Environ. Sci. Technol. 1996, 30, 2764-2775
Covalent Binding of Aniline to Humic Substances. 2. 15N NMR Studies of Nucleophilic Addition Reactions KEVIN A. THORN,* PENNY J. PETTIGREW,† AND WAYNE S. GOLDENBERG‡ Water Resources Division, U.S. Geological Survey, Denver Federal Center, Mail Stop 408, Box 25046, Denver, Colorado 80225
ERIC J. WEBER Ecosystem Research Division, National Exposure Research Laboratory, U.S. Environmental Protection Agency, Athens, Georgia 30613
Aromatic amines are known to undergo covalent binding with humic substances in the environment. Although previous studies have examined reaction conditions and proposed mechanisms, there has been no direct spectroscopic evidence for the covalent binding of the amines to the functional groups in humic substances. In order to further elucidate the reaction mechanisms, the Suwannee River and IHSS soil fulvic and humic acids were reacted with 15N-labeled aniline at pH 6 and analyzed using 15N NMR spectrometry. Aniline underwent nucleophilic addition reactions with the quinone and other carbonyl groups in the samples and became incorporated in the form of anilinohydroquinone, anilinoquinone, anilide, imine, and heterocyclic nitrogen, the latter comprising 50% or more of the bound amine. The anilide and anilinohydroquinone nitrogens were determined to be susceptible to chemical exchange by ammonia. In the case of Suwannee River fulvic acid, reaction under anoxic conditions and pretreatment with sodium borohydride or hydroxylamine prior to reaction under oxic conditions resulted in a decrease in the proportion of anilinohydroquinone nitrogen incorporated. The relative decrease in the incorporation of anilinohydroquinone nitrogen with respect to anilinoquinone nitrogen under anoxic conditions suggested that inter- or intramolecular redox reactions accompanied the nucleophilic addition reactions.
* Corresponding author e-mail address:
[email protected]; telephone: 303-467-8281; fax: 303-467-9598. † Present address: University of Alabama, Huntsville, AL. ‡ Present address: University of Wisconsin, Madison, WI.
2764 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 30, NO. 9, 1996
Introduction One possible fate of aromatic amines in the environment is covalent binding to the naturally occurring organic matter, primarily humic substances, in soils, sediments, and natural waters (1-5). Binding is thought to occur through nucleophilic addition of the amine group with the carbonyl functionality of the humic substances, through phenol oxidase enzyme or metal-catalyzed reactions between the aromatic amines and humic substances, or a combination of all three. The phenol oxidase enzymes act by effecting free radical coupling reactions between the aromatic amines and phenolic moieties within the humic molecules or by creating additional substrate sites within the humic substances for nucleophilic addition by the amine groups. As a consequence, there has been a significant research effort both to understand the reaction mechanisms of the covalent binding and to determine the relative strength and persistence of the immobilization. Bartha and co-workers performed extensive studies on the covalent binding of 3,4dichloroaniline (DCA) to soil humic substances (5-12). They determined what percentages of DCA covalently bonded to humic acids could be released upon subsequent acid or base hydrolyses. They also measured rates of remineralization of DCA covalently bonded to humic substances. Based upon studies with model compounds such as 4-methylcatechol and toluquinone and an analysis of reaction conditions affecting the uptake of DCA by humic substances, they concluded that quinone groups in humic substances were the major substrate sites for nucleophilic addition by the anilines and that subsequent addition and oxidation reactions eventually locked the amino nitrogen into heterocyclic ring systems. Parris provided further support through kinetic studies that aromatic amines react with the quinone functionality in humic substances (13). Bollag and co-workers identified products from the reaction mixtures of chloroanilines with such model phenolic humus constituents as guaiacol, syringic acid, coniferyl alcohol, and ferulic acid in the presence of peroxidases, tyrosinases, laccases, and manganese dioxides (14-18). Among the forms of nitrogen identified in the reaction products were carbazole, iminodiphenoquinone, anilinodiphenoquinone, N-phenylpyrrolidine, and diarylamine. More recently, Hatcher et al. (19) demonstrated the peroxidase-catalyzed coupling of 2,4-dichlorophenol, the oxidation product of 2,4-dichloroaniline, to a peat humic acid using 13C NMR. Because direct spectroscopic evidence of how anilines react with humic substances is still lacking, however, and because an understanding of these mechanisms is necessary to determine the role of enzyme- or metal-catalyzed versus noncatalyzed reactions in the actual soil and aquatic environment, we have undertaken studies to further elucidate the binding of aniline to humic substances. In the first part of this study (1), we examined factors affecting the kinetics of the uptake of aniline by four well-characterized soil and aquatic humic substances: the IHSS soil fulvic and humic acids and the IHSS Suwannee River fulvic and humic acids. In this paper, we use liquid phase 15N NMR spectrometry to directly observe the incorporation of 15Nlabeled aniline into the IHSS samples, an Iowa soil humic acid, and the model compounds 4-methylcatechol and 1,4benzoquinone. The effect of anoxic conditions, treatment S0013-936X(95)00933-3 This article not subject to U.S. copyright. Published 1996 by the American Chemical Society.
with sodium borohydride and hydroxylamine prior to reaction with aniline under oxic conditions, and the chemical exchangeability by ammonia of aniline incorporated under oxic conditions were examined in the Suwannee River fulvic acid. A comparison of the nucleophilic addition, peroxidase, and birnessite-catalyzed reactions of aniline with the soil samples, and a study of the uptake of aniline by the organic matter of the whole soil are the subject of our next paper (20). We use the same approach in applying liquid phase 15N NMR to the aniline-reacted fulvic and humic acids as in our previous studies (21, 22), wherein the rationale behind our choice of pulse sequences was explained. Briefly, the ACOUSTIC (23) pulse sequence was recorded on samples doped with paramagnetic relaxation reagent to obtain quantitative distributions of the nitrogens incorporated into the samples. The ACOUSTIC sequence alleviates the severe acoustic ringing encountered in the normal single-pulse experiments when applied to humic samples. The INEPT (24) sequence was used to discriminate between protonated and nonprotonated nitrogens in the ACOUSTIC spectra and to provide additional signal enhancement in the protonated nitrogen region. Additionally, solid state cross polarization/magic angle spinning (CP/MAS) 15N NMR spectra were recorded on the aniline-reacted soil humic acids, which have limited solubilities in deuterated organic solvents.
