Limitations of Electrospray Ionization of Fulvic and Humic Acids as

Natural Organic Matter and the Event Horizon of Mass Spectrometry. N. Hertkorn , M. Frommberger , M. Witt , B. P. Koch , Ph. Schmitt-Kopplin and E. M...
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Anal. Chem. 2003, 75, 6275-6281

Limitations of Electrospray Ionization of Fulvic and Humic Acids as Visible from Size Exclusion Chromatography with Organic Carbon and Mass Spectrometric Detection Anja These and Thorsten Reemtsma*

Department of Water Quality Control, Technical University of Berlin, Sekr KF 4, Strasse des 17 Juni 135, 10623 Berlin, Germany

A method was developed to link size exclusion chromatography electrospray ionization mass spectrometry (SECESI-MS) analyses of fulvic and humic acids with SEC and organic carbon detection (SEC-OCD), the latter providing an absolute measure of the amount of organic matter eluting from the SEC column. This approach allows us to determine which molecular weight fraction of the complex polydisperse mixtures is detectable by ESI-MS. It could be shown that the cone voltage setting for the ESI interface has strong impact on ESI-MS detection. Using conventional settings for low molecular weight compounds, the high molecular weight (HMW) compounds are hardly amenable to ESI-MS. With increasing cone voltage, an increasing signal intensity is obtained for the HMW fraction that elutes at shorter retention times. However, mostly fragment ions are obtained under these conditions. Thus, the range of compounds amenable to ESI-MS analysis is restricted by the limited stability of the fulvic and humic acid molecules of higher molecular weight in the electrospray process rather than by the mass spectrometer used. Compounds above 1000 amu are hardly visible as intact ions. However, insight into structural characteristics of these compounds can be gained by investigating their fragment ions by SEC-ESI-MS. The use of SEC-OCD parallel to SEC-MS helps to assess and optimize the detection potential of ESI-MS for polydisperse mixtures. In recent years, electrospray ionization (ESI) has developed into the most widely used ionization method for the mass spectrometric (MS) investigation of aquatic fulvic and humic acids. It has been coupled to quadrupole1 and triple-quadrupole2-4 mass spectrometers, to ion traps3 and quadrupole time-of-flight mass spectrometers,5,6 and to Fourier transform ion cyclotron resonance mass spectrometers.6-9 These ESI-MS applications have led to * Corresponding author. E-mail: [email protected]. (1) Persson, L.; Alsberg, T.; Kiss, G.; Odham, G. Rapid Commun. Mass Spectrom. 2000, 14, 286-292. (2) McIntyre, C.; Batts, B. D.; Jardine, D. R. J. Mass Spectrom. 1997, 32, 328330. (3) Leenheer, J. A.; Rostad, C. E.; Gates, P. M.; Furlong, E. T.; Ferrer, I. Anal. Chem. 2001, 73, 1461-1471. (4) Reemtsma, T.; These, A. Anal. Chem. 2003, 75, 1500-1507. 10.1021/ac034399w CCC: $25.00 Published on Web 10/04/2003

