Graphical Method for Analysis of Ultrahigh-Resolution Broadband

Sep 6, 2003 - Electrospray ionization mass spectrometry (ESI-MS) is becoming an important tool in the study of natural organic matter (NOM) at the mol...
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Anal. Chem. 2003, 75, 5336-5344

Graphical Method for Analysis of Ultrahigh-Resolution Broadband Mass Spectra of Natural Organic Matter, the Van Krevelen Diagram Sunghwan Kim, Robert W. Kramer, and Patrick G. Hatcher*

Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210

Electrospray ionization mass spectrometry (ESI-MS) is becoming an important tool in the study of natural organic matter (NOM) at the molecular level.1 Ultrahigh-resolution ESI-MS analyses of NOM often produce very complicated spectra; therefore, visual presentation and structural interpretations of the spectra are difficult. To meet this analytical challenge, we herein propose and demonstrate an approach using the van Krevelen diagram. With this approach, complicated mass spectra can be visualized in a way that allows for (1) possible reaction pathways to be identified and presented, and (2) qualitative analyses on major classes of compounds that comprise ultrahighresolution spectra. The qualitative analyses are in a good agreement with results obtained from analyses by other analytical techniques. Additionally, the van Krevelen diagram can be expanded to a 3D plot by using peak intensities or relative intensities as the z-axis. The 3D van Krevelen diagram allows for an evaluation of the relative significance of structurally related compounds. The 3D plot can also be a useful tool for compositional differentiation among samples. Recently, a new technique has emerged which holds great promise in being able to provide both an overview of natural organic matter’s (NOM’s) composition and molecular scale details. This technique is electrospray ionization coupled to ultrahighresolution mass spectrometry afforded by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). FT-ICRMS is known for its high resolution capability2 and has been successfully applied to the characterization of natural organic mixtures.3-6 Natural organic matter (NOM) is a complex mixture of naturally produced or modified biochemicals in soil, air, or water. Understanding the chemical composition of NOM is of * Corresponding author. Fax: (614) 688-5920. E-mail: hatcher@ chemistry.ohio-state.edu. (1) Hatcher, P. G.; Dria, K. J.; Kim, S.; Frazier, S. W. Soil Sci. 2001, 166, 770794. (2) He, F.; Hendrickson, C. L.; Marshall, A. G. Anal. Chem. 2001, 73, 647650. (3) Brown, T. L.; Rice, J. A. Anal. Chem. 2000, 72, 384-390. (4) Fievre, A.; Solouki, T.; Marshall, A. G.; Cooper, W. T. Energy Fuels 1997, 11, 554-560. (5) Stenson, A. C.; Landing, W. M.; Marshall, A. G.; Cooper, W. T. Anal. Chem. 2002, 74, 4397-4409. (6) Kujawinski, E. B.; Hatcher, P. G.; Freitas, M. A. Anal. Chem. 2002, 74, 413-419.

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great importance to the field of environmental science because it has a huge impact on understanding numerous molecular and global-scale processes. Unfortunately, a significant fraction of NOM remains undefined.7 A troubling complication in structural studies of NOM has been its enormous complexity on a molecular scale. Accordingly, it is an understatement to say that few analytical tools are capable of providing a broad spectrum characterization of NOM. The ultrahigh-resolution FT-ICR spectra of NOM can be extremely complicated. These spectra routinely contain a multitude of peaks at each nominal mass and thousands of peaks in an entire spectrum. Each peak can represent a chemically distinct compound. This complexity poses an analytical challenge to studying the spectra for structural interpretation. Reducing and visualizing the complex ultrahigh-resolution mass spectra has not been trivial. Kendrick mass defect analysis8 has been successfully applied to ultrahigh-resolution mass spectra, allowing one to sort peaks in complicated spectra by their homologous relatives across the range of masses.9 Thus, compounds whose compositions differ by masses associated with a structural unit (e.g., CH2, COOH, CH2O, etc.) can be discerned by 2-dimensional plots whereupon the structurally related peaks plot on horizontal or diagonal straight lines. Such an approach allows one to extract peaks that are homologously related, and the method has been used effectively to identify groups of related compounds in FT-ICR-MS of petroleum samples.9 The same approach has been implemented with humic substances, especially one that can be expected to contain a range of related oligomers of lignin.10 Recently, this Kendrick mass defect approach was combined with calculations of the degrees of unsaturation for elemental formulas obtained from exact mass measurements of a sample of NOM from the Suwannee river.5,11 Kendrick mass defect analysis has greatly helped the interpretation of mass spectra. However, despite these efforts, it still is difficult to derive structural details for molecules that contribute to complex ultrahigh-resolution mass spectra. Therefore, it would be advantageous (7) Hedges, J. I.; Eglinton, G.; Hatcher, P. G.; Kirchman, D. L.; Arnosti, C.; Derenne, S.; Evershed, R. P.; Kogel-Knabner, I.; de Leeuw, J. W.; Littke, R.; Michaelis, W.; Rullkotter, J. Org. Geochem. 2000, 31, 945-958. (8) Kendrick, E. Anal. Chem. 1963, 35, 2146-2154. (9) Hughey, A. C.; Hendrickson, C. L.; Rodgers, R. P.; Marshall, A. G. Anal. Chem. 2001, 73, 4676-4681. (10) Kujawinski, E. B.; Freitas, M. A.; Zang, X.; Hatcher, P. G.; Green-Church, K. B.; Jones, R. B. Org. Geochem. 2002, 33, 171-180. (11) Stenson, A. C.; Marshall, A. G.; Cooper, W. T. Anal. Chem. 2003, 75, 12751284. 10.1021/ac034415p CCC: $25.00