Experimental Section Materials. The Suwannee River humic acid (SRHA) and reference soil fulvic and humic acids (SFA&SHA; isolated from the mollic horizon of the Elliot Silt Loam soil, Joliet, IL) were purchased from the International Humic Substances Society (IHSS). Suwannee River fulvic acid (SRFA), obtained from J. A. Leenheer, was described previously (21, 22). The Iowa soil was collected from the surface horizon of the Walnut Creek Watershed MSEA (management system evaluation area) site (25). Geologically, the soil is described as Des Moines Lobe Glacial Till. It is a poorly drained, nearly neutral pH, loamy soil of moderate carbon content (2.19%). Humic acid was extracted from the Iowa soil using the procedure recommended by the IHSS. Aniline, 99 atom % 15N, was purchased from ISOTEC. All other reagents were purchased from Aldrich. Preparation of Model Compounds for 15N NMR Chemical Shift Determination. 3-Anilino-2-chloro-1,4-dihydroxynaphthalene was prepared from the sodium dithionite reduction of 3-anilino-2-chloro-1,4-naphthoquinone. 4-Anilino-1,2-naphthoquinone was prepared by reacting 15N-labeled aniline (φ15NH ) with the sodium salt of 1,22 naphthoquinone-4-sulfonic acid in aqueous solution (26). The sodium salt of 7-hydroxy-3H-phenoxazin-3-one (resorufin) was converted to the H+-saturated form by precipitation from aqueous solution with HCl. Other condensation products were prepared by refluxing model carbonyl compounds with 15N-labeled aniline in methanol. Reaction of 4-Methylcatechol and 1,4-Benzoquinone with Aniline. 4-Methylcatechol (1.0 g) and 200 µL of φ15NH2 were dissolved in 200 mL of pH 6 phosphate buffer (0.05 M) and stirred for 9 days, open to the atmosphere. The reaction solution was filtered through a sintered glass funnel. The precipitate was washed with deionized distilled water, allowed to air dry, and then redissolved in DMSO-d6 for NMR analysis. The filtrate was passed through a C18 Mega-Bond Elut cartridge to recover the dissolved colored
reaction products. These reaction products were eluted from the cartridge with methanol, dried under a rotary evaporator, and combined with the precipitate in DMSOd6. 1,4-Benzoquinone (1.0 g) was added to 200 mL of pH 6 phosphate buffer, allowed to dissolve over 16 h, and then charged with 200 µL of φ15NH2. The reaction solution was stirred for 9 days. The reaction products were recovered as above and dissolved in 2 mL of DMSO-d6 for NMR analysis. Reactions of Fulvic and Humic Acids with Aniline. The H+-saturated fulvic or humic acids (400-500 mg) were added to 150-200 mL of H2O, adjusted to pH 6 with 1 N NaOH, and charged with 200 µL of φ15NH2. The reaction solutions were stirred at room temperature 4-5 days. The samples were then re-H+-saturated by passing the solutions through a Dowex MSC-1 cation exchange column and freeze-dried. The freeze-dried powders were redissolved in 2-3 mL of DMSO-d6 for NMR analysis. SRFA also was reacted with aniline at pH 6 in the absence of oxygen for 9 days. The anoxic reaction was carried out in an airless addition funnel, purged of atmospheric oxygen and blanketed under nitrogen with the aid of a Firestone valve. The re-H+-saturation on the MSC-1 resin was also performed under N2. Reduction of SRFA with NaBH4 followed by Reaction with O15NH2. SRFA (1.5 g), dissolved in 500 mL of H2O and adjusted to pH 8 with 1 N NaOH, was charged with 1.0 g of NaBH4 dissolved in 100 mL of 0.1 N NaOH. The reduction was allowed to proceed for 1 h and 45 min under a nitrogen atmosphere. The solution was then H+-saturated on the cation exchange resin and freeze-dried. A total of 500 mg of the NaBH4-reduced SRFA was reacted with the 15Nlabeled aniline as described above. Reaction of SRFA with Hydroxylamine Followed by Reaction with O15NH2. Hydroxylamine hydrochloride (236 mg; unlabeled) and 833 mg of SRFA were dissolved in 150 mL of deionized distilled water and titrated to pH 5 with 1 N NaOH. The solution was stirred for 6 days at room temperature. The solution then was sequentially Na+saturated and H+-saturated on the MSC-1 cation exchange resin to remove excess hydroxylamine, charged with 320 µL of φ15NH2, readjusted to pH 6 with 0.1 N NaOH, allowed to stir for 5 days, and then re-H+-saturated on the cation exchange resin, and freeze-dried. A total of 400-500 mg of the sample was dissolved in DMSO-d6 for NMR analysis. Reaction of SRFA with O15NH2 followed by 14NH4OH. SRFA was H+-saturated and freeze-dried after reaction with φ15NH2 for 5 days at pH 6 as described above. The sample was redissolved in 100 mL of H2O, titrated to pH 6 with unlabeled NH4OH and allowed to react for 5 days, and then titrated further to pH 9 and allowed to react for 2 more days. The sample was then H+-saturated on the MSC-1 resin and freeze-dried. NMR Spectrometry. Liquid phase 13C and 15N NMR spectra were recorded on a Varian XL300 NMR spectrometer at carbon and nitrogen resonant frequencies of 75.4 and 30.4 MHz, respectively, using a 10-mm broadband probe. Quantitative 13C NMR spectra of the unreacted fulvic and humic acid samples were recorded in DMSO-d6, 99.9 atom % 12C, as previously described (21). Two sets of 15N NMR spectra were recorded on the product mixtures from the reaction of 4-methylcatechol and 1,4-benzoquinone with aniline. The first spectrum was recorded using a 15 649.5Hz spectral window (514.8 ppm), 45° pulse angle, 0.5-s acquisition time, 5.0-s pulse delay, and inverse gated
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decoupling. The second was a refocused INEPT (proton decoupled) spectrum recorded using a 15 649.5-Hz spectral window, 0.5-s acquisition time, and 2.0-s proton relaxation delay. The polarization transfer time and refocusing delay were set equal to 1/4 J, or 2.78 ms (assuming 1JNH ) 90.0 Hz), values which have been reported optimal for signal enhancement of singly protonated nitrogens (27, 28). In the case of the fulvic and humic acids reacted with aniline, three sets of 15N NMR spectra were recorded. An INEPT spectrum was recorded using the above acquisition parameters, an ACOUSTIC (23) spectrum was recorded, chromium III acetylacetonate (100-200 mg) was then added to the sample, and the ACOUSTIC spectrum rerecorded. Acquisition parameters for the ACOUSTIC sequence included a 15 649.5-Hz spectral window, 0.5-s acquisition time, 45° pulse angle, 2.0-s pulse delay, and τ delay of 0.1 ms. Only the ACOUSTIC spectra recorded with the addition of the paramagnetic relaxation reagent are shown. Neat formamide in a 5-mm NMR tube, assumed to be 112.4 ppm, was used as an external reference standard for all spectra. 15N NMR chemical shifts are reported in ppm downfield of ammonia, taken as 0.0 ppm. Solid state CP/MAS 15N NMR spectra of the anilinereacted soil humic acids were recorded on a Chemagnetics CMX-200 NMR spectrometer at a nitrogen resonant frequency of 20.3 MHz using a 7.5-mm ceramic probe (zirconium pencil rotors). Acquisition parameters included a 30 000-Hz spectral window, 17.051-ms acquisition time, 2.0-ms contact time, 1.0-s pulse delay, and spinning rate of 3500 Hz. Chemical shifts were referenced to glycine, taken as 32.6 ppm.
