High-Resolution Fourier Transform Ion Cyclotron Resonance Mass

This work was supported by the St. Johns River (FL) Water Management District, ... FT-ICR Mass Spectrometry Facility (CHE-94-13008) at the National Hi...
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Energy & Fuels 1997, 11, 554-560

High-Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry of Humic and Fulvic Acids by Laser Desorption/Ionization and Electrospray Ionization Anne Fievre,† Touradj Solouki,‡ Alan G. Marshall,†,‡ and William T. Cooper*,† Department of Chemistry, Florida State University, Tallahassee, Florida 32306-3006 and Center for Interdisciplinary Magnetic Resonance, National High Magnetic Field Laboratory, Florida State University, 1800 Paul Dirac Drive, Tallahassee, Florida 32310 Received January 3, 1997. Revised Manuscript Received March 19, 1997X

High-field (9.4 T) Fourier transform ion cyclotron resonance (FT-ICR) mass spectra of standard Suwannee River humic and fulvic acids have been obtained by use of laser desorption (LDI) and electrospray (ESI) ionization. The LDI FT-ICR mass spectrum was similar to those observed previously, with ions at essentially every nominal value, 200 e m/z e 800. In contrast, the ESI FT-ICR mass spectrum, although still containing ions at most values in the 200 e m/z e 800 range, was dominated by a relatively few prominent species. ESI FT-ICR mass spectra of standard humic and fulvic acid isolates were similar. Although many ionic species appeared in both fulvic acid and humic acid ESI FT-ICR mass spectra, the fulvic acid mass spectrum contained more highly charged species. Subfractions of the fulvic acid mixture isolated by an HPLC procedure yielded similar mass spectra. The stability of high-mass ions produced by ESI combined with the high-mass resolution capability of FT-ICR MS allow for precise determination of molecular masses, from which molecular formulas may be obtained by mass alone. Future two-dimensional FT-ICR MS2 determinations of humic and fulvic acid structures should be feasible by use of collisionally induced and multiple-photon dissociation techniques.

Introduction Humic Substances. Humic substances are ubiquitous in virtually all terrestrial and estuarine environments and comprise between 50 and 80% of the dissolved organic matter (DOM) in surface waters.1 These geomacromolecular compounds are amorphous, acidic substances of molecular weights estimated to range from several hundred to tens of thousands. The ability of naturally-occurring organic matter in general, and humic substances in particular, to absorb, bind, and/or complex environmentally-significant substances such as pesticides, polychlorinated biphenyls, polyaromatic hydrocarbons, and other nonpolar organics has been well documented.2 Complexation of heavy metals by humics has also been reported. The effects of binding to organic matter on the bioavailability of chemicals is a particularly interesting but poorly understood phenomenon. Several studies have suggested that such binding reduces bioavailability.3-5 At pH 1, the insoluble com†

Department of Chemistry. National High Magnetic Field Laboratory. Abstract published in Advance ACS Abstracts, May 1, 1997. (1) Malcolm, R. L.; Aiken, G. R.; Bowles, E. C.; Malcolm, J. D. In Humic Substances in the Suwanee River Georgia: Interactions, Properties, and Proposed Structures; Averett, R. C., Leenheer, J. A., McKnight, D. A., Thorn, K. A., Eds.; U.S. Geological Survey, 1989; pp 2326. (2) Pignatello, J. J.; Boashan, X. Environ. Sci. Technol. 1996, 30, 1-11. (3) Carlberg, G. E.; Martinsen, K.; Kringstad, A.; Gjessing, E.; Grande, M.; Kallqvist, T.; Skare, J. U. Arch. Environ. Contam. Toxicol. 1986, 15, 543-548. (4) Kukkonen, J.; Oikari, S.; Johnsen, E.; Gjessing, E. Sci. Total Environ. 1989, 79, 197-207. ‡

ponent of humic substances is known as humic acid and the soluble component is called fulvic acid. In spite of advances in understanding the extent to which organic matter in aquatic systems influences the bioavailability and geochemical behavior of chemical contaminants, little is known about the relationship between the composition of humic substances and their chemical and biological reactivity. It is known that humics produced in different environments have different biogeochemical reactivity, and these differences can be related to some extent to gross structural differences (e.g., aromaticity, carboxyl content, etc.). However, up to this point, analytical techniques capable of correlating biogeochemical reactivity with specific geomacromolecular structures in humic/fulvic acid mixtures have not been routinely available. Mass Spectrometry of Humic Substances. Mass spectrometry has been used extensively in the study of humics, primarily as a means of obtaining information about fragments after chemical6,7 or thermal8,9 degradation. Of particular interest were studies using timeresolved pyrolysis-field ionization (PFI) coupled to high-