© 2003 American Chemical Society Published on Web 09/06/2003

to have more approaches to display and facilitate the interpretation of complex mass spectra. We present here a new approach for examining ultrahighresolution mass spectra, the van Krevelen diagram. The van Krevelen diagram, used extensively in the geochemistry literature to study the evolution of coals or oil samples,12-14 is constructed using the molar ratio of hydrogen to carbon (H/C ratio) as the ordinate and the molar oxygen-to-carbon ratio (O/C ratio) as the abscissa. Major biogeochemical classes of compounds (such as lignin compounds, lipids, carbohydrates, etc.) have their own characteristic H/C or O/C ratios. As a result, each class of compounds plots in a specific location on the diagram. It is well recognized that types of compounds can be identified from the location of points in the van Krevelen plot.15,16 In addition, the diagenetic history of these compounds can be traced from the van Krevelen plot17 because of the fact that typical biogeochemical reactions (e.g., oxidation, condensation, etc.) involve loss or gain of integral amounts of C, H, O or N atoms, and this will lead to a unique projectory on the plot.15 Ultrahigh-resolution mass spectrometric data can be readily transposed to van Krevelen diagram. The elemental compositions for each peak can be calculated from mass spectra, allowing the H/C and O/C ratios of each peak to be calculated and plotted on the diagram. In this paper, we demonstrate how this new method can be used to extract structural information from complex FT-ICR-MS data. EXPERIMENTAL METHODS Samples and Preparation. A water sample from a black-water stream located in the Pinelands of New Jersey [McDonalds Branch, dissolved organic carbon (DOC) content averages 1618 mg/L] was filtered and acidified, and dissolved organic matter (DOM) was extracted and recovered using C18 solid-phase extraction disks. Detailed information about the extraction and recovery procedures can be found elsewhere.18 A peat humic acid sample was treated to remove lignin components by a bleaching process, as described in a previous study.19 A humic acid was extracted from a fossil wood from the Morwell Open Cut from the Latrobe Valley (Australia). This particular sample was observed in hand specimen to contain a charred exterior, suggesting that it was subjected to a swamp fire at the time of deposition more than 20 million years ago.20 International Humic Substances Society (IHSS) standard humic acid extraction protocols21 were employed. Humic acid samples were dissolved in 70% HPLC grade methanol (Fisher Scientific, Itasca, IL) and 30% nanopure water (12) Hatcher, P. G.; Lerch, H. E.; Bates, A. L.; Verheyen, T. V. Org. Geochem. 1989, 14, 145-155. (13) Bostick, N. H.; Daws, T. A. Org. Geochem. 1994, 21, 35-49. (14) Curiale, J. A.; Gibling, M. R. Org. Geochem. 1994, 21, 67-89. (15) van Krevelen, D. W. Fuel 1950, 29, 269-284. (16) Hedges, J. I. In Organic acids in aquatic ecosystems; Perdue, E. M., Gjessing, E. T., Eds.; Wiley: New York, 1990; pp 43-63. (17) Reuter, J. H.; Perdue, E. M. Mitteilungen aus dem Geologisch-Palaontologischen Institut der Universitat Hamburg 1984, 56, 249-262. (18) Kim, S.; Simpson, A.; Kujawinski, E. B.; Freitas, M. A.; Hatcher, P. G. Org. Geochem, in press. (19) Chefetz, B.; Salloum, M. J.; Deshmukh, A. P.; Hatcher, P. G. Soil Sci. Soc. Am. J. 2002, 66, 1159-1171. (20) Hatcher, P. G. Energy Fuels 1988, 2, 48-58. (21) Swift, R. S. In Methods of soil analysis; Sparks, D. L., Page, A. L., Hemke, P. A., Loeppert, R. H., Soltanpour, P. N., Tabatabai, M. A., Johnston, C. T., Sumner, M. E., Eds.; Soil Science Society of America: Madison, WI, 1996; Vol. 3, pp 1018-020.