Results Reaction of Aniline with Carbonyl Groups. Aniline undergoes 1,4-addition (Michael addition) to both 1,2- and 1,4-quinones in aqueous solution (29-31). The reaction of aniline with 1,4-benzoquinone, from the oxidation of hydroquinone, is illustrated here (30): O
OH
OH
OH
O
O NHφ
φNH2
O2
NHφ
O2
OH
O
anilinohydroquinone
anilinoquinone
The 1,2-addition of aniline with quinones or ketones to form imines (Schiff bases) is less favorable in aqueous solution than in organic solvent, because the overall equilibrium favors hydrolysis in aqueous or partially aqueous solvents. This is in contrast to hydroxylamine, which almost exclusively undergoes 1,2-addition with both ketones and quinones in aqueous solution to form oximes (32). In the case of sterically hindered quinones in aqueous solution, however, 1,2-addition by aniline is more favorable and in some instances becomes the dominant mode of attack. As an illustration, 4-methylaniline was reported to undergo both 1,4- and 1,2-addition to 2,6-dimethyl-pbenzoquinone, resulting in a 3:1 product ratio of anilinoquinone to imine (33): Aniline also undergoes 1,4-addition to R,β-unsaturated ketones, and reacts with β-dicarbonyl compounds to form enamines. Aniline undergoes aminolysis reactions with esters and possibly amides to form anilides. 15N NMR chemical shifts of these various adducts and other classes
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NH2
O H3C
CH3
+ O
CH3 O O
H3C
CH3
H3C
CH3
+ H
N
N
O imine anilinoquinone CH3
CH3
of nitrogen compounds pertinent to this study, compiled from the literature (27, 28, 34, 35) or determined in this laboratory, are shown in Figure 1. Noteworthy is the difference in chemical shift between the reduced (anilinohydroquinone) and oxidized (anilinoquinone) forms of the Michael addition products of aniline with quinones. For example, the 15N NMR chemical shift of 2-anilino-1,4-naphthoquinone is 103 ppm whereas the chemical shift of 2-anilino-1,4-dihydroxynaphthalene is 74 ppm. For convenience sake, we will use the term anilinohydroquinone in this paper to refer to the reduced form of the Michael addition products of aniline with both 1,2- and 1,4-quinones. 13C NMR Spectra of Unreacted Fulvic and Humic Acids. Quantitative liquid phase 13C NMR spectra of the unreacted samples are shown in Figure 2. Peak areas of the spectra and elemental analyses of the samples are listed in Table 1. Percentages of aromatic carbons (160-90 ppm) range from 31% for SRFA to 56% for the IHSS soil humic acid. The naturally occurring nitrogen contents range from 0.87% for the SRFA to 4.18% for the soil humic acid. Overlap of functional groups that may serve as substrate sites for nucleophilic addition by the aniline occurs within the major peak areas of the spectra. Quinone carbons (190-178 ppm) overlap with ketone carbons from 220 to 189 ppm, amides and esters (174-164 ppm) overlap with carboxylic acid carbons from 175 to 159 ppm, and phenolic carbons, including hydroquinone and catechol moieties, overlap with other substituted aromatic carbons in the range from approximately 165 to 135 ppm. The Suwannee River humic acid has the highest proportion of substituted aromatic carbons (165-135 ppm), and diazomethylation analysis in conjunction with 13C NMR shows that it also has the highest concentration of phenolic hydroxyls (36). The application of 13C NMR subspectral editing techniques to these samples indicates that aldehyde carbons, which would occur from approximately 202 to 192 ppm, are not present (21, 36, 37). This is significant in view of the fact that some researchers have considered aldehydes to be part of the carbonyl functionality in humic substances that reacts with aromatic amines. It is possible that latent aldehydes, e.g., aldose sugars that are free to convert from the cyclic (hemiacetal) to open chain form under the conditions of the reaction, are present in the samples and may condense with the aniline. Naturally Abundant Nitrogens. The naturally occurring nitrogens in the fulvic and humic acids were examined both to identify functional groups that might be involved in the reactions with aniline and to determine the potential for overlap between the naturally occurring nitrogens and the labeled aniline nitrogens incorporated into the samples.
FIGURE 1. 15N NMR chemical shifts of nitrogen compounds representing condensation products of aniline with carbonyl and other functional groups. Chemical shifts determined in DMSO-d6 in this lab unless otherwise noted. (a) From refs 27, 28, 34, and 35. (b) Determined in solid state. (c) Tentative assignment. TABLE 1
Elemental Composition and Peak Areas for Quantitative 13C NMR Spectra of Fulvic and Humic Acidsa,b sample Suwannee River FA Suwannee River HA IHSS soil FA IHSS soil HA Iowa soil HA
a
C
H
53.6 54.3 50.5 58.0 56.4
O
3.93 4.08 4.01 3.78 3.81
N
40.9 39.4 42.6 33.7 36.4
0.87 1.08 2.68 4.18 3.55
S
P
ash
0.66 0.68 0.62 0.41 0.55
NDc
0.58 3.38 0.79 0.90 1.57
0.01 0.05 0.32 ND
sample
220-180 ppm
180-160 ppm
160-90 ppm
90-60 ppm
60-0 ppm
Suwannee FA Suwannee HA IHSS soil FA IHSS soil HA
6 7 3 7
19 16 25 16
31 48 42 56
15 12 12 10
27 17 19 13
Elemental analyses reported on ash- and moisture-free basis.
b
Peak areas measured by electronic integration. c ND, not determined.