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(5) Landrum, P. F.; Reinhold, M. D.; Nihart, S. R.; Eadie, B. J. Environ. Toxicol. Chem. 1985, 4, 459-467. (6) Sedlacek, J.; Kallqvist, T.; Gjessing, E. In Aquatic and Terrestrial Humic Material; Christman, R. F., Gjessing, E. T., Ed.; Ann Arbor Science: Ann Arbor, MI, 1983; pp 495-516. (7) Aiken, G. R.; McKnight, D. M.; Wershaw, R. L.; MacCarthy, P. Humics Substances in Soil, Sediment and Water; Malcolm, R. L., Ed.; Wiley Interscience: New York, 1985; pp 181-209. (8) Saiz-Jiminez, C.; de Leeuw, J. W. J. Anal. Appl. Pyrolysis 1986, 9, 99-119. (9) Sorge, C.; Muller, R.; Leinweber, P.; Schulten, H.-R.; Fresenius, J. Anal. Chem. 1993, 346, 697-703.

© 1997 American Chemical Society

LDI and ESI FT-ICR MS of Humic and Fulvic Acids

resolution mass spectrometry (Py-FIMS).10 Those experiments provided direct molecular characterizations and confirmed degradation products previously identified by GC-MS experiments. Virtually all early mass spectrometric studies of humic substances were characterized by the extensive fragmentation produced by conventional electron impact ionization, leading to fragment ions at virtually every nominal mass below mass-to-charge ratio, m/z ∼ 200, and few if any fragment ions above m/z 200. (m is ion mass in daltons and z is the ion charge in multiples of the elementary charge.) “Soft” ionization techniques, such as fast atom bombardment (FAB),11 field desorption (FD),12 and in-source PFI,13 are therefore of interest. Field ionization studies were particularly promising,12 with intact fragments observed up to m/z 670. Laser desorption (LD) and matrix-assisted laser desorption/ionization (MALDI) techniques have also been used as soft ionization methods for humics and related compounds such as lignins.14,15 Combining LDI with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS),16-19 Srzic et al. observed from lignins negative ions that ranged from m/z of several hundred to above 3000.15 In addition, Novotny and Rice recently reported on the use of low-power LDI and FT-ICR MS to characterize the number-average (Mn) molecular weights of five fulvic acid samples.20 Their calculations were based on the assumption that all ions observed in the FT-ICR mass analyzer are intact, singly-charged humic ions, and not fragments. However, their mass spectral results did not agree with Mn values obtained by osmometry techniques, suggesting that even at the relatively low laser power used in that study, significant fragmentation of parent humic acid geomacromolecules still occurs. Detection of such large fragments raises the possibility of reconstructing the structure of entire humic molecules by mass analysis alone. In this initial presentation, we report the use of what we believe to be the “softest” ionization technique available for analysis of these macromolecules, namely, electrospray ionization (ESI). We compare LD FT-ICR MS at 3.0 T to ESI FT-ICR-MS at the highest available magnetic field to date (9.4 T).21 The high-field ESI FT-ICR MS technique yields ultrahigh resolution mass spectra,22 (10) Abbt-Braun, G.; Frimmel, F. H.; Schulten, H. R. Water Res. 1989, 23, 1579-1591. (11) Saleh, F. Y.; Chanz, D.; Frye, J. S. Anal. Chem. 1983, 55, 826. (12) Andreux, F.; Constantin, E.; Gioia, B.; Traldi, P. Org. Mass Spectrom. 1988, 23, 622-623. (13) Schulten, H.-R.; Schnitzer, M. Org. Geochem. 1993, 20, 1725. (14) Srzic, D.; Martinovic, S.; Pasa, L.; Kezele, N.; Shevchenko, S. M. Rapid. Commun. Mass Spectrom. 1995, 9, 245-247. (15) Srzic, D.; Martinovic, S.; Pasa-Tolic, L.; Kezele, N.; Kazazic, S.; Senkovic, L.; Shevchenko, S. M.; Klasinc, L. Rapid. Commun. Mass Spectrom. 1996, 10, 580-582. (16) Marshall, A. G. Acc. Chem. Res. 1985, 18, 316-322. (17) Buchanan, M. V.; Hettich, R. L. Anal. Chem. 1993, 65, 245A259A. (18) Wilkins, C. L. Trends in Analytical Chemistry 13, Special Issue: Fourier Transform Mass Spectrometry; Wilkins, C. L., Ed., 1994; pp 223-251. (19) McLafferty, F. W. Acc. Chem. Res. 1994, 27, 379-386. (20) Novotny, F. J.; Rice, J. A. Environ. Sci. Technol. 1995, 29, 2464. (21) Marshall, A. G.; Guan, S. Rapid Commun. Mass Spectrom. 1996, 10, 1819-1823. (22) Senko, M. W.; Hendrickson, C. L.; Pasa-Tolic, L.; Marto, J. A.; White, F. M.; Guan, S.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 1824-1828.