Figure 1. Negative ion mode ultrahigh-resolution mass spectrum of McDonalds Branch DOM.

prepared from a water purification unit (UHQ, ELGA, Lowell, MA). A small amount of 30% NH4OH solution was spiked into samples to induce a negative charge on the humic acid components during the electrospray ionization process. Instrumentation. Samples were analyzed by a 9.4-T FT-ICR mass spectrometer at the National High Magnetic Field Laboratory (Tallahassee, FL). Samples were introduced by a syringe pump through a microelectrospray ionization source22 at a rate of 350 nL/min. All of the samples were analyzed in negative ionization mode with a needle voltage of -1.8 kV. Ions were stored in an octapole ion trap for 45 s before being transferred to a Penning trap.23 Approximately 200 time domain signals were added for a time period of ∼5 h. The summed FID signal was zero-filled once and Hanning-apodized before being processed by magnitude computation mode Fourier transformation. To obtain exact mass to charge values (m/z) and high mass resolution, experiments were performed in two steps. In the first step, an internal calibrant (poly(ethylene glycol) solution of 600 Da average molecular weight purchased from Sigma) was used as a mass internal calibration standard. Internal standard was injected into the FT-ICR cell along with the sample by use of dualspray injection.24 The peaks in the resultant spectrum were calibrated by reference to the exact m/z of internal calibrants. In the second step, the sample was analyzed without the poly(ethylene glycol). This was done to eliminate possible peak contributions from the calibrant material. The resulting spectrum’s m/z scale was first calibrated by use of an external calibrant material (G2421A electrospray “tuning mix” from Agilent) and, second, by the exact m/z numbers of major sample peaks obtained from the first spectrum. RESULTS AND DISCUSSIONS Mass Spectra and Peak Assignments. Figure 1 displays the calibrated mass spectrum of McDonalds Branch DOM. Over 5000 peaks with >4% relative abundance (corresponding to a signalto-noise ratio of 5) are detected in the mass range from 300 to 700 m/z. The peak resolving power (m/δm50%) is calculated to be over 300 000 at ∼300 m/z with an average resolving power of over (22) Emmett, M. R.; White, F. M.; Hendrickson, C. L.; Shi, S. D. H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1998, 9, 333-340. (23) Senko, M. W.; Hendrickson, C. L.; Emmett, M. R.; Shi, S. D. H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1997, 8, 970-976. (24) Hannis, J. C.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2000, 11, 876883.

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Table 1. List of Peaks Identified in the Expanded Spectrum (Figure 2) peak no.

Figure 2. The expanded view of the 469.0-469.3 m/z region of the ultrahigh-resolution mass spectrum of McDonalds Branch DOM. The numbers above peaks are used for identification in Table 1.

200 000 for the entire mass range (300 < m/z < 700). The complexity, also shown in previous studies,3,5,6,11,18 derives from the fact that DOM contains a multitude of natural products or their biodegraded residues, resulting in a multitude of peaks being observed at each nominal mass (see Figure 2). With the current instrument capabilities, we assume that the sharp peaks observed in Figure 2 are not further resolved. However, we must leave open the possibility that with higher field FT-ICR and attendant higher mass resolving power, we may, indeed, observe additional splitting of peaks. As previously observed,5,18 peaks in the vicinity of odd nominal m/z are dominant. Considering the low content of nitrogen (around 1%) in this sample, it is very unlikely that a significant part of even numbered peaks are from molecules with even numbers of nitrogen atoms. Rather, the majority of peaks at even m/z can be assigned to the 13C isotope of peaks at odd m/z, since in most cases, peaks at even m/z can be identified readily by adding the mass of a neutron to the mass of odd m/z peaks. A similar dominance of 13C isotope peaks was previously used to explain the even m/z peaks in high-resolution mass spectra of NOM.5 Accordingly, only peaks at odd m/z are considered for processing in this paper. By the same token, most of the observed peaks at odd m/z are found to be from singly charged ions, since corresponding 13C isotope peaks can be found at a unit mass difference. Therefore, mass instead of mass-to-charge ratio is used to indicate peaks in the spectrum through the rest of this paper. The elemental compositions of peaks exceeding a 4% of base peak threshold at odd mass are calculated from the corresponding exact mass numbers obtained from the calibrated spectrum. C, H, O, and N atoms are used to assign the most probable elemental formulas. The probability for molecules’ having two 13C atoms has been also considered in the calculation of elemental compositions, and no peaks with two 13C atoms have been observed. The compositions can be assigned with usually 480) portion of the spectrum, have more than one possible elemental formula. In those cases, Kendrick mass defect analysis is used to determine the assigned elemental formula, as was done in previous studies.5,9,11 To show the complexity of the spectrum, one mass unit region is selected and expanded (Figure 2). Eighteen peaks can be detected and assigned (Table 1). Therefore, examining individual peaks in the entire mass range (300 < m < 1000) and extracting information such as the distribution of classes of 5338 Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

