Exact Masses and Chemical Formulas of Individual Suwannee River

Anal. Chem. 2003, 75, 1275-1284

Exact Masses and Chemical Formulas of Individual Suwannee River Fulvic Acids from Ultrahigh Resolution Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectra Alexandra C. Stenson,†,‡ Alan G. Marshall,†,§ and William T. Cooper*,†

Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306-4390, and Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida 32310-3706

Molecular formulas have been assigned for 4626 individual Suwannee River fulvic acids based on accurate mass measurements from ions generated by electrospray ionization and observed by ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Formula assignments were possible because of the mass accuracy of FTICR MS at high field (9.4 T) and the regular mass spacing patterns found in fulvic acid mixtures. Sorting the 4626 individually observed ions according to Kendrick mass defect and nominal mass series (z* score) revealed that all could be assigned to 1 of 266 distinct homologous series that differ in oxygen content and double bond equivalence. Tandem mass spectrometry based on infrared multiphoton dissociation identified labile fragments of fulvic acid molecules, whose chemical formulas led to plausible structures consistent with degraded lignin as a source of Suwannee River fulvic acids. Humic substances are extremely complex mixtures of organic molecules that occur naturally in soil and water. They are generally believed to be degradation and condensation products of biomolecules. The complexity of humic mixtures is such that isolation of a single humic molecule is not possible by liquid-phase separation techniques (i.e., HPLC,1 SEC,2,3 UF,4,5 FFF,6 CE,7 and CIEF8). Gas chromatography of humics is limited because they are not naturally volatile. It is, however, possible to volatilize * Corresponding author: (tel) 850-644-6875; (fax) 644-8281; (e-mail) cooper@ chem.fsu.edu. † Department of Chemistry and Biochemistry. ‡ Current address: Vertex Pharmaceuticals, Inc., Cambridge, MA 02139-4242. § Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory. (1) Saleh, F. Y.; Chang, D. Y. Sci. Total Environ. 1987, 62, 67-74. (2) Chin, Y.-P.; Aiken, G.; O’Loughlin, E. Environ. Sci. Technol. 1994, 28, 18531858. (3) Wagoner, D. B.; Christman, R. F.; Cauchon, G.; Paulson, R. Environ. Sci. Technol. 1997, 31, 937-941. (4) Buesseler, K. O.; Bauer, J. E.; Chen, R. F.; Eglinton, T. I.; Gustafsson, O.; Landing, W.; Mopper, K.; Moran, S. B.; Santschi, P. H.; VernonClark, R.; Wells, M. L. Mar. Chem. 1996, 55, 1-31. (5) Guo, L.; Wen, L.-S.; Tang, D.; Santschi, P. H. Mar. Chem. 2000, 69, 7590. (6) Beckett, R.; Jue, Z.; Giddings, J. C. Environ. Sci. Technol. 1987, 21, 289295. (7) Garrison, A. W.; Schmitt, P.; Kettrup, A. Water Res. 1995, 29, 2149-2159. 10.1021/ac026106p CCC: $25.00 Published on Web 01/16/2003

© 2003 American Chemical Society

humics with soft ionization techniques such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). Humic ions can then be analyzed with high-resolution mass spectrometry. It has become apparent, however, that quadrupole time-of-flight (Q-TOF)9 and double-focusing10 mass spectrometers are not able to fully resolve individual humic ions. It was not until very recently that the full resolution of complex humic mixtures was accomplished with ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR11) mass spectrometry at 9.4-T magnetic field.12-14 We recently demonstrated that humic mass spectra contain repeating mass spacing patterns and that these patterns provide insight into the bulk composition of humic substances.12 In this study, we exploit those mass spacing patterns and the ultrahigh mass accuracy provided by 9.4-T FTICR mass spectrometry to identify molecular formulas for several thousand Suwannee River fulvic acid ions. The ability to determine molecular formulas of fulvic acid molecules by high-field FTICR mass spectrometry has been demonstrated previously for a small number of ions.14,15 This will be the first time, however, that molecular formulas have been determined for a representatively large number of individual fulvic acid molecules. The present approach, including Kendrick mass analysis, is modeled after prior successful resolution and identification of thousands of individual components of petroleum16-19 and its distillates.20-24 The principal difference between petroleum and (8) Schmitt, P.; Garrison, A. W.; Freitag, D.; Kettrup, A. Water Res. 1997, 31, 2037-2049. (9) Plancque, G.; Amekraz, B.; Moulin, V.; Toulhoatt, P.; Moulin, C. Rapid Commun. Mass Spectrom. 2001, 15, 827-835. (10) McIntyre, C.; Jardine, D. Rapid Commun. Mass Spectrom. 2001, 15, 19741975. (11) Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. Mass Spectrom. Rev. 1998, 17, 1-35. (12) Stenson, A. C.; Landing, W. M.; Marshall, A. G.; Cooper, W. T. Anal. Chem. 2002, 74, 4397-4409. (13) Kujawinski, E. B.; Hatcher, P. G.; Freitas, M. A. Anal. Chem. 2002, 74, 413-419. (14) Kujawinski, E. B.; Freitas, M. A.; Zang, X.; Hatcher, P. G.; Green-Church, K. B.; Jone, R. B. Org. Geochem. 2002, 33, 171-180. (15) Llewelyn, J. M.; Landing, W. M.; Marshall, A. G.; Cooper, W. T. Anal. Chem. 2002, 74, 600-606. (16) Qian, K. N.; Robbins, W. K.; Hughey, C. A. Energy Fuels 2001, 15, 15051511. (17) Qian, K.; Rodgers, R. P.; Hendrickson, C. L.; Emmett, M. R.; Marshall, A. G. Energy Fuels 2001, 15, 492-498.

