The eect of laser wavelength and power density on the
laser desorption mass spectrum of fulvic acid
Teresa L. Brown
1, James A. Rice *
South Dakota State University, Department of Chemistry and Biochemistry, Box 2202, Brookings, SD 57007-0896, USA
Abstract
Fulvic acid (FA) is a water-soluble component of natural organic matter whose environmental and organic geo-chemistry is routinely correlated to its molecular weight. In an attempt to examine the usefulness of laser desorption (LD) mass spectrometry to unambiguously determine this characteristic, laser wavelengths of 10.6mm, 1.06 mm and
355 nm were evaluated using the four International Humic Substances Society FAs. Under the conditions employed, FA desorption and ionization were optimal when an infrared wavelength is used and thermal desorption conditions dominate. Mass distributions observed in the 10.6 and 1.06 mm FA positive-ion LD mass spectra were centered at
500±600m/z, and ranged from 200m/zto beyond 800m/z. Lowerm/zdistributions were generally observed in the corresponding negative-ion spectra. Increasing power density, or use of the UV wavelength, resulted in extensive fragmentation of the FA component molecules. The positive-ion spectrum at 355 nm, or at a higher power density, consisted predominantly of low-mass fragments (<200m/z). The corresponding negative-ion spectra displayed an ion distribution with greaterm/zratios, but them/zdistribution was still less than the negative-ion spectra obtained with an infrared wavelength. The complex pattern of mass distributions observed in the FA LD mass spectra re¯ect the inherent chemical complexity of these materials.#2000 Elsevier Science Ltd. All rights reserved.
Keywords:Fulvic acid; Laser desorption mass spectrometry; Humic substances
1. Introduction
Humic substances represent complex mixtures of organic compounds formed by the degradation and decomposition of organic materials in natural environ-ments. As a result, humic substances are ubiquitous in the soils, sediments and waters of the earth's surface. In each of these environments humic substances play a number of vital geochemical roles. For example, humic materials represent the diagenetic state of organic car-bon in the global carcar-bon cycle (Welte, 1970). In soils, they create and maintain soil aggregate structure, bind and transport mineral ions making them available for plant uptake, and act as a pH buer (Stevenson, 1982). The importance of humic materials in the solubilization
and transport of hydrophobic organic compounds in aquatic, soil and sediment environments has also been noted many times (Wershaw et al., 1969; Boehm & Quinn, 1973; Means and Wijayarante, 1982; Hassett and Millcic, 1985; Baker et al., 1986; Chin et al., 1990; Young and Weber, 1996).
Because they are apparently formed by nonspeci®c reaction pathways, a myriad of dierent mechanisms may contribute molecules to their nature (MacCarthy and Rice, 1991). Humic substances are as diverse in structural composition as they are ubiquitous. Humic materials are usually described as aromatic polycarboxylic acids which can be divided into three operationally de®ned fractions according to the fraction's solubility in an aqueous solution as a function of pH. Humin is insoluble at all pH values. Humic acid is soluble under alkaline condi-tions (pH>7), and fulvic acid (FA) is soluble at all pH values. Each of these fractions is itself a complex mix-ture that has de®ed all attempts to separate it into dis-crete compounds (MacCarthy and Rice, 1991). Because of its lower molecular weight, and ostensibly simpler
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* Corresponding author. Tel.: 605-688-4252; fax: +1-605-688-6364.
E-mail address:james_rice@sdstate.edu (J.A. Rice).
1 Current address: Chemistry Department Rochester
nature (Stevenson, 1982; Hayes et al., 1989), fulvic acid was chosen for the study described here.
In order to understand how humic materials ful®ll their functions in the natural environment, it is impera-tive that we be able to describe their molecular struc-ture. A basic component of a structural characterization is molecular weight. As pointed out by Wershaw and Aiken (1985), a method for the accurate molecular weight determination of humic materials will allow for the establishment of approximate molecular formulas when used in conjunction with other characterization data, determination of stoichiometric relationships between humic materials and other chemical species, and the accurate comparison of diagenetic dierences between humic materials from dierent environments. In fact, molecular weight is the one characteristic which is rou-tinely correlated with many of the environmental beha-viors and reactions of humic materials. Yet it is a characteristic of FA whose absolute nature has remained an enigma.