Naturally abundant 15N nuclei were not observed in liquid phase ACOUSTIC and INEPT 15N NMR spectra recorded on the unreacted samples at concentrations and numbers of transients comparable to the reacted samples. Therefore, the nitrogens observed in the liquid phase spectra of the reacted fulvic and humic acids represent only the labeled aniline nitrogens incorporated into the samples. Naturally abundant nitrogens were observed in solid state CP/MAS 15N NMR spectra recorded on the unreacted samples (20, 38). Amide nitrogens (amino acid nitrogens involved in peptide linkages) were observed in the Suwannee River and IHSS soil fulvic and humic acids; additionally, heterocyclic nitrogens were observed in the Suwannee River fulvic and humic acids and in the IHSS soil fulvic acid. In
the solid state spectra of the soil humic acids reacted with aniline, however, it is unlikely that the naturally abundant nitrogens distort the intensities of the labeled aniline nitrogens because the concentration of the labeled nitrogens is so much greater than the naturally abundant nitrogens. The detection of the naturally occurring amide nitrogens in the samples does substantiate the argument that aminolysis reactions with aniline in these samples is a real possibility. 15N NMR Spectra of Model Compound Reactions. The 15N NMR spectra of the product mixture from the reaction of aniline with 4-methylcatechol are shown in Figure 3. The top spectrum was recorded using inverse gated decoupling (IGD) and a 5-s pulse delay and should represent
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FIGURE 3. Liquid phase inverse gated decoupled and INEPT 15N NMR spectra of product mixture from reaction of O15NH2 with 4-methylcatechol.
FIGURE 2. Quantitative liquid phase 13C NMR spectra of unreacted fulvic and humic acids. LB ) line broadening in Hz.
a close to quantitative distribution of all nitrogens in the product mixture. The large number of resonances in the spectrum indicates a multitude of reaction products. The isolation and identification of the individual products is beyond the scope of this study, and so only the assignments for the types of nitrogens in the product mixture will be discussed. The bottom INEPT spectrum was recorded using polarization transfer times and refocusing delays based
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upon an assumed one bond nitrogen-proton coupling constant (1JNH) of 90 Hz and so shows only nitrogens directly bonded to protons. Comparison of the two spectra indicates that resonances downfield of the peak at 138 ppm in the IGD spectrum are nonprotonated nitrogens. There are two major groups of resonances clustered around 106 and 82 ppm, representing the anilinoquinone and anilinohydroquinone nitrogens, respectively. (Phenoxazine nitrogens also may overlap with the anilinohydroquinone nitrogens.) Peaks downfield of 138 ppm and through approximately 235 ppm represent a combination of heterocyclic and intermolecular condensation linkages. Further work will be necessary to more accurately assign these peaks. Three distinct peaks occur in the imine region at 314, 325, and 331 ppm. You et al. (8) identified N-(3,4dichlorophenyl)-3-hydroxy-6-methyl-p-benzoquinonimide from the product mixture of 3,4-dichloroaniline and 4-methylcatechol. It is possible that one of the peaks corresponds to the aniline equivalent of this reaction product. Alternatively, the peaks represent imines resulting from 1,2-additions to higher molecular weight condensation products. Spectra of the product mixture from the reaction of aniline with 1,4-benzoquinone are shown in Figure 4. In comparison with 4-methylcatechol, there are fewer peaks in the inverse gated decoupled spectrum and, with the exception of those at 154 and 173 ppm, no peaks representing nonprotonated nitrogens. The major band of resonances present corresponds to the anilinoquinone
FIGURE 4. Liquid phase inverse gated decoupled and INEPT 15N NMR spectra of product mixture from reaction of O15NH2 with 1,4benzoquinone.
nitrogens (104-112 ppm). Some anilinohydroquinone peaks also are present at about 87 ppm. The greater number of reaction products in the 4-methylcatechol mixture as compared to the 1,4-benzoquinone mixture is consistent with the fact that 1,2-quinones are generally more electrophilic than 1,4-quinones (31). The fact that the reduced form of the Michael addition product occurs in reaction mixtures of aromatic amines with quinones, hydroquinones, and catechols has not always been reported in published model compound studies. 15N NMR Spectra of Fulvic and Humic Acids Reacted with O15NH2. 15N NMR spectra of the Suwannee River fulvic and humic acids reacted with the labeled aniline at pH 6 are shown in Figure 5. The ACOUSTIC spectra represent the quantitative distribution of all nitrogens incorporated into the samples, whereas the INEPT spectra show only the nitrogens directly bonded to protons. Focusing on the fulvic acid spectra for the moment, there are several observations to be made. The INEPT spectrum exhibits three major peaks from approximately 58 to 95 ppm, from 95 to 125 ppm, and from 125 to 150 ppm, with maxima at 80, 117, and 137 ppm. These correspond primarily to anilinohydroquinone, anilinoquinone, and anilide nitrogens, respectively. Other forms of nitrogen may also overlap with these peaks. For example, phenoxazine (∼77 ppm), indole (∼133 ppm), quinolone (∼134 ppm), and enamine and enaminone (∼70-130 ppm) nitrogens may occur in this region. Phenoxazines have been reported to result from the reaction of chloroanilines with hydroquinones (40). Quinolones can
FIGURE 5. Liquid phase ACOUSTIC and INEPT 15N NMR spectra of suwannee river fulvic and humic acids reacted with O15NH2.