Analytical Chemistry, Vol. 75, No. 6, March 15, 2003 1275

humic/fulvic acids is that petroleum-derived samples typically contain numerous compounds with a single nitrogen atom,16,17 thereby providing a starting point for internal mass calibration and chemical formula assignment. It is the virtual absence of nitrogen in humic/fulvic acids that (until now) has prevented mass-based identification of their constituents. METHODS Samples. Suwannee River Fulvic Acid (SRFA) standard acquired from the International Humic Substances Society was stored in a darkbox in a freezer until use. For positive-ion ESI FTICR mass spectrometry, SRFA was dissolved in 65.4% Milli-Q distilled water, 32.7% Purge and Trap Grade methanol (Fisher, Pittsburgh, PA), and 1.9% TraceMetal Grade acetic acid (Fisher) directly before use. For negative-ion mass spectra, Optima Grade 2-propanol (Fisher) was used instead of methanol and TraceMetal Grade ammonia instead of acetic acid. SRFA concentrations ranged from 1.2 to 2.4 mg/mL. The internal standard, poly(ethylene glycol) (PEG) of 600 average molecular weight, was not added directly to the sample, but rather trace amounts were intentionally allowed to “contaminate” the syringe, the ESI source apparatus, or both before injection of the sample. That procedure was necessary because PEG is a competing electrolyte that electrosprays with significantly higher efficiency than fulvics. If PEG were added to the sample in anything but trace amounts, the signals from PEG ions would completely obscure the fulvic signals. FTICR MS. Mass spectra were obtained at the National High Magnetic Field Laboratory with a home-built 9.4-T FTICR mass spectrometer equipped with an ESI source.25,26 A quadrupole mass filter has recently been added to the front of the instrument.27 Here it was used to fill the ICR cell with only low-m/z ions. The ions were accumulated in the first octopole ion guide for 0.9 s, then sent through the quadrupole, and reaccumulated in a second octopole ion guide. This filtered accumulation was repeated 20 times (i.e., the sequence went through 20 loops) before the ions were sent on to the ICR cell through a third and final octopole ion guide. Instrument settings were similar to those used previously.12 However, for molecular formula identification, it was necessary to coadd a large number of spectra (1500 for Figure 1, 1000 for Figure 2). The low abundance of MS/MS fragment ions also required many scans to be coadded to produce an adequate (18) Hughey, C. A.; Rodgers, R. P.; Marshall, A. G.; Qian, K.; Robbins, W. R. Org. Geochem. 2002, 33, 743-759. (19) Hughey, C. A.; Rodgers, R. P.; Marshall, A. G. Anal. Chem. 2002, 74, 41454149. (20) Guan, S.; Marshall, A. G.; Scheppele, S. E. Anal. Chem. 1996, 68, 46-71. (21) Rodgers, R. P.; White, F. M.; McIntosh, D. G.; Marshall, A. G. Rev. Sci. Instrum. 1998, 69, 2278-2284. (22) Rodgers, R. P.; White, F. M.; Hendrickson, C. L.; Marshall, A. G.; Andersen, K. V. Anal. Chem. 1998, 70, 4743-4750. (23) Rodgers, R. P.; Blumer, E. N.; Freitas, M. A.; Marshall, A. G. Anal. Chem. 1999, 71, 5171-5176. (24) Hughey, C. A.; Hendrickson, C. L.; Rodgers, R. P.; Marshall, A. G. Energy Fuels 2001, 15, 1186-1193. (25) 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. (26) Senko, M. W.; Hendrickson, C. L.; Emmett, M. R.; Shi, S. D.-H.; Marshall, A. G. J. Am. Soc. Mass Spectrosc. 1997, 8, 970-976. (27) Hendrickson, C. L.; Quinn, J. P.; Emmett, M. R.; Marshall, A. G. In 48th American Society for Mass Spectrometry Annual Conference on Mass Spectrometry & Allied Topics, Long Beach, CA, 2000; Vol. MP083.

1276 Analytical Chemistry, Vol. 75, No. 6, March 15, 2003

Figure 1. SRFA, positive-ion ESI FTICR broadband mass spectrum, calibrated from a PEG 600 internal standard (1500 coadds).

signal-to-noise (S/N) ratio (700 coadds, Figure 6b, 400 coadds: Figure 7b; 600 coadds, Figure 7c). Infrared Multiphoton Dissociation (IRMPD). Ions within a 2-Da mass window were isolated in the ICR cell through quadrupole isolation followed by two stored waveform inverse Fourier transform (SWIFT)28,29 isolations to eject all other ions from the cell (see also ref 30). The most abundant ion within a quadrupole slice was chosen for dissociation by irradiation with a 40-W CO2 laser31 for 0.4 s at power densities of 126 or 314 mW/ mm2. In this way, ions at 410, 422, and 440 Da nominal neutral mass were dissociated. Data Analysis. Data were zero-filled once, Hamming apodized, subjected to fast Fourier transformation and magnitude calculation, and frequency-to-m/z converted by the usual quadrupolar electric field approximation.32,33 The spectra were calibrated with PEG internal standard ions in MIDAS34,35 Analyzer software. Subsequent spectra were also calibrated with previously identified SRFA ions. Residual root-mean-square mass error after calibration ranged from 0.10 to 0.27 ppm for calibration with PEG and from 0.08 to 0.27 ppm for calibration with SRFA. The average mass error for the 4626 ions identified in Figure 1 was 1.6 ppm, and 0.5 ppm for the 453 ions in Figure 2 that includes only relatively high-abundance ions below 440 Da. RESULTS AND DISCUSSION Identification of Low Molecular Weight Chemical Formulas. The positive-ion ESI FTICR broadband mass spectrum of SRFA (Figure 1) contains more than 9800 peaks. Since mass resolving power is best at low m/z,11 however, S/N ratio was maximized in that range by external quadrupole isolation of ions (28) Marshall, A. G.; Wang, T.-C. L.; Ricca, T. L. J. Am. Chem. Soc. 1985, 107, 7893-7897. (29) Guan, S.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1996, 157/ 158, 5-37. (30) Solouki, T.; Freitas, M. A.; Alomary, A. Anal. Chem. 1999, 71, 4719-4726. (31) Fievre, A.; Solouki, T.; Marshall, A. G.; Cooper, W. T. Energy Fuels 1997, 11, 554-560. (32) Ledford, E. B.; Rempel, D. L.; Gross, M. L. Anal. Chem. 1984, 56, 27442748. (33) Shi, S. D.-H.; Drader, J. J.; Freitas, M. A.; Hendrickson, C. L.; Marshall, A. G. Int. J. Mass Spectrom. 2000, (34) Blakney, G. T.; Hendrickson, C. L.; Emmett, M. R. In 50th American Society for Mass Spectrometry Conferenceon Mass Spectrometry & Allied Topics, Orlando, FL, 2002; Vol. MPJ 315. (35) Senko, M. W.; Canterbury, J. D.; Guan, S.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 1839-1844.