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from which chemical formulas of various species may be determined directly by accurate mass measurement. In addition, after stored waveform inverse Fourier transform (SWIFT)23,24 isolation of ions over very narrow (a few Da) mass ranges, MS/MS experiments based on collisionally induced dissociation (CID) and infrared multiple photon dissociation (IR/MPD) yield fragment ions that may be used for structural characterization of unknown molecules. Such tandem mass spectrometry experiments support the feasibility of determining the chemical formulas (and ultimately the structures) of primary and fragment ions, and lay the groundwork for future mass analysis of humic substances. Experimental Section Samples. Standard humic and fulvic acid, SRHA and SRFA, respectively, were supplied by the International Humic Substances Society (Golden, CO). They were isolated from the Suwannee River (GA), by the IHSS standard method for extraction and isolation of aquatic humic substances.1 SFRI is a Suwannee River (FL) fulvic acid collected and isolated by a similar procedure described in Standard Methods of Water and Wastewater.25 Electrospray FT-ICR Mass Spectrometry. ESI FT-ICR mass spectra were acquired with a homebuilt FT-ICR mass spectrometer equipped with a 9.4 T superconducting magnet, as described elsewhere.22 Briefly, ions are produced with an external electrospray source.26 The electrosprayed ions pass through a 1 mm diameter skimmer prior to their entrance into a first, 60 cm long, rf-only octupole ion guide. A second octupole, 200 cm in length, guides the ions into a 9.4 cm diameter cylindrical (∼30.4 cm long) open-ended three-section Penning trap.27,28 To increase the number of trapped ions inside the ICR cell, we allow ions to accumulate for about 10 s inside the first octupole prior to ion transfer into the second octupole. A Model 48-2 air-cooled carbon dioxide laser (SYNRAD, Bothell, WA), operated at a wavelength range of 10.55-10.65 µm and a maximum output of 40 W, was used for infrared multiphoton dissociation (IR/MPD) experiments. The CO2 laser is located outside the magnetic field and ∼135 cm away from the center of the ICR ion trap. The laser beam (beam diameter/divergence: 3.5 mm/4 mrad) is coaligned with the magnetic field and is directed through a flange-mounted barium fluoride (BaF2) window (BICRON, Solon, OH) on the ICR cell axis. A 30 cfm rotary pump (Varian, Lexington, MA) and three 1100 L/s hybrid turbo-drag pumps (Balzers, Hudson, NH) provide differential pumping of the vacuum system to maintain an operating base pressure of ∼2 × 10-8 Torr inside the ICR ion trap. Instrumental parameters are controlled by an Odyssey data system (Finnigan Corp., Madison, WI). Argon collision gas was introduced into the ICR chamber at ∼2 × 10-5 Torr, via a pulsed valve (General Valve, Fairfield, NJ). The fulvic and humic sample solutions were electrosprayed at a rate of 1-5 µL/min. Laser Desorption/Ionization Mass Spectrometry. Laser desorption/ionization (LDI) FT-ICR mass spectra were (23) Marshall, A. G.; Wang, T.-C. L.; Ricca, T. L. J. Am. Chem. Soc. 1985, 107, 7893-7897. (24) Guan, S.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1996, 137/138, 5-37. (25) Clesceri, L. S.; Greenberg, A. E.; Trussell, R. R.; Franson, M. A. Standard Methods for the Examination of Water and Waste Water; American Public Health Association: Washington, DC, 1989; Vol. 17, pp 5.37-5.41. (26) Chowdhury, S. K.; Katta, V.; Chait, B. T. Rapid Commun. Mass Spectrom. 1990, 4, 81-87. (27) Gabrielse, G.; Haarsma, L.; Rolston, S. L. Int. J. Mass Spectrom. Ion Processes 1989, 88, 319-332. (28) Beu, S. C.; Laude, D. A., Jr. Int. J. Mass Spectrom. Ion Processes 1992, 112, 215-230.