result from a series of reactions between aniline and β-ketoesters, as in the Knorr synthesis (41). Condensation of aniline with an R-hydroxyketone can result in formation of an indole, as in the Bischler indole synthesis (42):
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In a variation of the Nenitzescu synthesis, aniline condenses with a β-ketoester to form an enamine, which in turn reacts with a 1,4-benzoquinone moiety in a series of steps to form an N-phenyl indole structure (42):
O O EtO2C
C
CH2CO2Et
φNH2
quinolone
CO2Et
N H
OH O R
C H
C
CH2R′
O
R1
φNH2
R
N
O
indole H
R
C
CH2CO2R′
H
φNH2
NHφ C
′RO2C
The assignments of the peaks centered at 80, 117, and 137 ppm as primarily anilinohydroquinone, anilinoquinone, and anilide nitrogens, however, is consistent with our previous studies on the reaction of ammonia and hydroxylamine with humic substances. Quinone monoximes, from the reaction of hydroxylamine with quinones, and hydroxamic acids, from the reaction of hydroxylamine with esters or amides, were observed in 15N NMR spectra of oximated samples (21). Aminohydroquinones, from the reaction of ammonia with quinones, and primary amides, from the reaction of ammonia with esters (or transamination reactions with secondary amides), were observed in 15N NMR spectra of ammonia fixated samples (22). The increased resolution and signal enhancement within the protonated nitrogen region from ∼0 to 140 ppm in the INEPT spectrum as compared to the ACOUSTIC spectrum is significant. This will prove to be advantageous when examining the effects of prior treatments on the uptake of aniline and determining the chemical lability of the incorporated aniline, as will be seen further on. Comparison of the two spectra indicates that peaks downfield of ∼140 ppm in the ACOUSTIC spectrum are not bonded to protons and therefore represent nitrogens involved in heterocyclic, inter-, or intramolecular condensation linkages. Integration of the ACOUSTIC spectrum reveals that the nonprotonated nitrogens represent 55% of the total nitrogen incorporated into the fulvic acid. We are unable to make specific assignments for the various peaks (e.g., 163, 176, and 191 ppm) in the nonprotonated nitrogen region. However, some plausible reactions leading to the formation of heterocyclic nitrogens are illustrated in the following schemes. As an example of an intramolecular condensation reaction, aniline condenses with 1,4-diketones to form N-phenyl pyrrole structures (δ15N ) 175 ppm): O R1
φNH2
R
R O
N
R1
φ
As an example of an intermolecular condensation reaction, aniline forms an enamine with a β-ketoester and then condenses with an R-hydroxy ketone to form an N-phenyl pyrrole structure (Hantzsch synthesis) (41):
O
C R
CO2R′
HO N
R
φ
In general, the 15N NMR spectra in Figures 5 and 6 indicate that the Suwannee River humic acid and the IHSS soil fulvic and humic acids react with aniline similarly to the SRFA, although the distribution of the various reaction products varies. The INEPT spectra all show anilinohydroquinone, anilinoquinone, and anilide peaks. The three sharp peaks at 100, 106, and 112 ppm in the INEPT spectrum of the soil humic acid appear to represent reaction products of aniline with a discreet component or contaminant in the soil humic acid. (The peaks were also present in a duplicate sample, even after dialysis, as was the case with the peak at 315 ppm in the ACOUSTIC spectrum.) The intensities of the anilinohydroquinone peaks are less than the anilinoquinone peaks in the INEPT spectra of the Suwannee humic acid and the soil fulvic acid, in contrast to the SRFA. The ACOUSTIC spectra of the Suwannee humic and the soil fulvic and humic acids still suffer from ringing, as evident from the curvature in the baselines. Several peaks are common to the samples. For example, the peaks of greatest intensity in all four samples, possibly representing N-phenyl pyrrole nitrogens, occur at ∼175 ppm (SRFA, 176 ppm; SRHA, 173 ppm; SFA, 176 ppm; SHA, 175 ppm). Peak maxima occur at 163 ppm in the SRFA, 161 ppm in the SRHA, and 162 ppm in the SFA. Peak maxima also occur at 185 ppm in the SRHA, 186 ppm in the SFA, and 187 ppm in the SHA. Noteworthy in the ACOUSTIC spectrum of the SRHA is the peak at 215 ppm. The assignment of this peak is uncertain, but nitrogens that would occur in this region include the imino nitrogens of guanidines and some imidazole or pyrrole-like nitrogens. The ACOUSTIC spectra of the SRHA, SFA, and SHA also were integrated into protonated (0-140 ppm) versus nonprotonated (140-220 ppm) nitrogens (Table 2). In all four samples, approximately 50% or more of the aniline is incorporated into the heterocyclic linkages. These numbers do not take into consideration the possibility that some nonprotonated nitrogens in the form of intermolecular condensation linkages, as in the following triphenylamine structures, may occur in the approximate chemical shift range from 60 to 130 ppm:
O O H3C
C
CH2CO2Et
φNH2
C EtO2C
C
R
N
O
O R
N OH
φ
N OH
O
φ
O
CH3
φ
9
OH
R
CH3 CO2Et
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OH
NHφ R C CH2OH
H
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It is interesting that, in both pairs of samples, more aniline was incorporated into heterocyclic structures in the
TABLE 2
Percentages of Protonated versus Nonprotonated Nitrogens in ACOUSTIC 15N NMR Spectra of Fulvic and Humic Acids Reacted with O15NH2 and Summary of Assignments sample
nonprotonated nitrogen (200-140 ppm)
protonated nitrogen (140-0 ppm)
Suwannee River FA Suwannee River HA IHSS Soil FA IHSS Soil HA
55% 50% 68% 48%
45% 50% 32% 52%
chem shift range, ppm
assignmenta
60-100 100-122 122-148 148-200 300-350
anilinohydroquinone, phenoxazine anilinoquinone, enamine anilide, enaminone, quinolone, indole N-phenylindole, N-phenylpyrrole, heterocyclic N imine, phenoxazinone, quinoline
a
FIGURE 6. Liquid phase ACOUSTIC and INEPT 15N NMR spectra of IHSS soil fulvic and humic acids reacted with O15NH2.
fulvic acid than in the humic acid. Since fulvic acids have greater aliphatic character than humic acids, they are more likely to contain a greater concentration of the types of aliphatic ketone structures (dialkyl or enolizable ketones) that undergo condensation with aniline to form nitrogen heterocycles. Additionally, the quinone, hydroquinone, and catechol groups in fulvic acids are more likely to exist in less substituted and in less condensed aromatic structures than in humic acids and, therefore, are more favorably disposed to react with aniline to form the heterocyclic
Most probable assignment listed in italics.