Table 1. Measured Recurring Mass Differences between Ions in Suwannee River Fulvic Acid Mass Spectra11 mass differences pattern

origin

theor

measd

SDa (× 104)

nb

observable in

1 2 3 4 5

CH4 vs O NH vs CH2 13C vs 12C H2 vs DBc or ring (CH2)n vs. (CH2)n-1

0.0364 0.9953 1.0034 2.0157 14.0156

0.0364 0.9952 1.0034 2.0157 14.0164

1.3 8.9 5.5 2.5 2.3

131 141 156 125 146

Table 2 and Figure 3a,b (ions 1-7 and a-f) Figure 3a,b (ions 1-7 vs 1′-7′ & a-f vs a′-f ′) Figure 3a,b (ions 1-7 vs I -VII and a-f vs A-F) Table 4 Table 3 and Figure 4 (horizontal lines)

a

Standard deviation. b Number of calculated mass differences. c Double bond.

23Na

Figure 2. SRFA positive-ion ESI FTICR mass spectrum after quadrupole isolation of the low-m/z fraction, again with PEG 600 internal standard (1000 coadds).

of interest (Figure 2). Three slightly overlapping quadrupoleisolated mass spectra were taken spanning a region from 313 to 490 Da. For each ion in the lowest-mass quadrupole-isolated m/z range and all odd-mass ions in the other two ranges, all possible formulas were determined with mass calculator software. Because electrospray ionization can produce multiply protonated (positive) or deprotonated (negative) ions, the first step in assignment of elemental composition is the determination of the charge state of each ion. Fortunately, species in the present mass range contain a large enough number of carbon atoms that for each of many chemically distinct ions containing 12Cn, there is a detectable signal from ions of the same chemical formula but containing 13C12Cn-1. In the present spectra, all ions were singly charged, based on the absence of peaks at intervals of (1.0034/z) higher in mass than the monoisotopic ion.36,37 Therefore, from here on we shall discuss mass, not m/z value. For low-mass molecular formula calculations, the mass of a proton was subtracted from the measured mass of each ion. For that step, it does not matter if the charge originates from H+ or Na+, as long as Na is included in the list of elements to be considered in the formula calculation (and as long as the formula contains at least one H atom). MS Calculator software was used to calculate all possible formulas based on the requirement that the mass calculated for a given chemical formula agree with the measured mass to within (0.001 Da. The following elements (and number of atoms of each element) were considered in the calculation: 12C (0-100), 1H (0-200), 14N (0-10), 16O (0-50), (36) Henry, K. D.; McLafferty, F. W. Org. Mass Spectrom. 1990, 25, 490-492. (37) Marshall, A. G.; Hendrickson, C. L. Rapid Commun. Mass Spectrom. 2001, 15, 232-235.

(0-1), and 13C (0-1) (for even-mass ions). 32S (0-2), 31P (0-1), and 15N (0-1) were taken into consideration for a small excerpt of ions, but it soon became apparent that these elements are not present or, for 15N, may not have been observable. For some ions, only one chemical formula (elemental composition) containing only C, H, N, O, and Na was possible within 0.001 Da of the measured mass. Usually those ions were the higher mass species within any given 1-Da mass segment. In cases for which more than one formula matched the measured mass to within 0.001 Da, the mass spacing patterns previously described12 and reproduced here in Table 1 serve to identify the true formula. Those patterns were also used to check all assigned formulas from the first quadrupole slice for internal consistency. That is, it was confirmed that all assigned formulas differed from each other by the replacement of (multiples of) CH4 by O, 12C by 13C, or CH2 by NH, or by the addition/subtraction of H2 or CH2. Furthermore, the m/z windows of the three quadrupole slices overlapped, so that some ions are present in two quadrupole slices. Because chemical formulas were assigned for each slice independently, formula assignments for the redundant ions provided yet another internal consistency check. Identification of High Molecular Weight Molecular Formulas. Identifying the formula for each ion individually makes it possible to probe each assignment for internal consistency on several levels. However, the number of possible formulas for each measured mass increases with molecular weight, whereas mass resolving power decreases with increasing m/z. Those trends make individual formula assignments based on measured mass alone impossible for higher mass ions. High-mass ions (above ∼490 Da) were therefore identified based on their membership in molecular families, by reasoning similar to that described by Hughey et al.38 for crude oil. All measured masses from Figure 1 were entered into an Excel spreadsheet. Odd- and even-mass ions were then separated, and the mass of a proton was subtracted from each measured mass. Formula identification was based on odd-mass ions. Formulas for even-mass ions could be assigned from the odd-mass ions by replacing one 12C with 13C or one CH2 group with NH, depending on the mass difference (Table 1, Figure 3). For example, one of the repeating mass spacing patterns observed for humic substances is the 14-Da pattern, the result of ions that differ from each other in number of CH2 groups. Although that pattern is not necessarily any more prevalent than others, as has been noted by Kujawinski et al.,13, it spans the widest (38) Hughey, C. A.; Hendrickson, C. L.; Rodgers, R. P.; Marshall, A. G.; Quian, K. N. Anal. Chem. 2001, 73, 4676-4681.

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

1277

Table 2. Molecular Formulas (as Neutral Molecules) of Ions Identified in Figure 3a and ba label

formula

DBEb

calcd mass

1 2 3 4 5 6 7

C16H10O13 C17H14O12 C18H18O11 C19H22O10 C20H26O9 C21H30O8 C22H34O7

12 11 10 9 8 7 6

410.0121 410.0485 410.0849 410.1213 410.1577 410.1941 410.2305

a b c d e f

C20H10O10 C21H14O9 C22H18O8 C23H22O7 C24H26O6 C25H30O5

16 15 14 13 12 11

410.0274 410.0638 410.1002 410.1366 410.1729 410.2093

a Two series of formulas, both corresponding to a substitution of CH4 for O, are evident, and they are distinguished by the labels; numeral (1-7) or letter (a-f). b Double bond equivalence (number of rings plus double bonds).

of digits after the decimal point (Table 3, column 6) and are thus readily recognized as belonging to the same homologous series. The Kendrick mass defect (KMD) is the difference between the accurate KM and the nominal IUPAC mass (NM) (eq 2). All

KMD ) [NM - KM ]

Figure 3. ESI FTICR mass spectrum of SRFA ions at 410 and 411 neutral mass, quadrupole and SWIFT isolated: (a) positive ions at 411 and 412 actual mass; (b) negative ions at 409 and 410 actual mass. Molecular formulas for the ions are listed in Table 2. Two series of formulas, both corresponding to a substitution of CH4 for O, are evident in these spectra, and they can be distinguished by the labels; numeral (1-7) or letter (a-f). In the odd neutral mass spectra (lower right), heavy isotope ions in which one 12C of the odd-mass ion has been replaced with a 13C are indicated by an apostrophe (i.e., 2', b'). Odd-mass ions arising from substitution of NH for CH2 are indicated by Roman numerals (i.e., II) or capital letters (i.e., B).