© 2003 American Chemical Society

(sub-) structure proposals for fulvic acids3,5 and to the detection of molecular masses of individual fulvic acid molecules.6,7 In the majority of these examples, aqueous solutions of the fulvic acids were infused into the ES interface without a previous separation. Obviously, not all of the very large number of analytes infused simultaneously into an electrospray interface are equally well ionized and detected by mass spectrometry. But with the infusion approach it was impossible to clarify which part of the mixture was detected and which was overlooked. Correspondingly, some researchers were optimistic that infusion-ESI-MS is suitable to determine the average molecular weight of fulvic acids, whereas others discussed systematic difficulties at the same time, such as multiple charging,3 in-source fragmentation,2 or mass dependent ionization.9 Method optimization for natural organic matter and, more specifically, fulvic and humic acids is complicated by the fact that these are polydisperse mixtures of several and only partly known compound classes. Molecularly defined standard compounds are not available, nor are relevant model compounds widely accepted. This even holds true after the pioneering work of Cooper and co-workers, who have very recently identified the molecular formulas of several thousand individual fulvic acid molecules by Fourier transform ion cyclotron resonance mass spectrometry.7 Without external information it would, therefore, be impossible to decide whether the information provided by a certain method reflects the whole mixture or is relevant only for a minor fraction of it. Recently, humic and fulvic acid analysis by ESI-MS was improved by a preceding on-line size exclusion chromatography (SEC).4 Compared to the infusion approach, SEC-MS provides separation according to the hydrodynamic volume, which reduces the polydispersity of the mixtures10 being introduced into the mass (5) Plancque, G.; Amekraz, B.; Moulin, V.; Toulhoat, P.; Moulin, C. Rapid Commun. Mass Spectrom. 2001, 15, 827-835. (6) Kujawinski, E. B.; Freitas, M. A.; Zang, X.; Hatcher, P. G.; Green-Church, K. B.; Jones, R. B. Org. Geochem. 2002, 33, 171-180. (7) Stenson, A. C.; Marshall, A. G.; Cooper, W. T. Anal. Chem. 2003, 75, 12751284. (8) Brown, T. L.; Rice, J. A. Anal. Chem. 2000, 72, 384-390. (9) Stenson, A. C.; Landing, W. M.; Marshall, A. G.; Cooper, W. T. Anal. Chem. 2002, 74, 4397-4409. (10) Hunt, S. M.; Sheil, M. M.; Derrick, D. J. Eur. Mass Spectrom. 1998, 4, 475-486.

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spectrometer at a given time. With SEC-MS, a distinction could be made between low molecular weight (LMW) molecular ions and low molecular weight fragment ions of high molecular weight (HMW) compounds.4 A comparison of the SEC-MS and SEC-UV chromatograms suggested that ESI-MS with a quadrupole mass spectrometer detected LMW components of humic and fulvic acids more sensitively than HMW compounds.4 This could be due to (a) a more effective ionization of the LMW compounds in the electrospray process, (b) fragmentation of HMW compounds during the ionization process, (c) a more effective transfer of LMW ions generated at atmospheric pressure into the mass spectrometer, or (d) a systematic decrease of the transmission of the mass analyzer for ions with increasing m/z ratio toward the upper limit of the instrumental mass range. However, the UV response of the analytes in a fulvic acid mixture can vary considerably, and this could have been responsible for the intensity differences observed between UV and MS detection. An absolute measure of the amount of organic material eluting from a SEC column is provided by the so-called organic carbon detection (OCD).11-13 Therefore, we developed an approach to combine the well-established SEC-OCD analysis and the recently developed SEC-MS analysis. With this approach, information is provided on (i) what can be detected from fulvic and humic acids by ESI-MS and (ii) how instrumental conditions affect the range of fulvic and humic acids that is detected by the mass spectrometer. This combination of SEC-MS and SEC-OCD provides insight into the width of the analytical window opened by ESI-MS for dissolved fulvic and humic acids. Such a combination of organic carbon detection and mass spectrometric analysis could be helpful in any situation, where complex and ill-defined polymer mixtures are to be analyzed by ESI-MS and in which an assessment is to be made on what is amenable to ESI-MS. MATERIALS AND METHODS Materials. All studies were performed with Suwannee River fulvic acid (SRFA) and humic acid (SRHA) from the International Humic Substances Society (IHSS) and lignin sulfonates (SigmaAldrich, Taufkirchen; Germany). Standard solutions of 1 g/L were freshly prepared in ultrapure water before use. Reference compounds used for retention time correction were poly(styrene sulfonates) with average molecular weights (Mw) of 4400, 6620, 13 400 and 20 700 amu (Polymer Standards Service, Mainz; Germany) and trimellitic acid, 3,4-dihydroxyphenylacetic acid, and benzene-1,2,4,5-tetracarboxylic acid (Sigma-Aldrich). Instrumentation. SEC-MS. These analyses were performed at an LC-MS system consisting of a HP 1100 (Hewlett-Packard) liquid chromatography system with a diode array detector that was coupled to a Quattro LC triple-quadrupole mass spectrometer (Micromass, Manchester, U.K.) with a Z-Spray electrospray ionization source and a 4000 amu mass range. The mass spectrometer was operated in the negative mode. A PL AquagelOH 30 SEC column from Polymer Laboratories (Shropshire, U.K.) (11) Gloor, R.; Leidner, H.; Wuhrmann, W.; Fleischmann, T. Water Res. 1981, 15, 457-462. (12) Huber, S. A.; Frimmel, F. H. Environ. Sci. Technol. 1994, 28, 1194-1197. (13) Her, N.; Amy, G.; Foss, D.; Cho, J.; Yoon, Y.; Kosenka, P. Environ. Sci. Technol. 2002, 36, 1069-1076.