556 Energy & Fuels, Vol. 11, No. 3, 1997 acquired with an FTMS-2000 Fourier transform ion cyclotron resonance mass spectrometer (Finnigan Corp., Madison, WI) equipped with a 3 T superconducting magnet, dual cubic Penning traps, and an Odyssey data system. Laser desorption/ ionization was performed with a Nd:YAG laser (Model Surelite I-10, Continuum, Santa Clara, CA) operated at a wavelength of 1064 nm (fundamental output) with a pulse width of 7 ns. The laser beam is focused onto the probe tip by a 2:1 telescope (typical laser pulse energy was 1 mJ measured before the telescope, corresponding to an estimated laser power density of ∼2 × 107 W cm-2 on the probe tip). The laser beam is directed through a quartz window on the analyzer side of the main vacuum chamber, passing through a 2 mm diameter conductance limit to a spot size of ∼400 µm × 600 µm on the probe tip behind the source trap plate. The fulvic and humic sample solutions were applied to a thin stainless steel plate (sample probe tip) and air-dried. The sample probe tip was inserted into the vacuum chamber and mass analyzed. All LD FT-ICR mass spectra were acquired in the source compartment of the dual trap at a background pressure of ∼2 × 10-8 Torr. After the laser desorption/ionization event, ion z-axis translational energy was minimized by use of gated deceleration, as previously described.29,30 The trapped ions were excited by dipolar frequency sweep excitation (∼132 Vp-p amplitude, 1-500 kHz at a sweep rate of 1100 Hz/µs). Fourier transformation of the resulting discrete time-domain signal (32 K data, 1 MHz Nyquist bandwidth), without zero-filling, followed by Hamming apodization, magnitude calculation, and frequencyto-mass conversion yielded an LD FT-ICR mass spectrum. MSn Experiments. The experimental event sequences for successive FT-ICR MSn experiments are published elsewhere.30 Briefly, ESI-generated ions of initially high kinetic energy are decelerated and trapped. The trapped ions are allowed to relax axially to the center of the trap for several seconds. Ion cyclotron motions of the trapped ions are then excited by dipolar frequency-sweep irradiation, for which the sweep rate and radiofrequency voltage amplitude are optimized for each sample. Fourier transformation of the resulting discrete timedomain signal (32-256 K data, 1 MHz Nyquist bandwidth), without zero-filling and with Hamming apodization, followed by magnitude calculation and frequency-to-mass conversion, yields an FT-ICR parent ion mass spectrum. We used storedwaveform inverse Fourier transform (SWIFT) radial ejection to remove parent ions of all but a selected m/z ratio(s). In CID experiments, the mass-selected parent ions are then translationally excited to dissociate by means of collisional activation provided by sustained off-resonance irradiation (SORI)31 at ∼800 Hz below the reduced ion cyclotron frequency of the parent ion. In IR/MPD experiments,32 a CO2 laser beam, coaligned with the magnetic field and directed through a flange-mounted barium-fluoride (BaF2) window on the ion trap axis, dissociates the selected ions inside the ICR cell. The fragment ions are then excited by dipolar frequency sweep excitation (∼132 Vp-p amplitude, 1-500 kHz at a sweep rate of 700 Hz/µs). Fourier transformation of the resulting discrete time-domain signal (32-512 K data, 1 MHz Nyquist bandwidth), without zero-filling and with Hamming apodization, followed by magnitude calculation and frequency-to-mass conversion yields an MS2 FT-ICR product ion mass spectrum. Ultrahigh-Resolution Mass Spectra. A large number of parent ions can be generated and trapped by optimizing the ESI parameters. Following ion transfer to the ICR ion trap, the electrostatic trapping potential is lowered adiabatically to +0.6 V. A long time delay (5-50 s) before signal detection (29) Castoro, J. A.; Wilkins, C. L. Anal. Chem. 1993, 65, 2621-2627. (30) Solouki, T.; Pasa-Tolic, L.; Jackson, G. S.; Guan, S.; Marshall, A. G. Anal. Chem. 1996, 68, 3718-3725. (31) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Anal. Chim. Acta 1991, 246, 211-225. (32) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Cornnor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815.