(2)

members of a homologous series have the same KMD (Table 3, column 7). Because it is possible for different homologous series to have very similar KMDs, it is necessary to presort the ions based on another, independent parameter. Hsu et al.40 suggested presorting based on membership in a nominal mass series, z* (eq 3). In eq

z* ) (modulus[NM/14]) - 14

(3)

mass range of any of the patterns in Table 1 and is thus the most useful for identifying high-mass formulas. The easiest way to identify ions that differ from each other only in number of CH2 groups (i.e., members of a homologous series) is by converting from measured, IUPAC mass, to Kendrick mass.13,15,38-40 The Kendrick mass is based on assigning the mass of CH2 to be 14.0000 Da instead of the IUPAC 14.015 65 Da. To convert to Kendrick mass (KM), the measured IUPAC mass is simply multiplied by 14.0000/14.01565 (eq 1). Because members

3, the modulus is simply the remainder of the division of nominal mass by 14. Presorting based on z* ensures that ions assigned to the same homologous series differ from each other in mass by 14 Da, because ions that differ from each other by multiples of 14 will produce identical remainders. Ions of identical KMD and z* values therefore can differ from each other only in number of CH2 groups. Once ions are sorted based on z* and KMD, formulas may be assigned simply by adding the appropriate number of CH2 groups to the previously identified low molecular weight formulas (i.e., those between 313 and 490 Da). Finally, ions can be characterized by the number of rings plus double bonds (double bond equivalence, DBE) according to eq 4.

KM ) IUPAC massmeasured[14.0000/14.01565]

DBE ) c - 1/2h + 1/2n + 1

(1)

of a homologous series differ from each other by multiples of 14.0000 in the Kendrick mass system, they have identical series (39) Kendrick, E. Anal. Chem. 1963, 35, 2146-2154. (40) Hsu, C. S.; Qian, K.; Chen, Y. C. Anal. Chim. Acta 1992, 264, 79-89.

1278


(4)

In eq 4, c, h, and n are the numbers of carbon, hydrogen, and nitrogen atoms in the chemical formula, CcHhNnOoSs. Table 3 contains a representative assignment of one such species above 490 Da for each of the three homologous series included in the table.

Table 3. Kendrick Masses and Molecular Families peak location

formula

DBE

calcd massa

mass error (ppm)

KMb

KMDc

z*d

m/∆m50%e

355.0660 369.0816 383.0973 397.1131 411.1286f g 593.3318

C15H14O10 C16H16O10 C17H18O10 C18H20O10 C19H22O10

9 9 9 9 9

354.0587 368.0743 382.0900 396.1056 410.1213

0.1 0.0 0.2 0.4 0.1

353.6634 367.6633 381.6634 395.6635 409.6634

0.337 0.337 0.337 0.337 0.337

-10 -10 -10 -10 -10

1.7 × 105 1.6 × 105 1.2 × 105 1.5 × 105 1.4 × 105

C32H48O10

9

592.3247

0.4

591.6631

0.337

-10

8.0 × 104

397.0917 411.1074f 425.1231 439.1386 453.1540 g 593.3110


14 14 14 14 14

396.0845 410.1002 424.1158 438.1313 452.1471

0.2 0.2 0.0 0.4 0.9

395.6422 409.6422 423.6422 437.6421 451.6418

0.358 0.358 0.358 0.358 0.358

-10 -10 -10 -10 -10

1.5 × 105 1.4 × 105 1.4 × 105 1.3 × 105 1.0 × 105

C35H44O8

14

592.3037

0.1

591.6423

0.358

-10

1.0 × 105

383.0609 397.0766 411.0921f 425.1079 439.1237 g 607.3112


10 10 10 10 10

382.0536 396.0693 410.0849 424.1006 438.1162

0.0 0.2 0.1 0.1 0.4

381.6270 395.6271 409.6269 423.6270 437.6272

0.373 0.373 0.373 0.373 0.373

-10 -10 -10 -10 -10

1.5 × 105 1.5 × 105 1.9 × 105 1.4 × 105 1.3 × 105

C32H46O11

10

606.3040

0.2

605.6269

0.373

-10

1.0 × 105

a True mass refers to calculated mass for the neutral molecule. b Kendrick mass, KM ) [IUPAC mass - 1.007 276 (mass of proton)] × 14/ 14.01565. c Kendrick mass defect, KMD ) [nominal mass - KM]. d z*, nominal mass series ) [modulus of NM/14] - 14. e Mass resolving power. ∆m50% is the mass spectral peak full width at half-maximum peak height. f Ions that also occur in Figure 3 and Table 2. g -, denotes the place where members of a family were omitted for brevity.

Because both the oxygen content and DBE of SRFA ions increase with mass, not all high-mass homologous series could be identified from the low-mass quadrupole isolated spectra. To identify homologous series that were not present in the first three quadrupole slices, the ions were resorted based on measured mass. Ions differing from each other by 0.0364 Da (CH4 vs O) were identified, and the appropriate CH4 groups were replaced with O (Table 1, pattern 1). That procedure identified some members of homologous series with high O-content but without homologues in the first three quadrupole slices (i.e., between 313 and 490 Da). The process of grouping first based on z* and KMD and then based on measured mass was repeated iteratively until the majority of fulvic acid ions (4626/5550 peaks) were identified. The above iteration provided an additional method to ensure that formula assignments were internally consistent, because ions identified from one pattern (i.e., either homologous series or CH4 vs O) had to fit into the second pattern when resorted, which they did in every case. The ions that remained unidentified after several iterations were electronic and chemical noise peaks, internal standard ions (PEG), and some ions that appeared to belong to certain homologous series but whose measured masses did not fall within 5 ppm (arbitrarily chosen limit because most ions fell within that limit) of the mass calculated from a given elemental composition. The spectrum in Figure 1 contained in excess of 9800 peaks. Of the 5050 odd-mass ions, 266 odd-mass homologous series (4626 odd-mass elemental compositions) were identified between 316 and 1098 Da (Table 4). The number of carbon atoms ranged from 14 to 58, the number of oxygens from 5 to 29, and the number of rings plus double bonds from 6 to 33. A listing of lowest molecular weight formula, highest molecular weight formula, z*, true KMD, average measured KMD, DBE, number of oxygens, DBE minus number of oxygens, and total percent abundance relative to all

