Environ. Sci. Techno/. 1995, 29, 2464-2466
Characterization of fuhric Acid by Laser-Desorption Mass Spectrometry F R A N K J . NOVOTNY' A N D JAMES A. RICE* Department of Chemistry & Biochemistry, South Dakota State University, Box 2202, Brookings, South Dakota 57007-0896
DAVID A. WEIL 3M Corporate Research Laboratories, 3M Center, St. Paul, Minnesota 55144-1000
Introduction Fulvic acid is a complex, heterogeneous mixture of organic substances formed by the profound alteration of organic materials in natural environments. The ubiquity of fulvic acid and its role in many geochemical processes has made it the subject of a substantial amount of fundamental and applied research. Despite this interest, fulvic acid's molecularweight (or more appropriately because it is amixture, its molecular weight distribution) has never been unambiguously determined. Yet, it is often this fundamental, enigmatic characteristic that is specifically correlated to many of the environmental interactions of fulvic acid. Its molecular weight has been related to the ability of fulvic acid to complex trace metals and radionuclides and aid in their transport through or removal from an aquatic system ( I ) ,the extent to which it will bind to organic contaminants (2-41, its ability to interact with inorganic colloids (5-7) and mineral surfaces (8, 9), its tendency to form trihalomethanes during drinking water treatment (IO),and its tendency to aggregate with other organic molecules (11 ) . In fact, the one characteristic of fulvic acid that seems to be identified as an important variable in many aspects of its chemistry in natural systems is molecular weight (12). The purpose of this paper is to report the molecular weight distributions of a suite of well-characterized fulvic acids as determined by laser-desorption (LD) Fourier transform mass spectrometry (FTMS) and compare them to the molecular weight distributions obtained by gel filtration chromatography and vapor pressure osmometry. These results provide definitive proof that low molecular weight substances dominate the components of the mixture which comprise fulvic acid. Techniques for molecular weight determination such as gel filtration chromatography (GFC) or vapor pressure osmometry W O ) each have their own limitations that compromise their utility when they are applied to the characterization of fulvic acid (13, 14). For example, GFC can be employed to determine a distribution that can be used for relative comparisons of the molecular weights of fulvic acid obtained under identical chromatographic * Corresponding author e-mail address:
[email protected]. Present address: Chemistry Department, Adams State College, Alamosa, CO 81102, +
2464
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29. NO. 9, 1995
conditions, but the problematic choice of a suitable calibration standard prevents GFC from being used to determine absolute molecularweight distributions (1516). Vapor pressure osmometry yields a number-average molecular weight (M,) that is affected by the polydispersity, the extent of dissociation, and the nonideal nature of fulvic acid (14). Unlike these techniques, molecular weight calibration in LD FTMS is performed by a procedure that is independent of the material being characterized. Thus, the molecular weight distributions reported here are absolute determinations.
Materials and Methods The materials that have been characterized in this paper include a fulvic acid isolated from the Big Sioux Aquifer (BSA)located in eastern South Dakota and the extensively studied International Humic Substance Society (IHSS) reference materials; Nordic, peat, soil, and Suwannee River fulvic acid samples. The BSA fulvic acid was isolated by the method of Thurman and Malcolm (17). The reference fulvic acid samples were purchased directly from the IHSS. Chemical characteristics of these samples are given elsewhere (18). Laser-desorption FTMS experiments were performed with an W e 1Fourier transform mass spectrometer coupled to a high-power, pulsed COZ(10.6pm) laser. The output energy of the laser was controlled by adjusting an aperture and set to -0.05 Jllaser pulse. The FA samples were prepared for analysisby dissolving the material in deionized, distilled water to give a FA concentration of -100 mg/L. Several drops of solution were placed onto a stainless steel probe tip, and the water evaporated. Desorption was accomplished with a single laser shot at the probe tip. Positive-ion mass spectra were recorded using a SWIFT excitation and detection pulse sequence (19). Mass spectra were recorded over a mass range of 100 to at least 1200 daltons. Laser-desorption FTMS-derived M , values were calculated from the mass spectra using the method described by Kahr and Wilkins (20). For comparison purposes, number-average molecular weights were determined for these FA samples by GFC and VPO. The VPO measurements were performed by a commercial laboratory (Huffman Laboratories, Golden, CO) using a single-point calibration method. The GFC measurements were perfonnedwith an isocraticHPLC system and a Supelco G-3000 PWXL high-performance GFC column. For comparison purposes, the retention volumes were converted to daltons using commercial poly(ethy1ene glycol) GFC molecular weight standards. These methods are described in detail by Novotny (18).