structures. There is some evidence that the same correlation also holds true for the reaction of ammonia with fulvic and humic acids (38). The same types of condensation reactions occur in both cases. It is instructive to compare the distribution of peak intensities in the inverse gated decoupled 15N NMR spectra of the 4-methylcatechol and benzoquinone (Figures 3 and 4) with the ACOUSTIC spectra of the fulvic and humic acids (Figures 5 and 6). Although a significant number of peaks occur in the region from ∼140 to 200 ppm in the case of 4-methylcatechol, in neither the benzoquinone or 4methylcatechol spectra do the proportions of intensities of nonprotonated versus protonated nitrogens match the fulvic and humic acid spectra. The implication is that, in humic substances, the quinone, hydroquinone, and catechol functionality alone is not sufficient to account for the distribution and quantities of heterocyclic condensation products formed upon reaction with aniline. Indeed, it is difficult to explain the formation of the heterocyclic structures in the fulvic and humic acids without the participation of a complex array of carbonyl structures. Assignments for the 15N NMR spectra of the aniline reacted fulvic and humic acids are summarized in Table 2. These are the best assignments we are able to make at this time, based upon our knowledge of the 15N NMR chemical shift literature and of the nucleophilic addition chemistry of aromatic amines. One caveat to keep in mind is that humic substances are known to contain stable free radicals, generally agreed to be semiquinones (42, 43), and so free radical coupling reactions between aniline and the fulvic and humic acid molecules are a possibility even in the absence of catalysis by metals or phenol oxidase enzymes. In the quinone-hydroquinone model postulated to account for the ESR spectral properties of humic substances (42, 43), however, because the spin concentration of fulvic and humic acids is at a minimum at acidic pH and increases markedly above neutrality as semiquinone radicals are converted into their corresponding stable radical anions, the potential for free radical coupling reactions with aniline should be greatest at basic pH. Solid State 15N NMR Spectra of Humic Acids Reacted with O15NH2. Solid state CP/MAS 15N NMR spectra of the
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substituted aromatic structures than in fulvic acids. As discussed earlier, imine formation (1,2-addition) is favored over 1,4-addition in such cases. The recent suggestion however that binding of aromatic amines by humic acids should occur predominantly by imine formation is incorrect (33). Examination of Anoxic Conditions and Effect of Prior Treatments on Binding of φ15NH2 by SRFA. To gain further insight into the mechanisms of the covalent binding, SRFA was reacted with φ15NH2 at pH 6 in the absence of oxygen and with φ15NH2 at pH 6 under oxic conditions after separate prior treatments with sodium borohydride and unlabeled hydroxylamine. Only the liquid phase INEPT spectra are shown here (Figure 8). NMR experiments to observe all nitrogens are currently being performed. All three INEPT spectra show the same resultsa relative decrease in the proportion of anilinohydroquinone nitrogen with respect to anilinoquinone and anilide nitrogen. In the absence of oxygen, catechol and hydroquinone groups are prevented from oxidizing to quinones and therefore from undergoing nucleophilic addition by the aniline: N2
X
FIGURE 7. Solid state CP/MAS 15N NMR spectra of IHSS and Iowa soil humic acids reacted with O15NH2.
O
OH O
OH
IHSS and Iowa soil humic acids reacted with φ15NH2 are shown in Figure 7. Although analyses of the 15N and 1H spin dynamics were not performed, it is evident from a comparison of the liquid (Figure 6) and solid state spectra of the IHSS soil humic acid that under the acquisition parameters used, the intensities of the nonprotonated nitrogens are underestimated with respect to the protonated nitrogens in the CP/MAS experiment. In general, the chemical shift positions of the peak maxima in the solid state spectrum correlate with the ACOUSTIC spectrums187, 175, and 139 ppm, with some slight differences in the anilinohydroquinone and anilinoquinone regions. Major peaks occur at 102, 113, 138, 173, and 185 ppm in the solid state spectrum of the Iowa humic acid. The unreacted aniline occurs at 49 ppm. The outstanding feature of the spectrum of the Iowa soil humic acid is the imine peak centered at 307 ppm, which most likely arises from the 1,2-addition of aniline to quinones. The imine peak is easier to observe in the solid state spectrum because of the increased sensitivity (sample concentration) and absence of baseline distortions in the CP/MAS experiment. Vertical expansion of the ACOUSTIC spectrum of the IHSS soil humic acid (Figure 6) also revealed the possible presence of a broad, low-intensity imine peak underlying the contaminant peak at 315 ppm. In the solid state spectrum, the contaminant peak is broadened out, presumably because of chemical shift anisotropy, and the underlying imine peak is more clearly visible. Alternative assignments for the imine peak would include phenoxazinone nitrogens, also from the condensation of aniline with quinones; quinolines from the reaction of aniline with 1,3-dicarbonyl compounds as in the Combes synthesis (44); or ketimines and aldimines from 1,2-addition of aniline to ketones or aldoses, respectively. However, the formation of imines in the humic acids, but not in the fulvic acids, via 1,2-addition to quinones would be consistent with the view that the quinone, catechol, and hydroquinone moieties in humic acids are likely to occur in more condensed or more
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This explains the decrease in the amount of aniline taken up by the fulvic acid as indicated by the relative decrease in the anilinohydroquinone peak in Figure 8B. The fact that anilinoquinone and anilinohydroquinone peaks are still present however indicates that quinone groups exist in their oxidized state at pH 6; in other words even in the absence of oxygen the equilibrium is shifted enough toward quinones so that substrate sites for 1,4-addition by aniline are still available. What is not immediately apparent is why the anilinohydroquinone peak is reduced in intensity with respect to the anilinoquinone peak. Intuitively, one would expect the reverse. The initial condensation product of aniline with a quinone, the anilinohydroquinone moiety, should be prevented from oxidizing to the anilinoquinone moiety in the absence of oxygen: O
O
OH NHφ
φNH2
NHφ
N2
X
N2
O
OH
O
The results observed in the 15N NMR may be explained in part by the occurrence of inter- or intramolecular redox reactions within the mixture of fulvic acid molecules. Two plausible examples are illustrated in Scheme 1: In the first scenario, aniline does not react with the 1,4-quinone because of steric hindrance but does undergo Michael addition to the 1,2-quinone. The anilinohydroquinone is then oxidized to the anilinoquinone via an intermolecular redox reaction with the 1,4-quinone. In the second scenario, aniline undergoes Michael addition to the 1,2quinone in the terminal ring of the oxidized alizarin molecule to form the anilinohydroquinone moiety, which is then oxidized to the anilinoquinone moiety via an intramolecular redox reaction. Saxena and Bartha (11) determined that removal of oxygen from the system decreased the binding of DCA to a soil humic acid by 10%.
FIGURE 8. Liquid phase INEPT 15N NMR spectra of Suwannee River fulvic acid. (A) Reaction with O15NH2 at pH 6 (from Figure 5). (B) Reaction with O15NH2 at pH 6 under anoxic conditions. (C) Reaction with O15NH2 after prior treatment with sodium borohydride. (D) Reaction with O15NH2 after prior treatment with unlabeled hydroxylamine. (E) Reaction with O15NH2 followed by reaction with unlabeled ammonium hydroxide.
They concluded that intramolecular redox reactions could also account for the less than dramatic inhibition of binding under anoxic conditions.