Table 4. Characteristics of Homologous Series Identified in Kendrick Plots at All Even z* Valuesa number of distinct homologous series carbon number range oxygen number range double bond equivalence range

266 14-58 5-29 6-33

a Each of the 266 series is characterized according to these parameters in the Supporting Information.

odd-mass ions for each homologous series is available as Supporting Information. Current ordering information is found on any masthead page. Chemical Formula Validation Based on Heavy-Isotope Relative Abundance. As noted above, it was possible to check the assigned formulas for internal consistency at various stages of the assignment process. For ions of a given chemical formula, the abundance of 13C12Cc-1 relative to 12Cc can in principle provide an independent estimate for the number of carbon atoms. This estimate is usually most accurate for electron impact ionization spectra of small molecules. In ESI FTICR MS, however, relative abundances are unfortunately affected by several factors other than solution concentration of the analytes. To determine whether ESI FTICR MS relative abundances could be used to provide an estimate of the number of carbon atoms in a molecule, PEG 600 and PEG 10 000 were sprayed as positive ions under acidic spray solvent conditions. PEG 10 000 dissociates in the source under such conditions12 thereby providing [PEG + H]+ ions over a wide mass range (503-1075 Da (22-48 carbon atoms)). For the PEG 600 sample, [PEG + H]+ and [PEG + Na]+ ions between 503 and 943 Da (22-42 carbon atoms) were investigated at four different PEG concentrations. The absolute-value error in the number of assigned carbon atoms based on abundances of species Analytical Chemistry, Vol. 75, No. 6, March 15, 2003

1279

containing 13C relative to all 12C (i.e., absolute value of the difference between the measured number of carbon atoms and the actual number of carbon atoms) ranged from 0 to 24 carbon atoms for PEG 10k and from 0 to 93 carbon atoms for the PEG 600 samples. The poor reliability of these estimates can likely be ascribed to a combination of insufficient digital resolution41 and well-known difficulties in determining relative abundances of ions of closely spaced cyclotron frequencies.42 In any case, FTICR MS relative abundances of 13C12Cc-1, and 12Cc clearly do not provide a reliable estimate of the number of carbons in a molecule and were, therefore, not used to confirm the present SRFA formula assignments. Method Validation through Internal Standardization. A direct measure of the reliability of the assignments of elemental compositions to fulvic acid ions is provided by the internal mass calibrant (PEG) peaks. When analyzed as for fulvic acid species, the known PEG peaks yielded correct chemical formulas consisting of only C, H, and O, with no rings or double bonds. Elemental Composition. Figure 3 provides an example of the classes (a class consists of species containing a specific number of “hetero” atoms (O, N, S)) of ions whose masses fall within a 1-Da range. Corresponding molecular formulas are listed in Table 2. Interestingly, the same ion classes are present for ions of both charge signs. The molecular formulas identified for fulvic acids suggest that the bulk of its electrospray ionizable fraction consists of molecules containing only C, H, N, and O. Odd-mass ions dominate and contain only C, H, and O. The average H/C ratio for Figure 1 is 1.1 (1.0 for Figure 2), and the average O/C ratio is 0.5 (0.4 for Figure 2). The average N/C ratio is 0.05 for those molecules in Figure 2 that contain N (0.01 for Figure 2 overall)). Based on the 4626 identified oddmass ions in Figure 1, the weight percent carbon is 59, hydrogen 5, and oxygen 36, (59% C, 5.5% H, and 35% O, when relative abundances are taken into account). Those values agree reasonably well with combustion data for SRFA in the literature (53% C, 4.8% H, and 41% O)43 The good agreement between overall elemental composition determined by combustion and that provided by ESI FTICR MS suggests that the ions generated by ESI are at least partially representative of the entire sample, despite the selective nature of the ESI source. Visual Representation. A Kendrick plot (Kendrick mass defect vs nominal Kendrick mass, KMD vs NM) provides a compact visual representation of a high-resolution mass spectrum.38 In the Kendrick plot, a mass spectrum is divided into 1-Da segments, and ions within each segment are converted to their Kendrick mass defects and then rotated to the y-axis. The overall shape of the Kendrick plot for humics is diagonal, due to an overall increase in both DBE and number of oxygens as overall nominal mass increases. A Kendrick plot for an entire humic mass spectrum is crowded and difficult to analyze.13 However, if the display is limited only to ions with identical z* (i.e., ions of only every 14th nominal mass), visual interpretation is greatly simplified. For example, Figure 4 shows a Kendrick plot for odd-mass ions having z* ) -10. (Note that an even z* value corresponds (41) Marshall, A. G.; Verdun, F. R. Fourier Transforms in NMR, Optical, and Mass Spectrometry; Elsevier: New York, 1990. (42) Mitchell, D. W.; Smith, R. D. Phys. Rev. E 1995, 52, 4366-4386. (43) Leenheer, J. A.; Brown, G. K.; Maccarthy, P.; Cabaniss, S. E. Environ. Sci. Technol. 1998, 32, 2410-2416.

1280 Analytical Chemistry, Vol. 75, No. 6, March 15, 2003

Figure 4. Kendrick mass defect vs nominal Kendrick mass for oddmass SRFA ions (positive ions, broadband, Figure 1a) of z* ) -10 (i.e., peaks from only every 14th nominal mass). Spacing between vertically aligned ions is 0.0364 KMD (pattern 1, Table 1). Spacing between horizontally aligned ions is 14 Da nominal mass (pattern 5, Table 1; homologous series). Table 5. Effect of Presorting Based on z*a peak location

formula

DBE

true massb

KMD

z*

399.0709 401.0868 403.1023 405.1181 407.1336 409.1494 411.1650* 413.1805 415.1960

C20H14O9 C20H16O9 C20H18O9 C20H20O9 C20H22O9 C20H24O9 C20H26O9 C20H28O9 C20H30O9

14 13 12 11 10 9 8 7 6

398.0368 400.0794 402.0951 404.1107 406.1263 408.1420 410.1577 412.1733 414.1890

0.381 0.367 0.354 0.341 0.327 0.314 0.300 0.287 0.274

-8 -6 -4 -2 -14 -12 -10 -8 -6

357.0969 359.1126 361.1283 363.1438 365.1596 367.1752 369.1908


12 11 10 9 8 7 6

356.0896 358.1052 360.1209 362.1366 364.1522 366.1679 368.1835

0.308 0.295 0.281 0.268 0.254 0.241 0.228

-8 -6 -4 -2 -14 -12 -10

a z* decreases discontinuously from -2 to -14 with a concurrent continuous decrease in DBE. b True mass refers to calculated mass for the neutral molecule.