Results and Discussion The positive-ion LD FTMS spectrum of the BSA fulvic acid (Figure 1) shows an essentially continuous series of mass peaks. This result is consistent with fulvic acid being an extremely heterogeneous mixture and is typical of the appearance of the laser-desorption mass spectra of M v i c acids acquired at a laser wavelength of 10.6 pm. The spectrum in Figure 1 is dominated by high-intensity, lowmass peaks with intensity maxima at 370 and 530 Da. Table 1summarizes the number-averagemolecular weight values
0013-936X/95/0929-2464509.00/0
%. 1995 American Chemical Society
mixtures of a narrow series of homologous polymers or oligomerswith a specified average molecular weight.) The M. of 987 Da calculated from the positive-ion LD FTMS spectrum is in good agreement with the Mn of 1000 Da reported by the manufacturer for this standard. Finally, to confirm that the experimental conditions were capable of desorbing and ionizing intact higher molecular weight molecules actually found in fulvic acid, the M. of an oligosaccharide GFC molecular weight standard was measured. Oligosaccharides have been previously identified as a component of fulvic acid by LD FTMS (23). The
la0
2jC
,
3d0
400
560
6b0
7b0
BOO
I
900
lOd0
rnh FIGUREI. Positive-ion LO Flmass spectrumof BSAfulvicacid (FA) sample. Only the pottion of the spectrum from 1W to 1wO Oa is presented.
TABLE 1
Number-Average (Mn)Molecular Weights for Five Fulvic Acid (FA) Samples Obtained from Laser-Desorption FTMS (LD FTMS). Gel Filtration Chromatography (GFC), and Vapor Pressure Osmometry (VPO)a LO FTMS
GFC
VPO
RSA
dlR .._
l?fiq
i i S S Nordic IHSS peat IHSS soil IHSS Suwannee River
445
lnRR 1042
*ample
587
1380 1957
2377
405 463
1792 960
839 768
*All values are in daltans.
calculated from thelaser-desorptionmassspectrumofeach fulvic acid sample. For each fulvic acid sample characterized, the M. value obtained by LD FTMS is between 400 and 600 Da. These M. values are considerably less than the M, of the sample determined by GFC or VPO for that sample [Table 1) and is substantially lower than the 8002300 Da mass range typically reported for fulvic acid (21, 22). Thespectmm inFigure 1 shows alow-intensity, highmass "tail" on the mass distribution, which indicates that the BSA fulvic acid does contain some larger molecules in addition to the smaller molecules that dominate its LD FTMS spectra; this again is typical of the fulvic acids characterized. To confirm that the LD FTMS molecular weight distributions in Table 1 were not the result ofthe fragmentation ofhigh molecular weight compounds that might be present in these fulvic acids during the laser-desorption process, several experiments were run. First, the laser power used to desorb a sample from the probe tip was varied between 1.0 and 0.05 J. It was observed that at higher laser powers the molecular weight distribution could be completely converted to fragments with masses < 100 Da. Consequently, all spectra were recorded at low laser power (0.05 J). Second, to confirm that the experimental conditions are capable ofdesorbingand ionizingintact moleculeswith molecular weights in the mass range typically reported for fulvic acid, a poly(ethylene glycol) GFC molecular weight standard was analyzed using conditions identical to those used in the characterization of the fulvic acids. (The molecular weight standards used in GFC are typically
positive-ionspectrumofthisstandardgaveaseriesofmass peaks representing the individual oligomers present in the standard. The mass peaks representingeach oligomer were separated by 162Da, which is attributable to one monomer (C6HI2O6- H20). This pattern is characteristic of the desorption mass spectra of oligosaccharides(24,23. The M. of 623 Da observed for the oligosaccharide standard (correctedforH20loss) agreeswell with the manufacturer's reported M. value of 600 Da. These experiments indicate that the LD FTMS conditions used here are capable of desorbing and ionizing intact molecules in the molecular weight range typicallyreported for fulvic acid and that these molecules undergo little, if any, fragmentation. Thus, the lower Mnvalues reported in this paper are apparently not the result of the fragmentation of high molecular weight compounds, but representthe actual contributionsofintact small and large molecules to the molecular weight distribution of fulvic acid. High molecular weight molecules appear to represent only a small portion of the substances that comprise fulvic acid. Comparison of the M. d u e s in Table 1 clearly shows thatVPO andGFC M.valuesforfulvicacid aresignificantly higher than those obtained by LD FTMS. We believe that the higher M, values usually reported in VPO studies are the result of fulvic acid aggregating in the concentrated solutions used for analysis. Marinsky and Reddy (26)have provided direct evidence for the occurrence of this phenomenaduringtheVP0characterizationoffulvicacid.The higher Mn values reported for fulvic acid from GFC characterizations are probably the result of problems in calibrant selection cited earlier.