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of 250 × 4.6 mm i.d. and a particle size of 8 µm was used. The column had a nominal molecular weight range of 100-30 000. Separation was performed at a flow rate of 0.3 mL/min with an eluent consisting of 80/20 (v/v) water/methanol with 10 mM NH4HCO3. A more detailed description has been given elsewhere.4 SEC-OCD. A HPLC system consisting of a Rheodyne 7125 injector, a L6100A gradient pump, and a T-6300 column thermostat (both from Merck-Hitachi, Darmstadt, Germany) was connected to the detection unit of a commercial SEC-OCD system (DOC laboratory Dr. Huber, Karlsruhe, Germany). This unit consisted of a WellChrom K 200 UV detector (Knauer, Berlin, Germany) and the OCD detection system (oxidation reactor and IR detector). To reduce the dead volume of this system, the effluent of the UV detector was directly introduced into the oxidation section of the reactor, while phosphoric acid (0.34%) was introduced at the reactor top. The same model of SEC column as in SEC-MS analyses was used but with an aqueous buffer (18.4 mM KH2PO4 and 8.4 mM Na2HPO4) at a flow rate of 0.3 mL/min. Data Treatment. Adjustment of Retention Times. Chromatographic data of both systems, LC-UVOCD-OCD (Simplex Numerica 5.2 software) and the LC-UVMS-MS (Masslynx 3.5; Micromass), were transferred to a spreadsheet program. Retention times (Rt) obtained by the UV detector in the LC-UVOCD-OCD system (UVOCD) were used as the reference, whereas the retention times of the other three detectors were corrected for differences in the chromatographic separation, dead volumes, and the chemical reaction of the OCD system. The equations used for these Rt transformations are given below. Determination of Peak Areas. Chromatographic data of the OCD and the MS detector were available in spreadsheet format from previous retention time corrections (see above). The following procedure was used to detect the coverage of the SEC-OCD chromatogram by the SEC-MS chromatograms recorded at different cone voltages: (i) all total ion chromatogram were normalized to the maximum signal intensity of the whole series. (ii) Then, the retention time of the intersect of the OCD chromatogram and each MS chromatogram was located in the spreadsheet table. (iii) Using the Simplex Numerica software, the area of the SEC-OCD chromatogram up to its intersect with each of the MS chromatograms was calculated. RESULTS AND DISCUSSION Linking SEC-MS and SEC-OCD. The usual concept when two detection systems are required in a chromatographic system is to link both detectors either in parallel or in series after the chromatographic column. However, a coupling of OCD and MS detection with one SEC system is not possible, since SEC separation requires the addition of an inorganic modifier to reduce ionic interactions between analytes and the column, but a volatile salt without carbon does not exist. For example, the completely volatile methanolic ammonium bicarbonate buffer previously developed for SEC-MS4 is not suited for the OCD, due to its inorganic and organic carbon content and the phosphate buffer routinely used in SEC-OCD systems12,13 is not volatile and, thus, not ideal for MS detection. Thus, SEC-OCD and SEC-MS analyses had to be performed in parallel. However, using different eluents in SEC-OCD and SECMS may result in different separations with the two SEC systems

tively large volume. This resulted in a peak broadening of the OCD signal, that was stronger for its descending flank than for its ascending flank (Figure 2b). The average peak broadening of the ascending and the descending flanks of the OCD signals as compared to the UVOCD signal was determined for seven compounds, and one average factor was derived for each of the flanks to compensate the average peak broadening (step C in Figure 2b).