to odd-mass ions because the mass of a proton is subtracted from the measured mass before z* is calculated.) Ions that differ in degree of saturation (pattern 4, Table 1) also fall into different z* score plots, except for ions that differ by integer multiples of seven double bond equivalence (Table 5; rows 1 and 8 as well as rows 2 and 9). Homologous series are easily identified in Kendrick plots because the data points form horizontal rows corresponding to ions spaced 14 Da apart. To assign an individual chemical formula, the measured mass, DBE (eq 4) and the number of heteroatoms (oxygen, in the case of odd-mass humics) must be determined. In Figure 4, DBE and number of O are listed for four different homologous series, each with a z* value of -10. From this figure it is apparent that the number of oxygen atoms, as well as the number of rings and double bonds, increases monotonically with KMD. Remarkably, the difference between oxygen number and DBE remains constant, however, and is always either -1 or +6 for z* ) -10. All of the homologous series in Figure 4 can thus

Table 6. Relationship between DBE Minus the Number of Oxygens and z* for All Odd-Mass SRFA Ionsa DBE minus no. of oxygens

z*

DBE minus no. of oxygens

z*

-5 -4 -3 -2 -1 0 1

-2 -4 -6 -8 -10 -12 -14

2 3 4 5 6 7 8

-2 -4 -6 -8 -10 -12 -14

a Again, z* increases discontinuously whereas DBE minus the number of oxygens increases continuously. Formulas for all ions within homologous series for which DBE minus number of oxygens ranges from -5 to 1 can be expressed by eq 5a; those for which DBE minus number of oxygens ranges from 2 to 8 can be expressed by eq 5b. Only the z* ) -10 series is plotted in Figure 4.

be further divided into two subgroups, one for which the difference between DBE and number of oxygens is -1 and another for which that difference is 6. Further investigation shows that at least two such sets of homologous series are present at every even z* value! It is thus possible to classify the vast majority of odd-mass ions with just two equations relating DBE-O to z*.

DBE - O ) -0.5z* + 1, -5 < (DBE - O) < 1 (5a) DBE - O ) -0.5z* - 6, 2 < (DBE - O) < 8

(5b)

Even-mass formulas were not assigned except for the first quadrupole slice. Because the replacement of 13C by 12C or NH by CH2 does not alter either the DBE or the number of oxygen atoms, the trends observed for odd-mass ions are, however, expected to apply to even-mass ions as well. We verified that expectation for the 119 nitrogen-containing, even-mass ions that had been identified from the first quadrupole slice. Therefore, two more equations relating DBE minus number of oxygens to z*odd serves to classify the vast majority of even-mass ions.

DBE - O ) -0.5z* + 1.5, -5 < (DB - O) < 1 (6a) DBE - O ) -0.5z* - 5.5, 2 < (DB - O) < 8

(6b)

The observation that all 4626 assigned odd-mass formulas can be grouped into two sets based on the relationship between DBE minus the number of oxygens and z* might be interpreted to suggest that two independent sets of homologous series are present within the mass spectrum of SRFA. However, ions that are related by seven degrees of saturation (i.e., differ from each other by 14 H atoms) have identical z* values. Closer inspection of all the assigned formulas reveals that the difference between DBE and the number of oxygens per molecule increases monotonically by successive integers, with a concurrent decrease in z* by multiples of -2 until z* reaches a value of -14. After z* has reached its smallest possible value (-14), the next increase in DBE minus the number of oxygens by 1 brings the z* value back up to -2 (Table 6). From -2, z* values again decrease by integers of negative 2 as DBE minus the number of oxygens increases by 1 until z* values again reach -14, where the values will again

continue at -2. The small range of possible z* values is responsible for the apparent discontinuity. It thus becomes possible to include all even-z* species in one equation and all oddz* species in a second equation, demonstrating the unexpectedly close relationships between all observed chemical formulas.

DBE - 7x - O ) -0.5z* + 1, z* is even

(7a)

DBE - 7x - O ) -0.5z* + 1.5, z* is odd

(7b)

x ) -5 for < (DBE - O) < 1

(7c)

x ) 2 for < (DBE - O) < 8

(7d)

In which

Fulvic Acids as Lignin Degradation Products. To validate that the assigned formulas are consistent with expected fulvic acid characteristics, mass spacing patterns summarized in Table 1 were further evaluated based on known characteristics of humic substances. Degraded lignin is generally believed to be one of the major constituents of SRFA.13,44,45 The assigned formulas and observed mass patterns were therefore evaluated for consistency with expected formula patterns for degraded lignins. Lignins are complex, high-mass, primarily ether-linked, phenylpropanoid biopolymers found mostly in wood cells.46,47 The main building blocks for the phenyl portion of lignins are coumaryl, coniferyl, and sinapyl alcohols.46 The lignin biopolymer is degraded by fungi and eventually bacteria.47-50 That degradation can take place directly on land or in a water body. In the former scenario, water-soluble material is leached from soils and transported to a water body through surface runoff or groundwater infiltration. Lignin structure and chemical formulas vary from plant to plant, but only carbon, hydrogen, and oxygen atoms are involved. Bonding between the main building blocks (i.e., coumaryl, coniferyl, and sinapyl alcohols) also varies, providing for great complexity. Helm51 summarized nine typical reported bonding patterns that differ in number of O and C as well as degree of saturation. The numbers of O and CH2 groups per lignin monomer also vary depending on whether coumaryl, coniferyl, or sinapyl alcohol is the base unit. The most common lignin degradation pathways include depolymerization, demethylation, side-chain oxidation, and aromatic ring cleavage (Figure 5).47-50,52,53 (44) Haiber, S.; Herzog, H.; Burba, P.; Gosciniak, B.; Lambert, J. Environ. Sci. Technol. 2001, 35, 4289-4294. (45) Leenheer, J. A.; Rostad, C. E.; Gates, P. M.; Furlong, E. T.; Ferrer, I. Anal. Chem. 2001, 73, 1461-1471. (46) Killops, S. D.; Killops, V. J., Eds. An Introduction to Organic Geochemistry; John Wiley & Sons: New York, 1993. (47) Filley, T. R.; Cody, G. D.; Goodell, B.; Jellison, J.; Noser, C.; Ostrofsky, A. Org. Geochem. 2002, 33, 111-124. (48) Leonowicz, A.; Cho, N.-S.; Luterek, J.; Wilkolazka, A.; Wojtas-Wasilewska, M.; Matuszewska, A.; Hofrichter, M.; Wesenberg, D.; Rogalski, J. J. Basic Microbiol. 2001, 41, 185-227. (49) Higuchi, T. J. Biotechnol. 1993, 30, 1-8. (50) Christman, R. F.; Oglesby, R. T. In Lignins: Occurrence, Formation, Structure and Reactions; Sarkanen, K. V., Ludwig, C. H., Eds.; Wiley-Interscience: New York, 1971; pp 769-793. (51) Helm, R. F. In Lignin: Historical, Biological, and Materials Perspective; Glasser, W. G., Northey, R. A., Schultz, T. P., Eds.; Oxford University Press: Washington, DC, 2000; pp 161-171.