proposed mol formula C25H10O10 C22H14O12 C26H14O9 C23H18O11 C27H18O8 C24H22O10 C28H22O7 C25H26O9 C29H26O6 C22H30O11 C26H30O8 C30H30O5 C23H34O10 C27H34O7 C31H34O4 C24H38O9 C28H38O6 C29H42O5

obs

theor

difference from theor value (ppm)

469.020 18 469.041 18 469.056 46 469.077 63 469.092 88 469.114 01 469.129 3 469.150 42 469.165 76 469.171 51 469.186 81 469.202 01 469.207 89 469.223 16 469.238 38 469.244 23 469.259 49 469.295 84

469.020 12 469.041 25 469.056 51 469.077 64 469.092 89 469.114 02 469.129 28 469.150 41 469.165 66 469.171 54 469.186 79 469.202 05 469.207 92 469.223 18 469.238 43 469.244 31 469.259 56 469.295 95

-0.1 0.1 0.1 0 0 0 0 0 -0.2 0.1 0 0.1 0.1 0 0.1 0.2 0.2 0.2

value

compounds directly from the conventional display of spectra represents a tremendous time and effort involved. The van Krevelen Diagram of Ultrahigh-Resolution Mass Spectrometric Data. The van Krevelen diagram facilitates information retrieval from assigned formulas. The plot, constructed from the assigned elemental compositions of each peak in the mass spectrum, is displayed in Figure 3. Most of the oddnumbered peaks (>95% of all odd-numbered peaks detected) in the mass spectrum are visually displayed in a single plot. The outliers possibly derived from noise spikes were excluded in the diagram. For the DOM sample, data are distributed in the form of a pattern. In the pattern, there are obvious blank spaces (for example, line A in Figure 3). One of the reasons for the pattern can be attributed to limitations in numbers of carbon, oxygen, and hydrogen atoms in the observed peaks. Given the mass range, the maximum numbers of carbon, oxygen, and hydrogen atoms in the compositions are 41, 20, and 58, respectively. In addition, the numbers of hydrogens are only even numbers. Because of these limitations, points cannot exist in certain areas of the plot. For example, since there is a maximum of 41 carbon atoms, the nearest points from any point with O/C ratio of 0.5 are 20/41 and 20/39. In other words, there cannot be any points either between the lines defined by O/C ) 0.5 and O/C ) 20/41 or between lines defined by O/C ) 0.5 and O/C) 20/39 in the plot, resulting in the empty space parallel to O/C ) 0.5 line (line B in Figure 3). Obviously, the pattern evolves from the fact that elemental compositions of the variety of peaks differ from each other by quantized ratios of the elements C, H and O. In the van Krevelen plot, trends along the lines can be indicative of structural relationships among families of compounds brought about by reactions that involve loss or gain of elements in a specific molar ratio.15 Lines from each reaction path have characteristic slopes or intercepts that can be easily demonstrated from mathematical calculations.15 The characteristics of the lines are summarized in Table 2. From these lines, a series of peaks, possibly products from various chemical reactions, can be visually identified. For example, a trend line representing methylation/ demethylation reactions always intersects the ordinate at an H/C

Figure 3. The van Krevelen plot for elemental data calculated from the ultrahigh-resolution mass spectrum of McDonalds Branch DOM. Distinctive lines in the plot representing chemical reactions are noted as: (A) methylation, demethylation, or alkyl chain elongation; (B) hydrogenation or dehydrogenation; (C) hydration or condensation; and (D) oxidation or reduction. Table 2. Characteristics of Lines Connecting Products of Various Chemical Reactions in the Van Krevelen Plota chemical reactions

characteristic of the line

methylation or demethylation1 hydrogenation or dehydrogenation2 hydration or dehydration3 oxidation or reduction4 decarboxylation

b)2 vertical line a)2 b)0 a ) 0; b ) 2

a a and b designate slope and intercept of a line defined by the equation H/C ) -a(O/C) + b. 1, 2, 3, and 4 correspond to lines A, B, C, and D, respectively, in Figure 3.