for the ascending flanks: Rt,UV(OCD) ) R ′′t,OCD ) R ′t,OCD,max (R ′t,OCD,max - R ′t,OCD) × 0.62 (min) (4)

Figure 1. Retention times of the SEC-UVOCD versus SEC-UVMS for a set of standard compounds.

with R ′t,OCD,max being the retention time of the peak maximum corrected for the dead volume according to eq 3.

for the descending flanks: and this would hamper comparison of the OCD and MS chromatograms. Therefore, an UV detector was coupled in each system and a total of four detections were performed: SEC-UVMSMS and SEC-UVOCD-OCD. The UV chromatograms were used to compare the separation obtained with the two different buffer systems and, if possible, to link both separations. Since an adequate correction of retention times (Rt) between the two SEC systems and the four detectors was essential for a comparison of the SEC-MS and the SEC-OCD trace, a series of standard compounds (poly(styrene sulfonates), aromatic carboxylates, SRFA, SRHA, LSA) was analyzed on both systems. It was most important to check whether the SEC separations obtained with the two buffer systems (OCD and MS buffer) were comparable. Though the separation was not completely the same, a linear correlation existed for Rt in both systems (Figure 1; r2 ) 0.9959, n ) 9). Thus, the following linear calibration equation was used to transform Rt values of the UVMS system into Rt values of the UVOCD system (step B in Figure 2a).

Rt,UV(OCD) ) R ′t,UV(MS) ) Rt,UV(MS) × 1.4198-1.9707 (min) (1) However, additional factors that affect the retention time have to be compensated for, based on the analysis of the standard compounds. This is illustrated for one standard compound in Figure 2: Different dead volumes between the two detectors coupled in series led to a constant time shift between the two chromatograms of each system (UVMS and MS, UVOCD and OCD). This can be easily compensated for by a fixed Rt shift (step A in Figure 2).

for the MS trace: Rt,UV(MS) ) R ′t,MS ) Rt,MS - 0.13 (min)

(2)

for the OCD trace: R ′t,OCD ) Rt,OCD - 2.08 (min)

(3)

Organic carbon detection requires chemical reactions and physicochemical processes to occur in a reactor unit of compara-

Rt,UV(OCD) ) R ′′t, OCD ) R ′t,OCD,max (R ′t,OCD,max - R ′t,OCD) × 0.13 (min) (5)

In this way, it was possible to link the SEC-OCD chromatogram of a sample with its SEC-MS chromatogram and, then, to compare the signals obtained by each of the detection methods. Parallel SEC-MS and SEC-OCD Analysis of a Fulvic Acid Standard. The same adjustment process was applied to the SECMS and SEC-OCD chromatograms of SRFA (Figure 3). After that adjustment based on the average correction factors, the two UV traces (UVOCD and UVMS) of SRFA coincide, indicating that the retention time adjustment between the two SEC systems was performed correctly. However, the OCD and the MS chromatograms do not coincide, but the maximum of the MS chromatogram appears at longer retention times than the maximums of the UV and the OCD traces. Moreover, the early-eluting fractions of this SRFA mixture are not at all detected by ESI-MS under the selected conditions. Obviously, ESI-MS tends to detect components of moderate and low molecular weight more sensitively than HMW compounds, as previously suggested.4,9 There are several possible reasons for a lower sensitivity of the ESI-MS toward HMW compounds. One could suggest that singly charged anions of the HMW compounds simply exceed the m/z range of the mass spectrometer.9 However, no significant signal intensity was ever detected in the range m/z 1500-4000 under any conditions with the ESI-quadrupole MS, implying that other effects are more critical. Indeed, a part of the discrepancy in the sensitivity of OCD and ESI-MS toward HMW compound can be ascribed to the fact that the OCD is a mass proportional detector whereas the ESIMS is expected to be a number proportional detection system.14 Therefore, the ESI-MS must lose sensitivity as compared to the OCD with increasing molecular weight of the analytes (shorter retention times). Cone Voltage Effect on SEC-MS Chromatograms. Now, that the organic carbon detection clarified that ESI-MS detected a limited range of fulvic acid compounds only, attempts were made (14) Koster, S.; Mulder, B.; Duursma, M. C.; Boon, J. J.; Philipsen, H. J. A.; van Velde, J. W.; Nielen, M. F. W.; de Koster, C. G.; Heeren, R. M. A. Macromolecules 2002, 35, 4919-4928.