Sodium borohydride reduces ketones and quinones to secondary alcohols and hydroquinones/catechols, respectively. The 13C NMR spectrum of the sodium borohydridetreated fulvic acid (not shown here) prior to reaction with aniline indicated that the ketone and quinone functionality (220-180 ppm) was incompletely reduced. Treatment with sodium borohydride did reduce enough quinone groups however to noticeably decrease the uptake of aniline by the fulvic acid, as evident from the INEPT spectrum in Figure 8C. This is in agreement with results from the kinetic studies, in which prior treatment with the borohydride decreased the rate constant for binding (1). Saxena and Bartha (11) also observed a reduction in the binding of DCA by humic acid pretreated with sodium borohydride, although the reaction of the reduced humic acid with DCA was performed under anoxic conditions. Prior treatment of SRFA with hydroxylamine also should serve to decrease the binding of aniline since hydroxylamine blocks the ketone, quinone, amide, and ester functionality of the fulvic acid. Our previous work demonstrated however, as in the case of sodium borohydride reduction, that hydroxylamine incompletely derivatizes the ketone and quinone functionality of the fulvic acid (21). Nevertheless, the INEPT spectrum in Figure 8D clearly indicates once again a relative decrease in the proportion of anilinohydroquinone nitrogen with respect to anilinoquinone and anilide nitrogen. These results are consistent with the kinetic studies (1) and were also duplicated with the IHSS soil humic acid (38). The fact that aniline reacts with the hydroxylamine-treated fulvic acid to the extent it does is somewhat surprising in that hydroxylamine should be as reactive toward, and therefore block, the same functional groups that aniline reacts with. The observed reactivity of aniline toward the hydroxylamine-treated fulvic acid may be explained in terms of nucleophilic catalysis. Chemical Lability of O15NH2 Incorporated into SRFA. Several investigators have noted that a fraction of the aniline covalently bonded to humic substances could be displaced by other amines (11, 13, 45). The ability of ammonia to displace the covalently bonded aniline was examined in the SRFA. In the INEPT spectrum of the sample treated with ammonia (Figure 8E), there appears to be a reduction in intensity of both the anilide and the anilinohydroquinone peaks with respect to the anilinoquinone peak. The loss of anilide nitrogens can be explained in terms of amminolysis by ammonia. The reduction of the anilinohydroquinone nitrogen with respect to anilinoquinone nitrogen is again counterintuitive. In order for ammonia to displace the anilinohydroquinone nitrogen, the anilinohydroquinone must first be oxidized to the anilinoquinone, since the substitution reaction proceeds via a retro-Michael addition. The results observed here may also be accounted for by the occurrence of inter- or intramolecular redox reactions during the course of the nucleophilic substitutions. The displacement of both the anilide and the anilinohydroquinone nitrogens by ammonia was replicated in the soil humic acid (38).
Discussion The previous work of Bartha (5-12) and of others (13) concluded that quinone groups were the major substrate site for the nucleophilic addition of aromatic amines to humic substances. Observation of the anilinohydroquinone, anilinoquinone, and imine nitrogens in the 15N NMR spectra presented in this study constitutes the first
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SCHEME 1 NHφ
O
O φNH2
+ O O
N2
+
N2
O
O
NHφ
direct spectroscopic evidence for the condensation of aniline with quinone groups in humic substances. In the latter case, imine formation via 1,2-addition of aniline to quinones implies the presence of sterically hindered quinones. However, the 15N NMR results suggest that a variety of carbonyl groups in addition to quinones are involved in the nucleophilic addition reactions. The occurrence of anilide nitrogens in the spectra demonstrated the reaction of aniline with ester or amide groups. Consideration of the possible reaction mechanisms leading to the formation of heterocyclic nitrogen suggests the participation of such carbonyl groups such as ketones, 1,4diketones, and β-dicarbonyls, including β-ketoesters. More work with model compound systems and more specific assignments for the heterocyclic nitrogen regions of the 15N NMR spectra will be necessary to confirm these mechanisms. That the majority of aniline was incorporated into heterocyclic structures also is in agreement with previous studies. Parris (13) summarized the probable sequence of steps through which primary aromatic amines undergo nucleophilic addition reactions with humic substances, according to the work of Hsu and Bartha (5-7). The first step involves a reversible Michael addition to quinone structures. The addition is followed by tautomerization and oxidation yielding aminoquinones. The amino group may react further by essentially the same addition-tautomerizationoxidation sequence to form a variety of nitrogen heterocycles. Progression along this sequence is expected to make the amine moiety more resistant to removal from the humic substance. Prior to oxidation, the amine residue from the first step might be displaced from the humic substance by a suitable competing reagent. The picture that emerges from the 15N NMR studies is in general agreement with this scheme but with additional complexities. First, as just discussed, a variety of carbonyl groups are involved in the formation of the heterocyclic nitrogen adducts. Secondly the observation of both the oxidized and reduced form of the Michael addition products of aniline with quinone groups in the 15N NMR spectra was not anticipated. This observation coupled with results from the studies on the effects of prior blockage and on the exchange reactions implies that intra- or intermolecular redox reactions are involved in the nucleophilic addition chemistry of aniline with the humic substances. The NMR results also are in agreement with the fact that the first step in the Michael addition of aniline to quinones (formation of the anilinohydroquinone nitrogen) is a reversible step and that the anilinohydroquinone nitrogen represents a fraction of the covalently bound aniline that is chemically labile and subject to exchange by other amines. Additionally, the NMR experiments suggest that the anilide nitrogens also are subject to exchange by other amines.
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OH
NHφ
OH
O
N2
N2
O
O
OH
φNH2
2774
O
OH
O
O
+
OH
O
O
NHφ
OH
O
OH O
OH
Acknowledgments The authors thank Tom Steinheimer of the USDA National Soil Tilth Lab for collection and information on the Walnut Creek soil sample, Angus McGrath of Lawrence Berkeley Lab and Colleen Rostad of USGS for helpful reviews of the manuscript, and James Eck for technical assistance in the early stages of this work. This research was supported by U.S. EPA Grants DW14935164, and DW14935652, and by USDA Grant 92-34214-7352. (Use of trade names in this report is for identification purposes only and does not constitute endorsement by the U.S. Geological Survey or U.S. Environmental Protection Agency.)