value of 2 (e.g., line A in Figure 3). Hydration/condensation reactions induce changes along a trend line with a slope of 2 (e.g., line C in Figure 3). It is apparent from Figure 3 that numerous trend lines in DOM can be clearly discerned. Several series of peaks from along the trend lines A, B, C, and D in Figure 3 are selected and further analyzed independently by Kendrick mass defect analysis9 (Figure 4). The Kendrick mass defect of points along a trend line represents a characteristic difference in the elemental formula (e.g., CH2, COO, H2, H2O, etc.). If we label these trendline variations as F, then the following equations can be used to define the Kendrick mass defect for any F trendline.

Kendrick mass (F) ) observed mass × [(nominal mass of F)/(exact mass of F)] (1) Kendrick mass defect (F) ) |nominal mass - Kendrick mass (F)| (2) For one particular Kendrick mass defect analysis (Figure 4a), F ) CH2, and peaks are normalized by the CH2 series, since the trend line (line A) can represent methylation/demethylation reactions or alkyl chain elongation (difference of CH2). Many different overlapping CH2 series are observed as horizontal trends among the data. Further investigation reveals that each horizontal series is related to another along a diagonal by the replacement of two hydrogen atoms with one oxygen atom (e.g., oxidation of a primary alcohol to acid). Peaks in line A can be chemically related to each other by a combination of demethylation or

oxidation reactions. It is possible that two reactions can occur in sequence (e.g., formation of a primary alcohol by demethylation and oxidation of a primary alcohol to an acid). Other normalizing units (e.g., F ) H2, H2O, and O) can be selected for Kendrick mass defect analysis and applied to data lying along the respective lines (lines B, C, and D in Figure 3) of the van Krevelen diagram (e.g., H2 ) hydrogenation/dehydrogenation). Kendrick mass defect plots for these are shown in Figure 4b, c, and d. The points in the characteristic trend lines are further divided into different series, and the possible relationships between each series were identified. For the H2-discriminated plot (Figure 4b), the points exist as families of compounds separated horizontally by the difference in mass of two hydrogen atoms and diagonally by masses corresponding to C2H4O. The peaks in line B can be chemically related to each other by a combination of chemical reactions, such as formation of an aldehyde or a ketone functional group by a dehydrogenation reaction, and by an aldol condensation reaction with an acetaldehyde. The points in the H2O-discriminated plot (Figure 4c) exist as families of compounds separated horizontally by a difference of H2O and diagonally by CH2O. In the O-discriminated plot (Figure 4d), compounds separated by a difference in mass of an oxygen atom lie along horizontal lines and by masses corresponding to C2H2 diagonally. Whether the apparent trends originate from a series of transformations of compounds that could have contributed to the DOM or are inherently produced by different paths from the multitude of biosynthetic reactions cannot be absolutely ascertained. Nonetheless, one can use the van Krevelen diagram to initially sort compounds that could be related. Moreover, the diagram can also be used to sort compounds that may have common structural relationships. It is important to point out that the genetic relationships among compounds identified by their elemental formulas is tenuous at best unless one has prior knowledge of a diagenetic reaction pathway leading to the transformation of precursors to products. Van Krevelen15 used this diagram to examine reactions of a series of coals that could be viewed as diagenetic homologues. Thus, one could excise specific reaction pathways on the basis of the knowledge that product-precursor relationships existed by nature of the way coal is formed (e.g., sequential burial after deposition). In DOM, all reaction states (products and precursors) exist in the same sample because DOM is an integrated accumulation of Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

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Figure 4. Kendrick mass defect analysis of peaks on trend lines identified in Figure 3: (a) points in trend line A (Figure 3) analyzed by CH2 Kendrick mass defect analysis, (b) points in line B by H2 Kendrick mass defect analysis, (c) points in line C by H2O Kendrick mass defect analysis, and (d) points in line D by O Kendrick mass defect analysis.