1281

Figure 5. Schematic representation of common lignin biodegradation pathways and how they produce some of the elemental substitutions listed in Table 1. Adapted from ref 48.

Based on the different types of starting materials, different types of bonding patterns, and different types of degradation pathways, it is clear that degraded lignin should be a complex mixture of molecules, consisting of C, H, and O and differing in degree of saturation (based on degree of side-chain oxidation and bonding pattern), number of CH2 groups (based on degree of demethylation, bonding pattern, and phenolic building blocks), and number of O (based on number of aromatic ring cleavages, bonding pattern, and phenolic building blocks). That description fits the presently assigned SRFA chemical formulas exceptionally well. In particular, the mass spacing patterns 1, 4, and 5 in Table 1 that have been observed for fulvic and humic acids are largely explained according to the known characteristics of degraded lignin. The lignin-based character of SRFA is further corroborated by the identification of expected lignin degradation products. Lignin degradation involves ring cleavage, resulting in the concurrent addition of two oxygen atoms to a chemical formula. Therefore, formulas differing by O2 (e.g., one aromatic ring cleavage) and CH4 versus O2 (e.g., one aromatic ring cleavage after one demethylation and one side-chain oxidation) should be present among the assigned formulas. Figure 5 is a schematic representation of the influence of these reactions on the structures and chemical formulas of typical lignin components. To test for the presence of such patterns among the assigned formulas, the masses of O2 (31.9898 Da) and O2 minus CH4 (15.9585 Da) were added to a semirandom selection of SRFA ions. Then, if peaks were found at the resultant masses, the set was checked for (52) Nelson, M. J.; Montgomery, S. O.; Mahaffey, W. R.; Pritchard, P. H. Appl. Environ. Microb. 1987, 53, 949-954. (53) Radnoti de Lipthay, J.; Barkay, T.; Vekova, J.; Sorensen, S. J. Appl. Microbiol. Biotechnol. 1999, 51, 207-214.

1282


additional members that differed by O2 or by O2 replacing CH4. The selection of ions for this process was semirandom because species from Figure 3 were purposefully included in the set for consistency. Table 7 provides a small subset of ions that matched the predicted patterns. For most ions investigated, pairs of ions differing by two O or two O instead of CH4 could be found, indicating that the assigned formulas allow for ions that are related to each other through aromatic ring cleavage. Plausible Structures for SRFA Molecules Based on Tandem Mass Spectrometry. The assigned chemical formulas have been shown to be internally consistent and to correspond well to known characteristics of SRFA. A reasonable SRFA formula should consist of functional groups expected to be present in SRFA (i.e., carboxylic, hydroxyl, carbonyl, and aromatic groups).43,54 We therefore performed MS/MS experiments to identify some of the structural components of individual fulvic acid ions. Ideally, fulvic acid ions of a single mass should be isolated within the ICR cell before fragmentation. Unfortunately, humic ions are spaced less than 40 mDa apart, and even SWIFT massselective ejection55 cannot eject ions of other masses without also exciting the ions of the mass of interest. Therefore, two consecutive SWIFT excitations were used: one higher energy excitation at frequencies that were either significantly higher or lower than the cyclotron frequency of the ion of interest and another, lower energy, excitation that included the cyclotron frequencies of ions in close proximity to the ion of interest. It was then possible to isolate a narrow (∼2 Da) mass window of SRFA ions (Figures 6a and 7a) without losing too many ions targeted for MS/MS. (54) Murray, K.; Linder, P. W. J. Soil Sci. 1983, 34, 511-523. (55) Chen, L.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1987, 79, 115-125.

Table 7

peak location

a

(a) Predicted Chemical Formula Patterns Based on Lignin Degradation Pathways (CH4 vs O2) formula DBE true massa mass error (ppm) KMD z*

m/∆m50%.b

497.2888 513.2481 529.2057 545.1649 561.1223 577.0801 593.0397


11 12 13 14 15 16 17

496.2825 512.2410 528.1995 544.1581 560.1172 576.0751 592.0337

1.9 0.5 2.1 0.9 3.9 2.6 2.1

0.273 0.331 0.391 0.450 0.510 0.570 0.627

-8 -6 -4 -2 -14 -12 -10

7.9 × 104 1.2 × 105 1.1 × 105 1.1 × 105 1.1 × 105 1.0 × 105 8.0 × 104

395.1855 411.1438* 427.1024 443.0607

C24H26O5 C23H22O7 C22H18O9 C21H14O11

12 13 14 15

394.1780 410.1366 426.0951 442.0536

0.6 0.1 0.0 0.5

0.262 0.321 0.381 0.440

-12 -10 -8 -6

1.5 × 105 1.4 × 105 1.4 × 105 1.1 × 105

395.2068 411.1650* 427.1236 443.0818

C21H30O7 C20H26O9 C19H22O11 C18H18O13

7 8 9 10

394.1992 410.1577 426.1162 442.0747

0.8 0.0 0.1 0.4

0.241 0.300 0.360 0.419

-12 -10 -8 -6

1.5 × 105 1.4 × 105 1.4 × 105 1.3 × 105

411.1438* 443.1338 475.1234 507.1127 539.1020 571.0909

(b) Predicted Chemical Formula Series Based on Lignin Degradation Pathways (O2) C23H22O7 13 410.1366 0.1 0.321 C23H22O9 13 442.1264 0.3 0.367 C23H22O11 13 474.1162 0.2 0.413 C23H22O13 13 506.1059 0.9 0.460 13 538.0959 2.1 0.506 C23H22O15 C23H22O17 13 570.0857 3.6 0.553

-10 -6 -2 -12 -8 -4

144,000 133,000 124,000 116,000 88,000 83,000

347.1858 379.1753 411.1650* 443.1545

C20H26O5 C20H26O7 C20H26O9 C20H26O11

-4 -14 -10 -6

170,000 156,000 140,000 133,000

8 8 8 8

346.1780 378.1678 410.1577 442.1475

1.5 0.5 0.0 0.6

0.208 0.254 0.300 0.346

True mass refers to calculated mass for the neutral molecule. b Resolving power.