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Figure 1. FT-ICR positive-ion mass spectra of Suwannee River Fulvic Acid (SFRI) based on (a) laser desorption/ ionization (LDI) at 3.0 T and (b) electrospray ionization (ESI) at 9.4 T. alleviates unwanted interactions between ion clouds with similar cyclotron frequencies.33 Conventional frequency-sweep excitation from 1 to 500 kHz at a 600 Hz/µs sweep rate is followed by heterodyne detection to yield 512 K time-domain data points. Each ultrahigh-resolution FT-ICR mass spectrum resulted from a single scan, based on direct FFT (and magnitude calculation) of the time-domain data. Singly and multiply charged ions of peptides (e.g., bradykinin, [D-Pen2,5]-enkephalin, LHRH) and proteins (e.g., bovine ubiquitin) were used as external and internal calibrants. In ultrahigh-resolution mass spectra, observed mass measurement accuracy with external calibration for all known peaks (at 0.6 V trapping voltage, over the range 500 e m/z e 1200) was within 5 ppm of values calculated from known chemical formulas; with internal calibrants the mass measurement accuracy improved to within 2 ppm of the values calculated from known chemical formulas. HPLC Fractionation. IHSS humic and fulvic acid solutions were prepared by dissolving 1 mg of dry standard in 10 mL of distilled, deionized water, producing a 100 ppm (w/v) standard solution. This solution was then fractionated on a high-performance liquid chromatography system consisting of a Beckman 506 autosampler, Toso Haas TSK-6010 pump and gradient controller, Toso Haas TSK-6041 UV detector operating at 245 nm, and a Hewlett-Packard 3394 integrator. Separations were carried out with an Alltech Absorbosphere Phenyl column, 5 µm particles, 25 cm × 4.6 mm i.d., with a mobile phase consisting of 50% methanol and 50% water (v/v) containing 1% acetic acid (v/v). This mobile phase mixture had a measured pH of 3.5 and was pumped at a flow rate of 0.5 mL/min.

Results and Discussion Comparison of Laser Desorption/Ionization and Electrospray Ionization. Figure 1 shows FT-ICR positive-ion mass spectra of Suwannee River (Florida) fulvic acid (SFRI) obtained by (a) laser desorption (LDI) and (b) electrospray (ESI) ionization. Clearly, these two ionization techniques produce significantly different mass spectra. The LDI mass spectrum is similar to those observed by others by LDI for humic substances (e.g., see ref 15), with ions at essentially every m/z value (33) Solouki, T.; Emmett, M. R.; Guan, S.; Marshall, A. G. Anal. Chem. 1997, 69, 1163-1168.

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Figure 3. ESI FT-ICR positive-ion mass spectra of (a) humic acid and (b) fulvic acid mixtures. Table 1. Structural Characterization Based on ESI FT-ICR Accurate Mass Measurement no. of possible chemical formulas within a given mass tolerance

Figure 2. ESI FT-ICR positive-ion mass spectra of fulvic acid based on a single time-domain data set, truncated to just the initial (a) 8K, (b) 16K, (c) 32K, and (d) 64K data before Hamming apodization and Fourier transformation/magnitude calculation.