organic matter derived from a multitude of sources at various levels of diagenetic history. Thus, we might expect to visualize in the van Krevelen plot a complete diagenetic series and, no doubt, some of the trendlines observed may, indeed, reflect such series. The van Krevelen Diagram As an Indicator of Structural Characteristics. Clusters of peaks in high-resolution mass spectra can be structurally related to families of similar compounds. Historically, an approach employing “elemental mapping” has been used to identify major components of complex high-resolution mass spectra that were obtained by traditional electron impact techniques.25,26 This elemental mapping has not been applied to ESI spectra because, in the traditional sense, fragmentation information used in these previous studies is not readily available with ESI. We propose that the van Krevelen diagram, which can be used to identify the types of compounds that comprise different types of natural organic matter,15-17,27 achieves the same goal as elemental mapping has for EI spectra. This is possible because major biomolecular components of source materials, mainly the products derived from plants, occupy fairly specific locations on the plot. In previous studies,15-17,27 the positions of classes of biologically derived compoundsslipids, cellulose, lignins, proteins and condensed polyaromatic type carbonsshave been noted, and the positions are reproduced on the plot shown in Figure 5. A qualitative analysis of the major classes of components contributing to DOM can be made using the locations of the peaks on the van Krevelen diagram with certain assumptions. The first of these (25) Biemann, K.; Bommer, P.; Desiderio, D. M. Tetrahedron Lett. 1964, 26, 1725-1731. (26) Burlingame, A. L.; Schnoes, H. K. In Organic Geochemistry; Eglinton, G., Murphy, M. T. J., Eds.; Springer-Verlag: New York, 1969; pp 89-160. (27) Visser, S. A. Environ. Sci. Technol. 1983, 17, 412-417.

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is that the peaks are representative of the material being characterized. This requires that ionizing efficiency be the same for all molecules, an assumption we know is not entirely valid. However, we know that natural organic matter is mostly composed of highly oxygenated molecules, which have a tendency to spray and ionize well. Until such time that a calibration can be devised for ionization discriminations, we must view the above assumption with a certain skepticism. The second assumption is that we do not include molecules having two 13C atoms. This has already been addressed. The third assumption is that charged molecules do not dissociate or undergo ion-molecule reactions. Although there has been no conclusive study suggesting that ESI analysis of humic substances is free from those problems, ESI is generally considered to be a soft ionization technique inducing minimal or no fragmentation. Stenson et al.11 studied humic and fulvic acid mixtures and could not confirm that fragmentation is important. To test the usefulness or limitations of this methodology, we analyzed a humic acid sample that was bleached to remove substituted aromatic components, such as lignin. In a previous study,19 we demonstrated by solid-state CP/MAS 13C NMR that the bleached peat humic acid showed structural characteristics similar to those of long-chain fatty acids and esters. The FT-ICRMS data for this bleached humic acid was obtained, and the elemental compositions were calculated and plotted on a van Krevelen diagram (Figure 6a). Most of the peaks in the plot are located in the area where O/C ratios are between 0 and 0.2 and H/C ratios are between 1.6 and 2. The data points fall in the range expected for fatty acid-like (or lipid-type) structures (see Figure 5). It is also apparent that several lines converging at (0, 2) can be drawn. This indicates that this bleached humic acid sample

Figure 5. Regional plots of elemental compositions from some major biomolecular components on the van Krevelen diagram, reproduced from previous studies.15-17,27 The arrow designates a pathway for a condensation reaction.

contains a series of hydrogen-rich molecules that are different by integer numbers of CH2. From this and a previous study,19 we can conclude that the mass spectrum of this sample contains mainly a homologous series of fatty acid-type molecules differing by CH2, confirming that the approach yields information consistent with the NMR data. The specific details for components identified in this sample will be reported at a later date because it is beyond the scope of the present study. In yet another test, we obtained FT-ICR-MS data for a humic acid extracted from an ancient wood sample having associated charcoal. Reducing the data to elemental formulas and plotting on a van Krevelen diagram reveals three regions of the plot where the data cluster (Figure 6b). Previous studies12,28 have demonstrated that the coalified wood (brown coal) samples from the Morwell brown coal in Australia are enriched in lignin and ligninderived structures. The O/C and H/C ratios, clustering between 0.3 and 0.7 and between 1 and 1.5, respectively (B in Figure 6b), certainly plot in the range expected for lignin (refer to Figure 5). The other clustered region of peaks (A in Figure 6b) is most consistent with structures that are likely to be associated with the charcoal in the sample. Hydrogen-poor structures consisting of condensed aromatic rings having sufficient oxygen functional groups to render them soluble in basic polar solvent are most likely responsible for these data. The third region (C in Figure 6b) corresponds to cellulosic materials that are known to be present in such samples.12,28 Although the results from the van Krevelen analyses of the above two samples (the bleached peat humic acid and the humic acid extracted from an ancient wood sample) are consistent with expected compositions on the basis of analyses by other techniques,12,19,28 the possibility always exists that we are discriminating for molecules that spray and ionize well. Therefore, one should be cautious to conclude that major classes of components delineated by the van Krevelen analysis accurately reflect the major components of analyzed samples. We should accept the fact that the van Krevelen analysis deals primarily with readily ionizable components. The van Krevelen diagram of compounds in the DOM (Figure 3) is more complicated than the previous two diagrams. Peaks are located in a broader region with O/C ratios between 0.1 and 0.7 and H/C ratios between 0.4 and 1.7. Comparison with Figure 5 shows that this region corresponds to mainly lignin-type (28) Bates, A. L.; Hatcher, P. G. Org. Geochem. 1989, 14, 609-617.