A principal method for obtaining a substance's mole-cular weight (or more appropriately here the molemole-cular weight distribution) is mass spectrometry. Its use in the characterization of FA, and humic materials in general, has been limited by the requirement that a sample must be ionized and introduced into the gas phase for mass spectrometric analysis.
The applicability of desorption ionization methods to the study of high-mass biomolecules (Karas and Hillen-kamp, 1988; Karas et al., 1989) suggests that they may also be applicable to the mass spectrometric character-ization of fulvic acid and its molecular weight. It has been shown that laser desorption Fourier-transform ion cyclotron resonances mass spectrometry (LD FT-ICR MS) can be employed in the characterization of FA. Mass distributions (100±1100 m/z) with number average molecular weights ranging from 400 to 600m/zfor the four International Humic Substances Society (IHSS) reference fulvic acids have been reported (Novotny et al., 1995). However, since LD processes have been found to be dependent on a number of experimental parameters (i.e. laser wavelength, laser power, and analyte nature (van Vaeck et al., 1993)), it is likely that the molecular weight distributions observed by LD FTMS would be dependent on the desorption conditions employed. The purpose of this study was to compare the desorption mass spectra of FA obtained with laser wavelengths of 10.6mm, 1.06mm,
and 355 nm and two dierent laser power-densities.
2. Materials and methods
Four International Humic Substances Society refer-ence fulvic acids were used in this study; Nordic aquatic (NaFA), peat (PFA), soil (SFA), and Suwannee river (SuFA). The source material preparation (Campbell and
Malcolm, 1985) and fulvic acid extraction procedure (Swift, 1996) for each of these samples are described elsewhere. Chemical characteristics of each FA sample are given by Novotny (1993). The fulvic acid samples were prepared by individually dissolving each sample in deionized, distilled water to give a concentration of5 mg/ml. Several drops of sample solution were placed on a stainless-steel probe-tip and air dried. An alkali kraft lignin and tannic acid were purchased from Aldrich and Sigma, respectively, and used as received.
Positive±and negative-ion LD FT-ICR MS experi-ments with a laser wavelength of 10.6mm were performed
on an 3T Extrel FTMS 2000 mass spectrometer coupled to a pulsed CO2laser according to the conditions
speci-®ed by Novotny et al. (1995). Laser-desorption FT-ICR MS experiments at 1.06mm and 355 nm were performed
on a 3T Extrel FTMS 2000 coupled to a pulsed Nd:YAG laser with positive- and negative-ion detection at the National High-Field FT-ICR Mass Spectrometry Facility at the National High Magnetic Field Labora-tory (Florida State University, Tallahassee, FL) and Oak Ridge National Laboratory (Analytical Sciences Division), respectively. Generation of the 355 nm laser pulses was accomplished by frequency tripling. At each wavelength, the laser power and delay time before exci-tation were optimized to give maximum ion intensity and mass distribution for comparison purposes. The para-meters were considered optimized at the point at which the observed m/z range was maximized. Additional experimental details are given by Brown (1998).
At 10.6 mm, the power density was 107 W/cm2.
Experiments performed at 1.06mm utilized two dierent
power densities, 108 and 109 W/cm2 to speci®cally
demonstrate the eect that this variable has on the observed spectrum. Spectra produced with the higher power density will be designated as 1.06mm-H. At 355
nm the power density was 107±108W/cm2. To increase
the signal-to-noise ratio, ten laser events at dierent spots on the probe tip were averaged during the acqui-sition of the 10.6mm spectra. Low-mass ion ejection via
a chirp excitation was necessary in the 10.6mm and 355
nm desorption experiments to remove high-intensity low mass ions (<200 m/z) which were a signi®cant con-tribution to the overall ion signal. Swift excitation was performed prior to ion detection.
Number-average molecular weights were calculated from the sample spectra as described by Cooper (1989).
3. Results and discussion
3.1. Eect of laser wavelength on the mass spectrum of FA
for the PFA sample, discussion regarding the general appearance of spectra, comparisons between positive-and negative-ion spectra, and the eect of desorption wave-length and power density on the spectra are applicable to all spectra except as noted. Regardless of wavelength, the large number of mass peaks in the spectra are clear and indisputable evidence of a multicomponent nature for fulvic acid.