in the range, 200 e m/z e 700. The ESI mass spectrum, however, while still containing peaks throughout the 200 < m/z < 800 range, is dominated by a relatively few prominent peaks. The higher molecular weight species in the ESI mass spectrum are not seen in the LD mass spectrum, suggesting that the higher-mass species are fragmented by the LD process. Thus, it is not safe to base molecular weight distributions of the parent neutrals on LD mass spectra. The ESI mass spectrum, on the other hand, holds out the possibility of determining unequivocally the structures of at least some of the individual compounds that make up a “geopolymer” mixture of humic substances. Low-energy electron impact and LDI spectra similar to that of Figure 1a have previously been used to compute number average molecular weights of complex organic geochemical assemblages such as fulvic acids20 and crude oils.34 However, such analysis is based on the assumption that all compounds in such mixtures have equal ionization efficiencies and that each peak represents a parent ion and not a product fragment ion. Given the wide variation in compound types found in such macromolecular mixtures and the complexities of ion formation, we doubt that any mass spectrum, including even those produced by “soft” ionization techniques such as ESI (e.g. Figure 1b) is quantitatively representative of the composition of a humic or fulvic acid mixture before ionization. A further difficulty with quantitation of a complex geomacromolecular mixture by mass spectrometry alone is demonstrated by the spectra of Figure 2. These (34) DeCanio, S. J.; Nero, V. P.; DeTar, M. M.; Storm, D. A. Fuel 1990, 69, 1233-1236.

observed ion

10 ppm

5 ppm

1 ppm

543.0931 Da 559.1371 Da 575.1023 Da

49 52 58

25 33 29

6 6 6

spectra represent the time evolution of the ESI spectrum shown in Figure 1b. All of these mass spectra were constructed from the same single scan data, by truncating the time-domain data after 8K, 16K, 32K, or 64K before Hamming apodization and FT/magnitude calculation. It is clear that during the data acquisition period, the charge states of some species shift (from doubly-to-singly-charged) and ion-molecule reactions change the identities and masses of some of the originally trapped ions. For example, the data truncated at 8K exhibit a prominent “hump” comprised of overlapping peaks in the 1000 e m/z e 1500 range. However, this high-mass hump virtually disappears in the spectrum truncated after 64K scans, and is completely removed in the 512K spectrum (Figure 1b). We infer that reactive, high-mass ions and/or fragmens are progressively lost through collisions and reactions with background neutrals. Thus, although the final mass spectrum obtained after averaging 512K scans includes various stable ions and/or fragments, it is not a direct representation of the initial chemical mixture. Comparison of Fulvic and Humic Acid Mixtures. Figure 3, a and b, shows ESI FT-ICR mass spectra of IHSS standard fulvic acid and humic acid mixtures, respectively. Electrospray conditions (e.g., pressure, spray voltage, solvent, etc.) were identical for both Figure 3, a and b). In contrast to spectra obtained with laser desorption ionization, both ESI FT-ICR mass spectra exhibit a relatively small number of prominent peaks. Mass-to-charge ratios of a selected series of these ions, under ultrahigh resolution conditions, are summarized in Table 1. Many of those ions appear in both fulvic and humic acid mixtures, suggesting a certain degree of molecular similarity between the two classes of compounds. This result is not unexpected, given the common origin of these macromolecules. Fulvic acid mixtures, because of their greater solubility in acidic solutions, are thought to be composed of smaller, more highly charged compounds than humic acid mixtures. The ESI results shown in Figure 3 are consistent with this view: the fulvic acid mass spectrum

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Figure 4. ESI FT-ICR positive-ion mass spectrum of fulvic acid mixture with bradykinin and LHRH added as internal standard mass markers (32K data points). The m/z scale expansions clearly show singly and doubly charged gas-phase ions, whose signals are spaced at integral and half-integral m/z values, respectively.