molecules. The water sample from McDonalds Branch had a dark tea color and relatively high total organic carbon (TOC) values (averages 16-18 mg/L). Organic-rich soil materials leaching from the soils in the area29 are primarily responsible for the color and high TOC values. Therefore, humic substances associated with the surrounding terrestrial vegetation would compose the major part of the analyzed DOM sample. The strong contribution from lignin-type molecules to the mass spectrum is understandable, because lignin has been widely considered to be a major portion of humic substances.30 In fact, Stenson et al.11 examined fulvic acid from a similar black water river by FT-ICR-MS and found peaks that could be structurally tied to modified lignin molecules. The peaks in the area could also be derived from tannin-like molecules, since tannin molecules would have H/C and O/C ratios similar to those of lignin-type molecules. Some of the points in the diagram can also be related to condensed (e.g., dehydrated) cellulose-type molecules, because the condensation reaction would move the points in the cellulose region toward the direction noted as an arrow in Figure 5. There could also be contributions from lipid-type structures that have undergone extensive oxidation. Other major biomolecules, proteins, were not considered as major contributors because of the low nitrogen content of the analyzed DOM sample.18 Although a solid-state CP/MAS 13C NMR spectrum of this sample is not available, we have previously obtained a total correlation spectroscopy (TOCSY) NMR spectrum of the same sample18 for comparison. The major structural categories of protons found in the NMR spectrum are aromatic, carbohydrate, methoxy, aliphatic hydroxy, and aliphatic protons.18 The proton NMR signals can be easily explained as originating from lignin, condensed carbohydrate, and oxidized lipid-type molecules. Another aspect of the plot shown in Figure 3 that should be pointed out is the existence of molecules with apparently low H/C ratios (e.g., H/C ratio of ∼0.5). A significant number of points are found in that area of the plot. The low H/C ratio indicates a significant deficiency in H among the molecules that could indicate presence of condensed ring structures. One of the identified elemental compositions with a low H/C ratio was selected (C26H14O9), and a possible chemical structure for the molecule is presented in Figure 7. Although multiple isomeric structures can (29) Maurice, P. A.; Leff, L. G. Water Res. 2002, 36, 2561-2570. (30) Stevenson, F. J. Humus Chemistry, 2nd ed.; John Wiley & Sons: New York, 1994.

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Figure 6. The van Krevelen plot for elemental data obtained from the ultrahigh-resolution mass spectrum of (a) peat humic acid that has been bleached19 and (b) humic acid extracted from a Morwell brown coal wood sample. Refer to text for assignment of peaks in circles A, B, and C.

Figure 7. A possible chemical structure for an observed peak (C26H14O9) in the mass spectrum of McDonalds Branch DOM.

be assigned to this molecular formula, it is apparent that the molecular formula contains a condensed ring system not unlike what might be expected for black-carbon derived material. Further studies are being conducted to evaluate this portion of DOM for possible presence of molecules derived from black carbon or charcoal. The 3D van Krevelen Diagram: a Visual Tool To Understand and Compare Complicated Mass Spectra. In addition 5342

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to the mass-to-charge ratios and calculated elemental formulas, the peak intensities or relative intensities are important pieces of information offered by mass spectrometric analysis. Intensities or relative intensities can be used in a semiquantitative way to differentiate between similar types of compounds or samples with same conditions. Therefore, it is beneficial to retain peak intensity information in the display. The relative intensity of peaks in the mass spectrum can be added to the van Krevelen diagram as a z axis, resulting in a 3D display. The 3D van Krevelen plot of ultrahigh-resolution data from McDonalds Branch DOM was constructed and displayed in Figure 8a. Colors of points are varied according to their relative peak height in the mass spectrum to make the plot more readable. A plan view of the 3D plot is also presented (Figure 8b). A possible application of the 3D van Krevelen plot is for an intersample comparison. Conventional 2D van Krevelen plots that appear to be very similar to one another may, in fact, be different