Desorption at 10.6 mm produced spectra (Fig. 1)
which display higher mass distributions than the corre-sponding negative-ion spectra. The positive-ion spectra exhibited mass distributions extending out to1000m/z
with the most intense ions in the 500±600m/zportion of the spectrum. The negative-ion spectra exhibited mass distributions extending out to700m/z with the most intense ions in the 400±500m/zportion of the spectrum. The negative-ion spectra produced measurable ion signals with one laser shot, and were highly reproducible. The positive-ion spectra, though obtained under identical des-orption conditions to Novotny et al. (1995) required the signal averaging of ten laser events in order to achieve a reasonable signal-to-noise ratio of 3:1.
Desorption at 1.06mm produced spectra (Fig. 2) with no
clear pattern in the mass distribution dierences between positive-and negative-ion spectra. The positive-ion spectra
exhibited mass distributions extending out to800m/z
with the most intense ions in the 400±500m/zportion of the spectrum. The negative-ion spectra exhibited mass distributions extending out to700m/zwith the most intense ions at400m/z. While both the positive- and negative-ion spectra shown are that of one laser event, the positive-ion spectra were obtained after the third laser irradiation of a given sample spot while the nega-tive-ion spectra were obtained on the ®rst irradiation of a given spot. The spectra were acquired in this way because no positive-ion signal was observed during the ®rst two experimental sequences on a sample spot. Visi-ble inspection of the sample ®lm showed its thickness decreasing with each laser event. Apparently during the ®rst two laser events, the sample was desorbed but not
Fig. 1. Positive-ion (a) and negative-ion (b) LD FT-ICR mass spectra of the IHSS peat fulvic acid (PFA) acquired with a laser wavelength of 10.6mm.
eectively ionized. Viewing the sample after the third laser event on a sample spot when signi®cant ion signal had been observed revealed that the substrate had been exposed. During this experimental sequence, sample desorption could have coupled with substrate ablation to produce favorable ionization conditions. Therefore, it appears that laser ablation of the substrate surface was necessary in order for ionization of the FA to occur. The ablated metal ions most likely interacted with des-orbed sample species in the selvedge region resulting in ionization through charge transfer or cationization. With further laser events on a spot, signal intensity would eventually decrease as the sample was depleted in that area. As a result, shot-to-shot variability was very high for these samples at this wavelength in the positive-ion mode. With close inspectpositive-ion of the spectra acquired at this wavelength, three peaks at 750±820m/zappear to be present in all positive- and negative-ion spectra. These peaks are instrumental background resulting from environmental vibrations (Dr. Touradj Souluki, NHMFL sta, personal communication, 1997).
The positive-ion and negative-ion spectra obtained for the IHSS PFA at a desorption wavelength of 355 nm are shown in Fig. 3. All spectra shown are the result of a single laser shot. The positive-ion spectrum consists of a single envelope of ions ranging from 100±300 m/z. There are few ions evident above 300 m/z suggesting that there has been extensive fragmentation of the FA components. The ion signal varies greatly from sample to sample, and shot to shot. Negative-ion spectra at this wavelength resulted in m/z ranges of 200±500 with distributions similar in shape to the negative-ion spectra recorded at 10.6 and 1.06mm.
3.2. Eect of power density
The FA spectra acquired at 1.06mm under high power
density conditions (Fig. 4) dier greatly from those acquired with the same wavelength under lower power density conditions (Fig. 2). The positive-ion spectra exhibited bimodal mass distributions of 150±250 m/z
and 250±450 m/z. The negative-ion spectra exhibited
Fig. 3. Positive-ion (a) and negative-ion (b) LD FT-ICR mass spectra of the IHSS peat fulvic acid (PFA) acquired with a laser wavelength of 355 nm.
mass distributions extending out to400m/zwith the most intense ions at 250 m/z. This decrease is the result of the relatively high laser power density employed since desorption at this wavelength under lower power density conditions resulted in higher m/z
distributions. These distributions are not only lower in
m/zthan those previously recorded, but also vary in the shape of their distribution.