contains more highly charged species than does humic acid. For example, inspection of the data truncated at 32K show that electrospray produces singly and doubly charged ions (see Figure 4). It should be noted that the exact appearance of an ESI mass spectrum (e.g., variations in charge state relative abundances) varies with the electrospray conditions and solvent. Biomolecules such as peptides and proteins cospray well with fulvic acid solutions (1% by volume) and therefore are suitable as internal calibrants for accurate mass measurement. Figure 4 shows a low-resolution (32K time-domain data points) ESI FT-ICR positive-ion mass spectrum of a sample mixture consisting of fulvic acid compounds (HPLC purified) and small peptides. Bradykinin (monoisotopic mass ) 530.787 98 Da) and human luteinizing hormone releasing hormone (LHRH, monoisotopic mass ) 591.794 06 Da) doubly-charged ions are present in the mass spectrum. (The monoisotopic mass corresponds to the species for which all carbons are 12C, all hydrogens are 1H, all nitrogens are 14N, all oxygens are 16O, and all sulfurs are 32S.) Mass scale expansions (Figure 4, bottom) are shown for 760 < m/z < 780 and 1520 < m/z < 1540. Although massresolving power is not sufficient to resolve species of different chemical formula, singly- and doubly-charged fulvic acid species are clearly resolved. For example, the higher mass ions in the range, 1520 < m/z < 1540, are singly-charged, whereas doubly-charged ions are evident in the range, 760 < m/z < 780. Without ejecting most of the ions from the ion trap and ion isolation, it is not possible to obtain an ultrahigh-resolution ESI FTICR mass spectrum for accurate mass measurement and structural identification of these species (see time evolution of the ESI FT-ICR mass spectra in Figure 2). However, comparison of the mass spectra for the two ranges suggests that at least some species may be present both as singly- and doubly-charged ions. ESI Spectra of HPLC-Fractionated Fulvic Acid. Figure 5 is an HPLC fractionation of the IHSS standard fulvic acid, SRFA. We believe that this separation is based principally on charge, because the first peak elutes well before the solvent (the negative, or “vacancy”, peak), whereas the second peak is composed of solutes which have been retained to some extent by the hydro-

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Figure 5. HPLC chromatogram of SRFA fulvic acid mixture. See Experimental Section for details of the separation. The negative peak at ∼5.5 min is a “vacancy” peak caused by the solvent (water).

Figure 6. ESI FT-ICR positive-ion mass spectra of a fractionated fulvic acid mixture: (a) spectrum for the first HPLC peak eluting at 2-4 min, (b) spectrum of second HPLC peak eluting at 5.5-6 min.

phobic stationary phase. Elution before the solvent peak suggests that the first peak is a mixture of highlycharged molecules which, because of their hydrophilic character, are excluded from the hydrophobic stationary phase and the “stagnant” mobile phase associated with it. Conversely, the second peak must be composed of relatively hydrophobic molecules resulting from neutralization of their ionic sites through proton association, ion-pairing, or possibly polymerization- or aggregationtype associations. The ESI FT-ICR positive ion mass spectra of the mixtures isolated by this HPLC procedure are presented in Figure 6. Again, there does not appear to be any difference in the molecular weight distributions of observable ions in the two mixtures. Indeed, the spectra are remarkably similar, with clusters of ions occurring in similar ranges: 540 e m/z e 580, 620 e m/z e 680, 690 e m/z e 740, 760 e m/z e 800, and 840 e m/z e 880. The differences in HPLC behavior are therefore most likely due to the nature of ionic sites on molecules in each fraction. However, an explanation for the exact molecular basis of this behavior will require more sophisticated high-resolution and MSn experiments of the type described in the next sections. High-Resolution Mass Spectrometry of a Fulvic Acid. The ESI FT-ICR positive-ion mass spectrum in Figure 7 demonstrates the use of FT-ICR for ultrahigh-

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Figure 7. Ultrahigh-resolution (resolving power, m/∆m50% > 450 000 for all observed ions) ESI FT-ICR positive-ion mass spectrum of a selected m/z region for fulvic acid at 0.6 V trapping voltage and 512K data points. Prior to ion detection, SWIFT m/z-selective ejection removed the ions of 100 e m/z e 550 and 580 e m/z e 3000.