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Figure 2. Process of adjusting the four chromatograms obtained for benzene-1,2,4,5-tetracarboxylic acid as standard. (a) SEC-UVMS-MS system: step A, dead volume correction (according to eq 2); step B, adjustment of retention times to those of the SEC-OCD system (according to eq 1 derived from Figure 1); (b) SEC-UVOCD-OCD system: step A, dead volume correction (according to eq 3; step C, correction of peak broadening during organic carbon detection (according to eqs 4 and 5).

Figure 3. Four chromatograms of a fulvic acid standard obtained by SEC-UVMS-MS and SEC-UVOCD-OCD after retention time adjustment as in Figure 2: (top) the two UV chromatograms; (bottom) OCD and MS chromatogram. The SEC-MS chromatogram was obtained by scan analysis from m/z 150 to 750; cone voltage 20 V.

to extend the detection range of ESI-MS toward HMW compounds. It has previously been shown for synthetic polymers such as poly(ethylene glycol) (PEG) that the signal intensity of the higher homologues continuously increased with increasing cone voltage.15 The authors outlined that the strong dependency on the cone voltage was not due to competiton in the ionization process. Rather they showed that the degree of deflection of the analyte ions orthogonal to the electrospray beam axis and toward (15) Hunt, S. M.; Sheil, M. M.; Belov, M. B.; Derrick, P. J. Anal. Chem. 1998, 70, 1812-1822.

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the aperture into the low-pressure region of the mass analyzer is the critical process. Since this deflection is affected by the massto-charge ratio of a given analyte ion, the mass dependency of the optimum cone voltage was strongest for singly charged ions.15 A similar dependency may also exist for fulvic and humic acids. Indeed, a stepwise increase of the cone voltage led to an increase in the TIC and to a shift of the peak maximum toward shorter elution times in SEC, which are occupied by HMW compounds (Figure 4). This trend was found for all three mixtures investigated, SRFA, SRHA, and lignin sulfonic acids (LSA). For SRFA, the TIC came very close to the OCD chromatogram at a cone voltage of 140 V, suggesting that a proper optimization of the cone voltage makes the full mass range of SRFA accessible to ESI-MS (Figure 4a). Increasing signal intensities in ESI-MS in the HMW range with increasing cone voltage were also found for SRHA (Figure 4b) and LSA (Figure 4c). For these mixtures with a larger HMW contribution as compared to SRFA, a larger difference remained between the OCD and the MS maximums. As a first estimate of the amount of organic matter not amenable to ESI-MS detection at a given cone voltage, the area of the OCD chromatogram was calculated that was not covered by the MS chromatogram in a chromatogram overlay (Table 1). The organic carbon not amenable to MS detection drastically decreases with cone voltage increase from 20 to 140 V for all three compound classes, from ∼60 to ∼10% for both the SRFA and the SRHA and from 90 to 30% for LSA. For the mixture with the lowest average molecular weight, the SRFA, 100 V appears to be an optimum with respect to organic carbon coverage. These results suggest that a higher cone voltage improves the accessibility of HMW compounds to ESI-MS and that it may finally be the mass range of the mass analyzer that determines the HMW cutoff of an ESIMS system. Cone Voltage Effects on Mass Spectra. This view is, however, not confirmed by the mass spectra that were recorded at different cone voltages. As shown here for SRFA, the spectrum obtained for the HMW fraction at a low cone voltage that would be used for LMW compounds (Figure 5a, 20 V) shows a

