmometry21 or to fragmentation3, multiple charging,9,17 or selective ionization in ESI-MS.6 Despite all of these discrepancies and the limited validity of absolute Mn and Mw values derived from (SEC-) ESI-MS spectra, such data can be used to determine relative differences between samples and to detect trends as long as all the samples are analyzed under identical experimental conditions. Structure Analysis by Tandem Mass Spectrometry. Tandem mass spectrometers, either triple-quads,3 Q-TOFs,4 or iontrap mass spectrometers,9 can be used to fragment molecular ions of fulvic and humic acids and to obtain insight into their structure. Triple-quadrupole mass spectrometers allow three kinds of experiments to be performed that help to elucidate structural characteristics of fulvic and humic acids. However, an unequivocal assignment of molecular formulas to the fragment ions is not attained with quadrupole and ion-trap mass spectrometers because of their limited resolution power. Product Ion Spectra. Figure 6a shows the product ion spectrum of m/z 267, an intense signal in the scan spectra of the second and third peak of the fulvic acid (Figure 5). Three consecutive losses of carbon dioxide from carboxylate moieties are seen (-44 amu), and parallel losses of water (-18 amu). Elimination of carbon monoxide (-28 amu) was also detected once. The very same fragmentation sequence determined here for the SR fulvic acids, multiple decarboxylation and parallel dehydration, has previously been determined for fulvic acids of different origin3,4,9,17. Starting with molecular anions of m/z 339 and 371, a total of four decarboxylation steps has been determined with either a quadrupole MS or a Q-TOF,3,17 and up to five losses of CO2 have been detected from m/z 537 of SRFA by an ion-trap MS.9 As outlined above, the mass resolution of the quadrupole MS used here as well as of the ion-trap used elsewhere is insufficient to resolve the isobaric compounds at a given m/z value. (21) Novotny, F. J.; Rice, J. A.; Weil, D. A. Environ. Sci. Technol. 1995, 29, 24642466.

Figure 6. Product ion spectra obtained by SEC-ESI-MS/MS of selected anions of the fulvic acid LMW fraction of m/z (a) 267, (b) 265, (c) 269, and (d) 281.

Thus, the product ion spectra obtained with a triple quadrupole MS or with an ion-trap MS may record product ions of all isobaric precursors. However, it is reasonable to assume that the SEC separation reduced the number of isobaric interferences because it separated fragments of HMW compounds from molecular anions of LMW compounds (see above). After the decarboxylations, the fragmentation sequence of the SRFA continued through some losses of 12-14 amu, probably alkyl chain fragments, and ended with ions in the range of m/z 103-109. These final low-mass ions may represent the defunctionalized skeleton (the core) of these fulvic acid molecules. These results coincide with others, who have reported either m/z 105 or 109 as final fragment ions of fulvic acid that are not amenable to further fragmentation.4,9 Besides m/z 267, the two adjacent signals (m/z 265 and 269; Figure 6b,c) were also investigated to determine their structural difference as compared to m/z 267. The fragmentation of both these anions is identical to that of m/z 267, leading to fragment ions 2 amu below (from m/z 265) or above (from m/z 269) those Analytical Chemistry, Vol. 75, No. 6, March 15, 2003

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of m/z 267. If the 2 amu spacing is due to a degree of saturation different from these, double bonds must be located in the core/ skeleton of the molecule. Otherwise, these unsaturations would be expected to induce different fragmentation reactions. The next alkyl chain homologue of m/z 267, the m/z 281 anion, showed the same fragmentations. In this case, all fragments determined have a mass shift of 14 amu, as compared to the m/z 267 fragments (Figure 6d). Thus, a very regular composition of the fulvic acid molecules is reflected in these product ion spectra. Interestingly, an anion of m/z 265 has been detected as a fragment ion of m/z 371 by Plancque and co-workers,4 and all the fragments of m/z 265 given in Figure 6b, namely m/z 221, 203, 177, and 133, have also been observed. On the basis of fragmentation studies of different molecular anions with the same fragmentations occurring and, hence, slightly different fragment ions being produced, two different structures were recently proposed for this fraction of fulvic acids.4,9 Reviewing the published data and combining them with those obtained by the new SECMS/MS method clarifies that all these data perfectly agree. This is quite astonishing because it implies that these components of aquatic fulvic acid from very different sources, from various groundwaters,3,4 from the SuwanneeRiver (ref 9, this paper) and from another surface water,17 not only belong to the same class of compounds, but also exhibit the same regular composition of the individual molecules. The exact structure of these polycarboxylated components, however, will remain a matter of debate. Further effort is required to clarify the structural basis of the repeated 2-amu spacings in the scan spectra of this fraction. The 14-amu spacing has been shown to be due to a homologous series differing in the number of methylene groups in an alkyl chain.6 If all 2-amu spacings between these 14 amu would be attributed to different degrees of unsaturation, then the structure of each alkyl homologue must allow accomodation of up to six double bonds. According to the fragmentations determined in the daughter ion spectra (Figure 6), these double bonds would have to be situated in the core of the molecule that has a total mass of around 100 amu only. This seems unlikely. FTICR-MS may help to identify the source of the 2-amu spacings. Product ion spectra of HMW components are more difficult to obtain, because this material is detected less sensitively. Nevertheless, the fragment ion signals determined from the anions m/z 507 and 511 of the HMW fraction of fulvic acids could be easily identified as decarboxylation and dehydration reactions and correspond to the fragmentations of the LMW fraction. This supports the previous suggestion that the HMW fraction (first SEC peak) consists of a number of subunits of the LMW fraction (third SEC peak). Neutral Loss Spectra. As outlined above, the expulsion of several CO2 molecules is the major fragmentation reaction observed for the well-ionizable fraction of fulvic acids. Performing neutral loss scans for 44, 88, and 132 amu selectively detects fulvic acid components that can eliminate one, two, and three CO2 molecules. Using model compounds, decarboxylation was obtained from aromatic carboxylic acids, but not with simple aliphatic or alicyclic carboxylic acids. A selective detection of (aromatic) carboxylates by these neutral losses indicated that each unit mass bears this functional 1506 Analytical Chemistry, Vol. 75, No. 6, March 15, 2003