Acknowledgments This work was supported by the US. Environmental Protection Agency through Grant 816962-01-0.
literature Cited 11) McKnighl, D.: Feder. G.;Thurman. E.; Wershaw. R.; Weslail. I. in Biological Availabiliry of Trace Metals; Wildung. R., lenne. E., Eds.; Elsevier: New York. 1983: pp 65-76. I21 Chin. Y.;Weber, W. Enuiron. Sci. Technol. 1989, 23. 978-984. (3) Kile, D.: Chiou. C. In Aquatic Humic Substances: Influence on Fare and Teatment of Pollurnnts; Suffet, 1. H.. MacCanhy, P.. Eds.; ACS Advances in Chemistly Series 219: American Chemical Society: Washington. DC. 1989 p p 131-157. I41 Caner.C. W.: Suffet. I.H. In FateofChemicalrin theEnwironment: Swam, R . L.. Eschenroeder. A.. Eds.; ACS Symposium Series 225:AmericanChemicalSociety: Washington. DC, 1983:pp215229. I51 Davis, J. Geockim. Cosmochim. Acta 1982, 46, 2381-2393. (6)Tipping, E. Geochim. Cosmockim. Acta 1981. 45, 191-199. (71 Tipping. E.; Higgins. D. C. Colloids Surf. 1982. 5. 85-92. (8) Wershaw. R. L.; Pinckney, D. In Contaminants and Sediments. Vo1.2;Baker, R.A..Ed.;AnnArhorScience:AnnArhor, Mi, 1980 pp 207-219. (9) Hedges, 1.1. Geochim.Cosmockim. Acta 1977.41. 1119-l123.
VOL. 28. NO. 9,1885 I ENVIRONMENTAL SCIENCE &TECHNOLOGY. 2465
(10) Rook, J. Environ. Sci. Technol. 1977, 11, 478-482. (11) Schnitzer, M.; Khan, S. U. HumicSubstances in theEnvironment; Marcel-Dekker: New York, 1972; 327 pp, (12) Orlov, D. S. HurnusAcidsofSoils, Kothekar,V., Transl.; Balkema: Rotterdam, 1985; 378 pp. (13) Wershaw, R.; Aiken, G. In Humic Substances in Soil, Sediment, and Water;Aiken, G., McKnight, D., Wershaw, R., MacCarthy, P., Eds.; John Wiley: New York, 1991; pp 477-492. (14) Aiken, G. R.; Gillam, A. H. In Humic Substances II. In Search of Structure; Hayes, M. H. B., MacCarthy, P., Malcolm, R. L., Swift, R. S., Eds.; John Wiley: Chichester, 1989; pp 515-544. (15) Swift, R.; Posner, A. 1.Soil Sci. 1971, 22, 237-249. (16) De Nobili, M.; Gjessing, E.; Sequi, P. In Humic Substances II. In Search of Structure; Hayes, M. H. B., MacCarthy, P., Malcolm, R. L.,Swift, R. S., Eds.; John Wiley: Chichester, 1989; pp 515544. (17) Thurman, E. M.; Malcolm, R. L. Environ. Sci. Technol. 1981, 15, 463-466. (18) Novotny, F. J. Reevaluation of the molecular weight of fulvic acid. Ph.D. Dissertation, South Dakota State University, Brookings, SD, 1993. (19) Marshall, A. G.; Wang, T.-C. L.; Ricca, T. L. 1.Am. Chem. SOC. 1985, 57, 7893-7897.
2466
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29, NO. 9,1995
(20) Kahr, M. S.; Wilkins, C. L. 1.Am. SOC.Mass Spectrom. 1993, 4, 453-460. (21) Stevenson, F. Humus Chemistry;John Wiley: New York, 1982; 443 pp. (22) Hayes, M. H. B.; MacCarthy, P.; Malcolm, R. L.; Swift, R. S. In Humic Substances Il. In Search of Structure; Hayes, M. H. B., MacCarthy, P., Malcolm, R. L., Swift, R. S., Eds.; John Wiley: Chichester, 1989; pp 689-733. (23) Rice, J.A.; Wed, D. In HumicSubstances in theGloba1Environment and Implications on Human Health; Senesi, N., Miano, T. M., Eds.; Elsevier B. V.: Amsterdam, 1994; pp 355-360. (24) Carroll, J. A.; Ngoka, L.; McCullough, S.; Gard, E.; Jones, A. D.; Lebrilla, C. B. Anal. Chem. 1991, 63, 2526-2529. (25) Puzo, G.; Prome, J. C. Org. Mass Spectrom. 1985, 20, 288291. (26) Marinsky, J. A.; Reddy, M. M. Anal. Chim. Acta 1990,232, 123130.
Received for review January 26, 1995. Revised manuscript received June 6, 1995. Accepted June 12, 1995.
ES950060I