Due to the high power density employed and the ability of the samples to absorb the energy at 355 nm, it is likely that desorption/ionization at 1.06mm-H occurs
via a shock-wave-type mechanism rather than a thermal desorption-like mechanisms (Brown et al., 1998). Under these conditions, the selvedge region is short lived and gas phase ion-molecule reactions are less likely to occur (Hillenkamp, 1989; van Vaeck et al., 1993). This would account for the diculty encountered in forming and/or detecting positive-ions for FA in these experiments. Further evidence for a shock-wave type of mechanism is that cationization was enhanced through the addition of alkali-metal salts to the sample solution at 1.06 mm-H
(Brown, 1998). Increasing the probability of cationization through addition of cations is a commonly employed practice under such desorption conditions.
3.3. Eect of laser wavelength on the Mnof FA
Tables 1±3 provide the number-average molecular weight (Mn) values calculated for each FA from the
positive-ion and negative-ion spectra acquired at each wavelength. These Mnvalues are less than those reported
by Novotny et al. (1995) for the same FA samples using vapor-pressure osmometry. Number-average molecular weights were not determined for the positive-ion spectra acquired at 355 nm due to the low ion abundances and variability observed in the spectra.
It must be noted that we recognize that the applica-tion of Mn values to these spectra is limited by the
potential possibility of multiply-charged ions formed because of FA's multifunctional nature, and fragmen-tation of molecules during the desorption/ionization process. As such, it is not our intent to propose that the
Mn values presented here represent ``the'' average
molecular weights of these FAs. Given the molecular heterogeneity, and structural ambiguity, that surrounds FA it may never be possible to unequivocally determine this quantity. But with this caveat in mind, the number-average molecular weight is a convenient tool to sum-marize and compare the mass distributions produced under a particular set of experimental conditions. It is within this latter context that we employ the Mn.
While the Mnvalues calculated at 10.6 and 1.06mm
are similar for all samples (Tables 1 and 2), most of the values obtained at 1.06mm are slightly higher than those
calculated from spectra at 10.6mm. The Mnreported for
the samples with desorption at 355 nm are much lower (Table 3). The low mass-to-charge distributions observed at 355 nm, and to a lesser extent at 10.6mm, are most
likely due to the ability of the FAs to strongly absorb radiation with UV (355 nm), and mid-IR (10.6 mm)
wavelengths (MacCarthy and Rice, 1985). Species that strongly absorb the incident laser wavelength tend to have lower irradiance thresholds (Karas et al., 1985). Above this threshold, the ratio of fragment ions to molecular ions greatly increases (Karas et al., 1985). Due to FA's strong absorbance at 355 nm, it seems unlikely that desorption experiments can be carried out at this wavelength without giving rise to signi®cant molecular fragmentation.
While the dierences in the FA spectra from one wavelength to another can be explained, a closer look at
Table 2
Number-average molecular weights calculated from the posi-tive-ion and negaposi-tive-ion LD FT-ICR mass spectra of each IHSS fulvic acid acquired with a laser wavelength of 1.06mma
Fulvic acid sample Positive ion Negative ion
Nordic aquatic 525 581
Peat 537 490
Soil 410 427
Suwannee river 502 487
a Number-average molecular weights are in m/z ratios.
Values are reproducible to within5%.
Table 1
Number-average molecular weights calculated from the posi-tive-ion and negaposi-tive-ion LD FT-ICR mass spectra of each IHSS fulvic acid acquired with a laser wavelength of 10.6mma
Fulvic acid sample Positive ion Negative ion
Nordic aquatic 445 370
Peat 587 491
Soil 405 391
Suwannee river 463 425
a Number-average molecular weights are in m/z ratios.
Values are reproducible to within5%.
Table 3
Number-average molecular weights calculated from the nega-tive-ion LD FT-ICR mass spectra of each IHSS fulvic acid acquired with a laser wavelength of 355 nma
Fulvic acid sample Negative ion
Nordic aquatic 285
Peat 375
Soil 331
Suwannee River 251
a Number-average molecular weights are in m/z ratios.
the dominate desorption/ionization mechanism at each wavelength is necessary to address the variances in the tendency of FA to form positive- and negative-ions (Brown et al., 1998).