Figure 6. FTICR mass spectra following infrared multiphoton dissociation (tandem) of electrosprayed SRFA positive ions. (a) SRFA ions of equivalent neutral mass, 410 and 411 Da, after quadrupole and double SWIFT isolation; (b) fragment ion spectra after IRMPD irradiation for 0.4 s at 126 mW/mm2.

Figure 7. FTICR mass spectra following infrared multiphoton dissociation (tandem) of electrosprayed SRFA negative ions. (a) SRFA ions of equivalent neutral mass, 410 and 411 Da after quadrupole and double SWIFT isolation; (b) fragment ion spectra after IRMPD irradiation for 0.4 s at 126 mW/mm2; (c) fragment ion spectra after IRMPD irradiation for 0.4 s at 314 mW/mm2.

Fortunately, SRFA ions whose masses fall within 1 Da of each other tend to lose identical neutral fragments. Therefore, fragment ions exhibit the familiar humic ion mass spacing patterns (patterns 1 and 2) making them stand out from the noise (Figures 6b and 7b,c). Unfortunately, even after several hundred coadds, the S/N ratio for the fragment ions is still very low. Therefore, it is not possible to assign unequivocally any given fragment to a particular parent ion within the 1-Da window. However, it is still possible to

follow the general reaction pathways of some subset of ions within the 1-Da mass window by calculating the masses and chemical formulas of the neutral fragments that are lost. Figures 6 and 7 include both negative and positive fragment ion spectra after IRMPD of the ions identified in Figure 3a and b. SRFA ions of both charge signs tend to lose several molecules of H2O. Negative ions can lose CO2 alone as well as both CO2 and H2O. Those results correspond well with previously published Analytical Chemistry, Vol. 75, No. 6, March 15, 2003

1283

negative-ion MS/MS data.9,45 Positive ions lose CO but only concomitantly with H2O. IRMPD of SRFA ions of 440 Da (equivalent neutral) mass (data not shown) produce the same fragments as the 410-Da SRFA except for the additional losses of H2O plus CO (negative ions) and three H2O plus CO (positive ions). Only the fragment resulting from the loss of one H2O molecule was observed in IRMPD fragment spectra of SRFA acid ions at 422 neutral mass (data not shown). Increasing the laser voltage (i.e., the energy provided for fragmentation, Figure 7c vs b) did not lead to the formation of new fragments for ions at 410 Da (or ions at 422 Da) but did shift fragmentation toward the loss of larger fragments, most likely due to secondary fragmentation. It therefore appears that the presently observed fulvic acid ions lose only H2O, CO, and CO2. That behavior thus accords with the assigned formulas, because the assigned odd-mass formulas contain only C, H, and O. Also encouraging is that the highest observed losses, two H2O plus one CO (positive ions) and H2O plus two CO2 (negative ions), are possible for all formulas within the 1-Da segment at 410 Da (Table 2). The MS/MS data thus suggest that SRFA molecules at 410 and 440 Da contain two carboxylic groups, as well as one additional IRMPD-labile hydroxyl group. Recent NMR results confirm that dibasic (i.e., dicarboxylic) acids are abundant in SRFA.43 CONCLUSION For the first time, molecular formulas have been determined for a large and representative number of individual fulvic acid molecules. In all, approximately 5000 formulas have been identified. Those formulas are internally consistent by several independent criteria and are also consistent with expected molecular formulas for degraded lignins, one of the main constituents of SRFA. Furthermore, the formulas are consistent with tandem MS (IRMPD) data and allow for structures that are consistent with fulvic acid characteristics previously reported. All mass spacing patterns in Table 1 are consistent with degraded lignin, except pattern 2. That pattern appears to originate from the nitrogen-containing fulvic acids. The inclusion of nitrogen into humics is still poorly understood, since lignin, for instance, contains no nitrogen. The presence of these N-containing ions in a mixture that otherwise appears to be almost exclusively made up of degraded lignin is therefore very interesting and warrants further investigation. (56) Trubetskoj, O. A.; Trubetskaya, O. E.; Afanas’eva, G. V.; Reznikova, O. I.; Saiz-Jimenez, C. J. Chromatogr., A 1997, 767, 285-292.

1284


The data also show that SRFA is a complex but highly ordered system with an almost polymeric character. Each ion within this apparently continuous series seems to be related to other ions by the patterns in Table 1 (or combinations thereof). This observation is consistent with the broad, continuous separation profiles that are observed in liquid-phase separation of humic mixtures.1,7,8,56 Finally, we find that Kendrick plots presorted based on z* significantly simplify the analysis of humic spectra. Their obvious and readily interpretable patterns make them useful tools for comparing humics from different sources or humic substances that have undergone different chemical treatments. It should be noted that the initial molecular formula assignments presented here took several months. However, now that a spreadsheet relating KMD to fulvic acid molecular formula exists (Supporting Information), future assignments should take only a few days or a few seconds if a computer program were used to perform the task. It should be possible, therefore, to base all future comparisons between humics on direct molecular information. ACKNOWLEDGMENT This work was supported by a Cornerstone Program Enhancement Grant from the Florida State University Research Foundation. Mass spectra were obtained at the National High-Field Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Facility (NSF CHE-99-09502) at the National High Magnetic Field Laboratory in Tallahassee, FL. The authors greatly appreciate the assistance of Drs. Chris Hendrickson and Ryan Rodgers of the ICR laboratory. Discussions with Professor Pat Hatcher of The Ohio State University were very helpful in identifying appropriate lignin degradation pathways. Finally, Dr. Chrisi Hughey provided invaluable guidance in the implementation of Kendrick mass sorting in Excel format. SUPPORTING INFORMATION AVAILABLE A listing of lowest molecular weight formula, highest molecular weight formula, z*, true KMD, average measured KMD, DBE, number of oxygens, DBE minus number of oxygens, and total percent abundance relative to all odd-mass ions for each homologous series. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review November 15, 2002. AC026106P

September

3,

2002.

Accepted

Exact Masses and Chemical Formulas of Individual Suwannee River

Recommend Documents