Figure 4. Combined SEC-OCD and SEC-MS chromatograms of (a) fulvic acids, (b) humic acids, and (c) ligninsulfonic acids at cone voltages of 20, 60, and 140 V. SEC-MS chromatograms were obtained by scan analyses from m/z 150 to 750. Table 1. Area Percentage of the SEC-OCD Chromatogram Not Covered by SEC-MS at Cone Voltages of 20-140 V for Fulvic Acids (SRFA), Humic Acids (SRHA), and Ligninsulfonic Acids (LSA) voltage

SRFA SRHA LSA

20

40

60

100

140

56 57 87

47 45 71

33 31 67

11 17 50

20 12 28

multimodal “wavy” intensity distribution with maximums around m/z 200, 500, and 900. This distribution has been interpreted as being the sum of HMW anions and their fragments.4 This spectrum already suggests that HMW molecules of fulvic and humic acids are labile and prone to fragmentation into their LMW subunits. With increasing cone voltage (Figure 5b), the ion intensity distribution was shifted toward the lower mass range, reflecting a much stronger in-source fragmentation of molecular anions and of larger fragment ions and the generation of small product ions. For SRHA and LSA, the same shift toward lower m/z ions in the mass spectra with increasing cone voltage was

found (not shown). This cone voltage effect is in agreement with but more pronounced than in previous reports.1,2 In principle, a signal increase in the low-m/z region could also be caused by an increasing charge (z) of the analyte ions instead of a decreasing mass (m). Singly charged ions would not be resolved from doubly or even triply charged ions in these mass spectra, but an increase in the charging at a cone voltage of 140 V would result in a broadening of the signals as compared to the 20-V spectrum. Such a signal broadening is not visible (see insets in Figure 5), and it is, thus, unlikely that multiple charging is the reason for the shift of the intensity distribution toward low-m/z values. Indeed, increasing the cone voltage usually reduces multiple charging and favors the formation of singly charged ions. Concerning the cone voltage influence, two other effects are more likely:15 (i) a more effective deflection of heavier anions toward the aperture of the mass spectrometer with increasing cone voltage and, thus, an increasing total ion intensity; (ii) a higher degree of in-source fragmentation with increasing cone voltage caused by increased energy of collision with the background gas. This leads to the detection of fragment ions of low molecular weight. In the case of fulvic acids, these two effects give rise to a ion intensity maximum at an intermediate cone voltage of 60 V (Figure 6a). Such an “optimum” was not visible for the humic acid mixture. Instead, the total ion intensity continuously increased with increasing cone voltage, based on increasing ion intensities in the mass ranges m/z 150-400 and 400-700 (Figure 6b). This different interrelationship between the cone voltage and the total ion intensity does not indicate a higher stability of the molecular anions of humic acids as compared to fulvic acids. Rather it is proposed that the difference is due to the large portion of HMW compounds in the humic acid mixture: with an increasing cone voltage, increasing amounts of low molecular weight fragments can be generated from HMW precursors without exhausting this reservoir of HMW compounds. Consequences for the Molecular Weight Determination by ESI-MS. The determination of number-average (Mn) and weight-average (Mw) molecular weights of polymer mixtures by means of mass spectrometry is very attractive since a mass spectrum could possibly provide an absolute measure of the mass distribution. Several reports on the use of ESI-MS to determine the mass distribution of synthetic polymers are available.10,15,16 However, three requirements must be fulfilled for an accurate determination of the average molecular weight of a polymer mixture by mass spectrometry: (a) molecular ions do not fragment, (b) only singly charged ions are formed or the envelop of multiply charged ions can be deconvoluted reliably, and (c) the signal response of a single molecule is independent of its mass or, at least, a known function of it.16 These conditions may be fulfilled for certain synthetic polymers such as polymethacrylates,16 PEG,15 or polyester resins.10 However, it has been recognized that the determination of average molecular weights is more reliable, when the samples were fractionated by SEC to reduce the polydispersity before MS analysis.10,17,18 (16) Simonsick, C. N. McEwen W. J.; Larsen, B. S.; Ute, K.; Hatada, K. J. Am Soc. Mass Spectrom. 1995, 6, 906-911. (17) Prokai, L.; Simonsick, W. J. Rapid Commun. Mass Spectrom. 1993, 7, 853856. (18) Nielen, M. W. F. Rapid Commun. Mass Spectrom. 1996, 10, 1652-1660.