Desorption at the infrared wavelengths (10.6 and 1.06
mm) occurred with relatively low power densities. Thus it
is likely that the operant mechanism was thermal desorp-tion. Thermal desorption is thought to occur as energy
is transferred from the heated substrate (ie, the probe tip) to the the sample lattice. The resultant selvedge region is thought to exist for a time span that allows for gas phase reactions to occur, producing pseudomolecular ions. Posi-tive-ion formation under these conditions is likely to occur as a result of cation attachment (ie, cationization) via ion-neutral interactions in the selvedge, resulting in ionization through metal ion attachment to the sample species.
In both the 10.6 and 1.06 mm experiments, FA
appeared to more readily form negative-ions. Because FA is a polycarboxylic acid whose functional groups are thought to readily dissociate (Stevenson, 1982; Averett et al., 1989; Hayes et al., 1989), it would be expected to readily form negative ions. There is also a dierence in the Mnvalues of the respective positive- and
negative-ion distributnegative-ions (Tables 1±3). Organic acids are often found to readily form negative-ions during LD MS experiment (Van Vaeck et al., 1993). Fulvic acid's ten-dency to do this would be consistent with the aromatic, polyacidic nature that is usually attributed to it (Stevenson, 1982; Averrett et al., 1989; Hayes et al., 1989; Schultenet al.,1992; Schulten and Schnitzer, 1997). The proton bind-ing energies of these acids may be overcome in the deso-rption process while the aromatic portion of the molecules can serve to stabilize the negative charge thus formed.
This dierence in ion formation suggests that posi-tive- and negaposi-tive-ion spectra may be displaying the signature of dierent components of the FA samples. From the study of model compounds at 1.06 mm, it
appears that ions measured in the positive-ion mode may indeed dier structurally from the species observed in the negative-ion mode (Brown, 1998). Model com-pounds which share structural traits such as carbohy-drate units and substituted benzyl groups (both moieties are known to exist in FA (Stevenson, 1982; Averrett et al., 1989; Hayes et al., 1989; Schulten et al., 1992; Schulten and Schnitzer, 1997) varied in their tendency to form positive- and negative-ions. The results of the laser desorption of tannic acid and lignin (Fig. 5) may be sig-ni®cant in this respect. Both natural polymers, precursors to FA in the natural environment, dier in their ability to form positive- and negative-ions under these conditions. Tannic acid displays a distribution much like that of FA in the positive-ion mode while lignin displays a dis-tribution much like that of FA acid in the negative-ion mode. Neither tannic acid or lignin produces a sig-ni®cant spectrum in the negative-ion, or positive-ion mode, respectively. Perhaps tannic-like components of FA are measured in the positive-ion mode while lignin-like components are measured in the negative-ion mode.
4. Conclusions
At the three wavelengths studied (355 nm, 1.06mm, and
10.6mm), desorption conditions for FA are optimal when
an infrared wavelength is used and thermal desorption conditions dominate. The highest number-average molecular weight values were observed at a desorption wavelength of 1.06mm. Increasing incident power
den-sity at this wavelength, probably creating shock-wave type desorption conditions, resulted in extensive frag-mentation of the FA molecules and lower Mnvalues.
The ability of FA to eciently absorb the incident energy at 355 nm also contributed to a high degree of fragmentation at this wavelength. While fragmentation is extensive under these conditions (ie, resonant wave-length, or high power densities), some fragmentation is also likely under the thermal desorption mechanisms occurring with nonresonant (e.g. infrared wavelength) desorption. We believe, however, that fragmentation can be minimized by optimization of the experimental parameters at each wavelength so that the laser energy used to irradiate the sample just exceeds the desorption/ ionization threshold leading to better estimates of the molecular weight distribution of the components which comprise fulvic acid.
Finally, the ion distributions observed in the LD MS characterization are very complex, consisting of multi-ple ions at each nominal mass. However, the ion signals are dependent on a number of variables in the LD experiment as pointed by this work, making it dicult to make direct comparisons between samples and experiments.
Acknowledgements
We thank the NSF National High-Field FT-ICR Mass Spectrometry Facility at the National High Magnetic Field Laboratory (Tallahassee, FL) and Dr. Touradj Souluki, and Oak Ridge National Laboratory and Dr. Robert Hettich for providing access to their facilities and their assistance in performing these experiments. TLB also acknowledges the NHMFL for funding for travel expenses to their facilities. This work was supported in part by NSF CHE94-13008 and EPA R816962-01.
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