resolution mass spectrometry and chemical formula determination. The ultrahigh-resolution mass spectrum was obtained at 0.6 V trapping voltage during detection; mass measurement accuracy with external or internal calibrants (see Figure 4) were within 5 and 2 ppm, respectively. The mass-resolving power (m/∆m50%) for all observed ions was greater than 450 000. From the high-resolution data presented in Figure 7, we conclude that the unknown fulvic acid compounds do not contain any sulfur atoms (i.e., we do not see the expected 34S species at ∼4% abundancee33). It is possible to identify the chemical formulae for unknwon species based on accurate mass measurement alone. Assuming that fulvic acid compounds contain only carbon, nitrogen, oxygen, and hydrogen atoms, we constructed Table 1 for three most abundant ions observed in Figure 7 (m/z 543.0931, 559.1371, and 575.102 28). For singlycharged ions, we subtracted the proton mass (i.e., 1.0073 Da) from the experimental data to obtain the mass of the corresponding neutral molecule. Note that as the mass tolerance is reduced, the number of possible structures for unknown species decreases rapidly. For example, at 5 ppm mass tolerance, 33 compounds with various elemental compositions and mass of ∼558.1298 Da (i.e., 559.1371 - 1.0073) may be assigned to the neutral compound. Moreover, the relative abundance of the same molecule with one 13C in place of 12C (i.e., at the monoisotopic mass plus 1.003 35 Da) peak provides a check as to the number of carbons in the molecule. Although the unmatched ultrahigh-resolution capability of FT-ICR instruments offers a great advantage over other conventional methods for fulvic acid analysis, exact mass assignment of species from such complex mixtures nevertheless requires careful mass calibration. Final assignment of chemical formula should include accurate mass measurement of product ions formed by fragmentation of the parent ion (see below). MSn Spectra. Figure 8 depicts the series of events used to obtain two-dimensional mass spectra (MS/MS or MS2) of ions electrosprayed from a fulvic acid sample mixture. These spectra were obtained from the firsteluted fraction of the HPLC-fractionated SRFA sample. Briefly, the SWIFT technique is used to eject from the

Figure 8. Series of ESI FT-ICR positive-ion mass spectra obtained in an MS/MS experiment. Proceeding from top to bottom: full mass spectrum of fulvic acid mixture; SWIFT waveform ejection from the ICR cell of ions of all but a narrow m/z range; the resulting isolated parent ion mass spectrum, and the product ion mass spectra produced by collisionallyinduced dissociation (CID).

Figure 9. MS/MS product ions spectra produced by (a) CID and (b) infrared multiphoton dissociation (IR/MPD) of parent ions of m/z 633.

ICR ion trap all ions in the original spectrum except those in a very narrow m/z range (m/z 633 in this case). The parent ions isolated in this manner are then fragmented by the collisionally induced dissociation (CID) process described in the Experimental Section. The highest mass fragment ion at m/z 617 is the most abundant ion at low CID energy (Ecenter-of-mass < 5 eV). Thus, the lowest-energy fragmentation pathway (i.e., the major fragmentation pathway in a CID experiment) is the loss of a small neutral from the parent ion. Ultrahigh-resolution mass spectra and accurate mass measurement identify the neutral lost from the parent ion to be CH4 rather than an oxygen atom.

560 Energy & Fuels, Vol. 11, No. 3, 1997

CID and infrared multiphoton dissociation (IR/MPD) product ion mass spectra derived from the m/z 633 parent ion are compared in Figure 9. The fragment ion at m/z 617 observed by CID is also present in the IR/ MPD MS2 product ion mass spectrum. Figure 9 shows that IR/MPD produces product ions of higher abundance than obtainable by CID in this case. Moreover, the degree of ion fragmentation is easily controlled by changing the ion irradiation period or irradiating laser power. However, CID provides complementary fragment ions that are not produced by IR/MPD; therefore, complete identification of an unknown parent ion may require both CID and IR/MPD to induce parent ion fragmentation. Summary In this presentation we have demonstrated the ability of FT-ICR mass spectrometry to produce high-resolution spectra of humic and fulvic acid mixtures that include stable, high-mass ions uniquely formed by electrospray ionization. These high-resolution spectra can be used

Fievre et al.

to determine exact ion masses and thus unique molecular formulas. Clearly, unraveling the detailed structures of these molecules will require many additional MSn experiments of the type described here. However, it appears that ESI FT-ICR MS may now represent the most direct approach for probing the molecular basis of the environmental chemistry of humic substances. Acknowledgment. HPLC fractionation and isolation of the SRFA sample was carried out by Tim Keefe. The Suwannee River (FL) fulvic acid sample was provided by William Davis, Department of Environmental Engineering Sciences, University of Florida, Gainesville, FL. This work was supported by the St. Johns River (FL) Water Management District, N.S.F. (CHE93-22824), Florida State University, and the N.S.F. High-Field FT-ICR Mass Spectrometry Facility (CHE94-13008) at the National High Magnetic Field Laboratory in Tallahassee, FL. EF970005Q