Figure 7. ESI-MS spectra of fulvic acid: (a) scan spectrum, (b) spectrum of the neutral loss experiment for 44 amu (CO2), (c) spectrum of the neutral loss experiment for 88 amu (2 × CO2).

Figure 8. of 88 amu coltage 20 ammonium

Total ion chromatograms for the neutral loss experiment detected by SEC-ESI-MS/MS of fulvic acid: (a) cone V and (b) cone voltage 40 V (SEC eluent, 10 mM bicarbonate in water/methanol (80/20)).

group (Figure 7). Neutral loss scans, thus, support the results of the product ion scans, in which this could be shown for only some of the molecular anions detected in the single-MS mode. The chromatograms obtained by monitoring the neutral loss of 88 amu (Figure 8a) confirm that decarboxylation is important for the second and third SEC-fraction of the fulvic acid. Since neutral loss scanning is the least sensitive detection mode, the first fraction is only weakly detected. Performing neutral loss scans at different CV provided insight into fragmentation processes that can occur inside the electrospray interface (in-source fragmentation). Increasing the CV from 20 to 40 V increased the signal intensity at the front of the chromatogram and shifted the intensity maximum to shorter elution times (Figure 8b; 7.9 min). Obviously, the HMW fraction is now more effectively introduced into the MS. However, the spectrum obtained at the elevated CV for this increased peak did

not show increasing intensities for higher m/z ratios, as compared to the 20 V situation. This indicates that an elevated CV supported transfer of HMW compounds into the MS but induced fragmentation of these HMW compounds in parallel manner. This is in agreement with results obtained for model carboxylic acids that all exhibited in-source decarboxylation at elevated CVs (not shown). CONCLUSIONS After appropriate adaptation, SEC adds a new dimension to the ESI-MS analysis of fulvic and humic acids. The use of ESIMS for analyzing fulvic and humic acids had two major aims, the determination of average molecular weights and structure analysis of these materials. For several reasons, among them in-source fragmentation of HMW compounds and lack of knowledge of response factors for individual humic acid molecules, molecular weight calculations based on ESI-MS must be erroneous with a tendency to underestimate HMW compounds. Nevertheless, such calculations may help to characterize the ion intensity distribution of a given scan spectrum and to lump it into one figure. Moreover, ESI-MS proved helpful for structure analysis when coupled to SEC separation. Previous infusion-ESI-MS analyses had to face the whole fulvic acid mixture and detected its LMW fraction preferentially. The SEC separation allows distinguishing between

three fractions of fulvic and humic acids, from LMW to HMW compounds. Scan spectra made homologous series (+2 amu, +14 amu) in the LMW mixture visible. A nonstatistical frequency distributions occurs in the scan spectra of the HMW fraction and suggests that the HMW components are formed by a number of LMW subunits. The fragmentations, primarily decarboxylations and parallel dehydration, observed in the product ion spectra of the LMW fraction agreed with those previously reported for whole fulvic acid mixtures and suggest that this fraction consists of well-defined ubiquitous polycarboxylated compounds. SEC-ESI-MS/MS may become a helpful tool that complements existing methods of structure analysis of dissolved organic matter. It may be especially useful for detecting chemical alterations in the course of water treatment processes. ACKNOWLEDGMENT We gratefully acknowledge funding by the German Research Council (DFG, Bonn), RE1290/4-1.

Received for review September 12, 2002. Accepted January 14, 2003. AC0261294

Analytical Chemistry, Vol. 75, No. 6, March 15, 2003

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