Literature Cited (1) Weber, E. J.; Spidle, D. L.; Thorn, K. A. Environ. Sci. Technol. 1996, 30, 2755-2763. (2) Adrian, P.; Andreux, F.; Viswanathan, R.; Freitag, D.; Scheunert, I. Toxicol. Environ. Chem. 1989, 20-21, 109. (3) Marco, G. J.; Novak, R. A. J. Agric. Food Chem. 1991, 39, 2101. (4) Bollag, J.-M. Environ. Sci. Technol. 1992, 26, 1876. (5) Hsu, T.-S.; Bartha, R. Soil Sci. 1974, 116, 444. (6) Hsu, T.-S.; Bartha, R. Soil Sci. 1974, 118, 213. (7) Hsu, T.-S.; Bartha, R. J. Agric. Food Chem. 1976, 24, 118. (8) You, I. S.; Jones, R.; Bartha, R. Bull Environ. Contam. Toxicol. 1982, 29, 476. (9) Bartha, R.; You, I.-S.; Saxena, A. Pestic. Chem.: Hum. Welfare Environ., Proc. Int. Congr. Pestic. Chem., 5th 1983, 3, 345. (10) Saxena, A.; Bartha, R. Soil Biol. Biochem. 1983, 15, 59. (11) Saxena, A.; Bartha, R. Soil Sci. 1983, 136, 111. (12) Saxena, A.; Bartha, R. Bull. Environ. Contam. Toxicol. 1983, 30, 485. (13) Parris, G. E. Environ. Sci. Technol. 1980, 14, 1099. (14) Bollag, J.-M.; Minard, R. D.; Liu, S.-Y. Environ. Sci. Technol. 1983, 17, 72. (15) Simmons, K. E.; Minard, R. D.; Bollag, J. M. Environ. Sci. Technol. 1987, 21, 999. (16) Simmons, K. E.; Minard, R. D.; Bollag, J.-M. Soil. Sci. Soc. Am. J. 1988, 52, 1356. (17) Simmons, K. E.; Minard, R. D.; Bollag, J.-M. Environ. Sci. Technol. 1989, 23, 115. (18) Tatsumi, K.; Freyer, A.; Minard, R. D.; Bollag, J.-M. Environ. Sci. Technol. 1994, 28, 210. (19) Hatcher, P. G.; Bortiatynski, J. M.; Minard, R. D.; Dec, J.; Bollag, J. M. Environ. Sci. Technol. 1993, 27, 2098. (20) Thorn, K. A.; Goldenberg, W. S.; Younger, S. J.; Weber, E. J., Submitted for publication. (21) Thorn, K. A.; Arterburn, J. B.; Mikita, M. A. Environ. Sci. Technol. 1992, 26, 107. (22) Thorn, K. A.; Mikita, M. A. Sci. Total Environ. 1992, 13, 67. (23) Patt, S. L. J. Magn. Reson. 1982, 49, 161. (24) Morris, G. A.; Freeman, R. J. Am. Chem. Soc. 1979, 101, 760. (25) Proceedings of the Conference on Agricultural Research to Protect Water Quality, February 21-24, 1993, Minneapolis, MN; Soil and Water Conservation Society: Ankeny, IA, Aug, 1993. (26) Smith, J. R. L.; Smart, A. U.; Hancock, F. E.; Twigg, M. V. J. Chromatogr. 1989, 483, 341. (27) Witanowski, M.; Stefaniak, L.; Webb, G. A. In Annual Reports on NMR Spectrometry; Webb, G. A., Ed.; Academic Press, Harcourt Brace Jovanovich: London, 1986; Vol. 18. (28) Witanowski, M.; Stefaniak, L.; Webb, G. A. In Annual Reports on NMR Spectrometry; Webb, G. A., Ed.; Academic Press, Harcourt Brace Jovanovich: London, 1993; Vol. 25. (29) Finley, K. T. In Chemistry of the Quinonoid Compounds, Part 2; Patai, S., Ed.; John Wiley and Sons: London, 1974; Chapter 17.
(30) Kutyrev, A. A. Tetrahedron 1991, 47, 8043. (31) Monks, T. J.; Hanzlik, R. P.; Cohen, G. M.; Ross, D.; Graham, D. G. Toxicol. Appl. Pharmacol. 1992, 112, 2. (32) Reeves, R. L. In The Chemistry of the Carbonyl Group; Patai, S., Ed.; John Wiley and Sons: London, 1966; Chapter 12. (33) Ononye, A. I.; Graveel, J. G. Environ. Toxicol. Chem. 1994, 13, 537. (34) Levy, G. C.; Lichter, R. L. Nitrogen-15 Nuclear Magnetic Resonance Spectrometry; John Wiley and Sons: New York, 1979. (35) Martin, G. J.; Martin, M. L.; Gouesnard, J. P. 15N-NMR Spectroscopy; Springer-Verlag: New York, 1981. (36) Thorn, K. A. In Humic Substances in the Suwannee River, Georgia: Interactions, Properties, and Proposed Structures; Averett, R. C., Leenheer, J. A., McKnight, D. M., Thorn, K. A., Eds.; Open-File Report, U.S. Geological Survey: Denver, CO, 1989; No. 87-557, pp 251-309. (37) Thorn, K. A.; Folan, D. W.; MacCarthy, P. Characterization of the IHSS Standard and Reference Fulvic and Humic Acids by Solution State 13C and 1H NMR. Water-Resour. Invest. (U.S. Geol. Surv.) 1991, No. 89-4196. (38) Thorn, K. A. U.S. Geological Survey, unpublished results. (39) Adrian, P.; Lahaniatis, E. S.; Andreux, F.; Mansour, M.; Scheunert, I.; Korte, F. Chemosphere 1989, 18, 1599.
(40) Newkome, G. R.; Paudler, W. W. Contemporary Heterocyclic Chemistry; John Wiley & Sons: New York, 1982. (41) Brown, R. K. In Indoles Part One; Houlihan, W. J., Remers, W. A., Brown, R.K., Eds.; John Wiley & Sons: New York, 1972; pp 317-385. (42) Senesi, N.; Steelink, C. In Humic Substances II. In Search of Structure; Hayes, M. H. B., MacCarthy, P., Malcolm, R. L., Swift, R. S., Eds.; John Wiley and Sons: West Sussex, England, 1989; pp 373-408. (43) Steelink, C. In NMR of Humic Substances and CoalsTechniques, Problems, and Solutions; Wershaw, R. L., Mikita, M. A., Eds.; Lewis Publishers: Chelsea, MI, 1987; pp 47-72. (44) Joule, J. A.; Mills, K.; Smith, G. F. Heterocyclic Chemistry, 3rd Ed.; Chapman and Hall: New York, 1995. (45) Fuchsbichler, G.; Suss, A. Chemosphere 1978, 4, 345.
Received for review December 14, 1995. Revised manuscript received May 3, 1996. Accepted May 13, 1996.X ES9509339 X
Abstract published in Advance ACS Abstracts, July 1, 1996.
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