Figure 8. 3D display of the van Krevelen plot (a) of the peak intensities and elemental data obtained from the ultrahigh-resolution mass spectrum of McDonalds Branch DOM and plan view (b). Colors of points were varied according to relative peak intensities. The intensities increase in the order blue, green, yellow, orange and red.

when expanded to 3D by inclusion of the peak intensities. The relative significance of each class of compounds among samples can be different. This approach could be limited to compare similar types of compounds or samples until more is known about the electrospray ionization process. Because there are numerous parameters that could affect the relative intensities of peaks (ionization efficiencies, mobile phase composition, data acquisition parameters, etc.), specific use of intensities is at best a relative approach. Nontheless, 3D plots can provide another dimension when multiple spectra are compared. Therefore, interpretation and comparison of multiple spectra could be more complete with a 3D display. From the mass spectral data set that includes intensity and elemental composition, one can calculate a weighted mean H/C and O/C ratio. For the DOM from McDonalds Branch, we obtain a value of 1.13 and 0.34 for the H/C and O/C, respectively. These compare with values of 1.19 and 0.80 measured for DOM by combustion analysis. Although the H/C ratios agree well, the O/C ratios are vastly different. We attribute the elevated O/C from combustion analysis to the lack of sufficient material, which did not allow for an ash determination. The O content, calculated by difference, requires an ash determination that was not possible, so it is clearly suspect because any ash would be included. The discrepancy can also be related to possible ionization discrimination during the ESI process. For example, although carbohydratederived compounds are known to be a small but important part of DOM,32 peaks corresponding to elemental compositions of carbohydrate molecules are not observed in this study. This may (31) Leenheer, J. A.; Rostad, C. E.; Gates, P. M.; Furlong, E. T.; Ferrer, I. Anal. Chem. 2001, 73, 1461-1471. (32) Thurman, E. M. Organic Geochemistry of Natural Waters; Martinus Nijhoff/ Dr. W. Junk Publishers: Boston, 1985.

be attributed to the possible ionization discrimination for these types of molecules in the ESI process. CONCLUSIONS In this study, the van Krevelen diagram is shown to be an effective and informative graphical method for displaying complex ultrahigh-resolution mass spectrometric data of complex mixtures. By employing van Krevelen diagrams, it is possible to investigate and visually present plausible reaction pathways of molecules displaying resolved peaks in an ultrahigh-resolution mass spectrum. Qualitative or semiquantitative analyses of major classes of compounds that comprise the complicated spectra can be accomplished readily by use of visual displays on the van Krevelen diagrams. The van Krevelen analysis was shown to be consistent with NMR analysis with regard to qualitative identification of major classes of compounds contained in NOM samples. Peak intensities or relative intensities in mass spectra can be included in a 3D van Krevelen plot. The resulting plot would make the interpretation of ultrahigh-resolution more complete by adding one more dimension over conventional 2 D diagrams. By applying the van Krevelen analysis to ultrahigh-resolution spectra of DOM, we have characterized the types of molecules composing the main portion of the readily ionizable part of DOM. It is surprising to find that there are molecules in DOM that have structures like what might be expected for black-carbon-derived material. Existence of black carbon in DOM could be an important finding to understand the fate of the material in a natural environment. More research is currently being conducted on the subject. Further, van Krevelen analysis could help to delineate diagenetic processes for natural organic matter by providing a method to examine the chemistry of compounds undergoing biogeochemical changes. For example, ultrahigh-resolution mass Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

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spectrometry and van Krevelen analysis can be applied to study heterotrophic degradation of natural organic matter. The comparison between natural organic matter before and after biodegradation is currently under investigation in our laboratory. Clearly, one could employ this approach to examine a series of diagenetically altered plant materials to develop a molecular-level understanding of the pathways for degradation.

National High Magnetic Field Laboratory (CHE-9903528) for their assistance in FT-ICR analysis; and Hyung Min Cho for his help in programming. Dr. Michael A. Freitas, Dr. Sarah Pilkenton, and Dr. Rakesh Sachdeva provided helpful discussions on the interpretation of the DOM data. This work was supported by NSF Grants CHE-0089172, CHE0089147, and DEB-9904047 at The Ohio State University.

ACKNOWLEDGMENT The authors thank Dr. Louis A. Kaplan of Stroud Water Research Center for providing McDonalds Branch water samples; Dr. Alan G. Marshall, Dr. Ryan Rodgers, and Zhigang Wu at the

Received for review April 21, 2003. Accepted July 29, 2003.

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