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Figure 5. ESI-MS mass spectra of the HMW fraction of fulvic acids at a cone voltage of (a) 20 and (b) 140 V. The insets show extended mass spectra of m/z 220-244.

Figure 6. Total ion intensities (peak areas) determined at different cone voltages: (a) fulvic acids; (b) humic acids. The ion intensities were separately recorded for four mass ranges.

Figure 7. Calculated Mn values of the HMW fraction versus cone voltage for fulvic and humic acids.

In parallel, attempts have previously been made to determine average molecular weights of fulvic acids by infusion-ESI-MS.3,6,8 Based on the experimental data shown in Figure 4, average molecular weights for the HMW fraction of the fulvic and humic acids were calculated at the different cone voltages and a strong influence of the cone voltage is visible (Figure 7). By increasing the cone voltage from 20 to 140 V, the Mn values calculated from the mass spectra of the HMW fraction decreased for both, SRFA and SRHA. At 140 V, the value obtained for the humic acids approach those of the fulvic acids (400 amu). These declining Mn values clearly reflect the fragmention of ions of higher molecular 6280

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weight. This finding seems to be a paradox: the highest average molecular weight is obtained under those tuning conditions that provides the lowest ion intensity for the HMW fraction. On the basis of the results of this study with the parallel use of SEC-MS and SEC-OCD, it is now sure that for fulvic and humic acids two of the three prerequisites for average molecular weight determinations by ESI-MS are not fulfilled: neither is the molecular response constant nor known for all the oligomers of the mixtures, nor can fragmentation be avoided in the electrospray process. Therefore, this investigation substantiates previous doubts of whether ESI-MS is suitable for average molecular weight determinations of fulvic and humic acids. Nevertheless, ESI-MS spectra of fulvic and humic acids carry valuable information, and the intensity distribution of the ions, certainly, belongs to it. However, one should not call the average intensity distribution in an ESI mass spectrum the “average molecular weight”. Instead, the term “average ion distribution”, with a number-average (In) and a weight-average (Iw) variant, would be more appropriate. These values summarize the intensity distribution of ions in a mass spectrum obtained under certain analytical conditions without implying that this is similar to the average molecular weight. However, one can assume that differences in the average ion distribution between two samples analyzed under identical conditions reflect differences in the average molecular weight of these samples. If this hold true, the average ion distribution determined by SEC-ESI-MS can be used

as a relative measure to compare the quality of dissolved humic matter.4 CONCLUSIONS The use of SEC-OCD parallel to the SEC-MS provided valuable insight into the range of compounds ESI-MS can detect from fulvic and humic acids and into the instrumental conditions that are suited for this purpose. It could be shown that at low cone voltages the LMW compounds are preferentially detected by ESI-MS, while significant parts of the HMW fraction are not determined at all. With increasing cone voltage, a higher total ion current is obtained in the retention time range of the HMW fractions due to a more effective deflection toward the aperture of the mass spectrometer. Owing to the labile nature of the larger humic and fulvic acid molecules, fragment ions are mostly detected under these conditions but hardly any intact HMW molecular anions. These experiments suggest that the analytical window that ESIMS opens for fulvic and humic acids is limited to ∼1000 amu for molecular ions. However, insight into structural characteristics can be obtained for a much broader molecular weight range by

detecting and further analyzing the fragment ions of HMW compounds using SEC-ESI-MS at elevated cone voltages. As a consequence ESI-MS, even when coupled to SEC, is not suited to determine average molecular weights of fulvic and humic acids, since HMW compounds are either detected with low intensity or are fragmented in the ESI source. However, differences between fractions and samples may still be expressed by the average ion distribution that can be calculated from mass spectra obtained by ESI-MS. ACKNOWLEDGMENT We thank Thomas Meyn for skilful laboratory assistance and gratefully acknowledge funding by the German Research Council (DFG, Bonn), RE1290/4-1, 4-2.

Received for review April 17, 2003. Accepted August 26, 2003. AC034399W

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