Environ. Sci. Technol. 1907, 21, 791-798
Structural Characterization of Aquatic Humic Material. 2. Phenolic Content and Its Relationship to Chlorination Mechanism in an Isolated Aquatic Fulvic Acid Daniel L. Norwood" and Russell F. Chrlstman Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, North Carolina 27514
Patrick G. Hatcher United States Geological Survey, Reston, Virginia 22092
The complementarytechniques of solid-state 13Cnuclear magnetic resonance spectroscopy and chemical degradation were utilized to examine the lignin/phenolic substructure of an isolated aquatic fulvic acid capable of producing upon aqueous chlorination a number of organohalides typically found in municipal drinking water. Results indicate that while phenolic moieties are present in the fulvic acid, they account for only a minor fraction of the total carbon. A sequential chemical degradation experiment utilizing aqueous chlorine and CuO demonstrated that the lignin/phenolic substructure was attacked by the chlorine. It is concluded that while phenolic ring rupture mechanisms appear to be important in organohalide generation, other aqueous chlorination mechanisms involving aliphatic and other types of aromatic structures should also be considered.
Introduction It is well documented that disinfection of natural waters with aqueous chlorine results in drinking waters that contain organically bound halogen (referred to as total organic halide or TOX). A portion of this TOX is composed of volatile, relatively hydrophobic organohalides such as the trihalomethanes, principally chloroform ( I ) . Another portion is composed of hydrophilic organohalides, principally di- and trichloroacetic acids (2-4). It is attractive to hypothesize that rupture of phenolic rings contained in natural aquatic humic material by aqueous chlorine is the principal formation pathway for TOX. Indeed, such mechanisms have been invoked to rationalize the formation of certain organohalides produced from the aqueous chlorination of humic substances (5-7). Isolated aquatic humic substances, however, produce a significant number and variety of chlorinated organic compounds when subjected to aqueous chlorination (8-11). For certain of these organohalides, phenolic ring rupture is not the most likely formation pathway. Humic substances are complex, macromolecular organic structures formed during the diagenesis of plant material. They are ubiquitous throughout the environment and constitute the bulk of organic material in soil and approximately half of the dissolved organic carbon in terrestrial streams (12, 13). Lignin, a phenolic polymer and a major component of woody tissue, is thought to be an important precursor for humic substances. The lignin/ phenol content of humic substances has been extensively investigated via oxidative chemical degradation (14). Alkaline solutions of cupric oxide (CuO) (15, 16) as well as other oxidizing agents (14) have been used to degrade
* Address correspondence to Research Triangle Institute, Analytical and Chemical Sciences, Research Triangle Park, NC 27709. 0013-936X/87/0921-0791$01.50/0
lignin and humic substances with the resulting production of phenolic aldehydes and acids. Solid-state carbon-13 nuclear magnetic resonance spectroscopy (13C NMR) employing cross-polarization (CP), magic-angle spinning (MAS),and high-power proton decoupling is a relatively new technique that is being widely utilized to assess qualitative and quantitative carbon distributions in humic substances (17) and other macropolecular materials (18,19). The technique is capable of assessing oxygen-substituted aromatic carbon contents in lignins (19) and humic substances (20), providing a powerful complement to chemical degradation. This paper describes the application of NMR techniques [including CP/MAS 13C NMR, relaxation experiments, and proton (lH NMR)] and chemical degradation procedures to an isolated aquatic fulvic acid. Inferences are drawn from the combination of NMR and chemical degradation results regarding the overall macromolecular structure and mechanisms of chlorination of the aquatic fulvic acid. Experimental Methods Fulvic Acid Isolation. Aquatic fulvic acid was isolated from Singletary Lake according to the adsorption chromatography procedure of Thurman and Malcolm (21). Singletary Lake is a highly colored natural lake located on the North Carolina coastal plain near Black Lake. The latter was utilized as a source of humic substances in several prevous studies (3,4,8-11), including the first in this series (22). Lignin utilized in this study was extracted from Douglas fir according to Hatcher (23). Soil- and peat-derived humic substances were obtained according to procedures described by Hatcher et al. (20). Elemental Analysis. Elemental analysis of solid fulvic acid samples was accomplished by Huffman Laboratories, Inc., Wheat Ridge, CO, utilizing standard gravimetric procedures (24). lH NMR. lH NMR spectra were acquired at 250 MHz with a Bruker WM-250 Fourier transform spectrometer (Bruker Instruments, Inc., Manning Park, Billerica, MA). Two hundred scans were averaged with a total cycle time of 2 s to produce an acceptable spectrum. 13C NMR. 13C NMR spectra of aquatic humic substances in the solid state were obtained by the cross-polarization, magic-angle spinning (CP/MAS) technique with high-power proton decoupling. A Chemagnetics CMClOO spectrometer (Chemagnetics, Fort Collins, CO) equipped with a 2.35-T superconducting magnet (25.2 MHz for carbon) was utilized for these experiments. Each pulse experiment involved the basic sequence of events described by Hatcher et al. (25), with a 1-ms cross-polarization contact time according to the Hartman-Hahn condition (26) and 500-ms-1-s pulse delay. The solid fulvic acid was held in a 300-pL KEL-F rotor and spun at a rate of 3.2 kHz
0 1987 American Chemical Society
Environ. Sci. Technol., Vol. 21, NO. 8, 1987
791
0
4 40
9:31
14 16
10'00
Time (mln)
Figure 1. E1 total ion chromatogram of the ether-extractable aqueous chlorination products of Singletary Lake fulvic acld: 15 m X 0.2 mm DB-1 fused silica capillary column (Jaw Scientific, Rancho Cordova, CA); injector temperature 270 OC;initial column temperature 60 OC (2-min initial hold); 6 deg/min program; final column temperature 256 OC; 1O:i split injection.
at the magic angle (54.7' to the applied field) over the course of the experiment. CP/MAS 13CNMR relaxation studies involved alternate pulse sequences, which are described later. Relative amounts of various types of protons and carbons were determined by area measurement of the appropriate spectral regions. It has been determined in this laboratory that errors in such area measurements are approximately *5%. Aqueous Chlorination. Procedures for the chlorination and analysis of the resulting organohalide products from Singletary Lake fulvic acid were similar to those described previously for Black Lake fulvic acid ( 9 , I I ) . To summarize, 300 mg of fulvic acid was reacted in 450 mL of pH 11.2 NaOH solution at an initial chlorine-to-carbon (HOCl/C) molar ratio of 2:l under headspace-free conditions. No buffers were employed, and the pH was allowed to drop to 9.6 over the 26-h reaction time period. A quenched, acidified 300-mL aliquot was removed for ether extraction, concentration, derivatization (diazomethylation), and qualitative analysis by combined gas chromatography/mass spectrometry (GC/MS). A system blank including all components except the fulvic acid was chlorinated and subjected to an identical analytical procedure. The GC/MS system and analytical conditions employed were the same as those utilized in previous studies (VG 7070F GC/MS system) (3, 4, 8-11). Chromatography conditions are indicated in Figure 1. The operation of the VG 7070F instrument in the low-resolution electron ionization (EI) mode has been described (11). Isobutane positive chemical ionization (PCI), negative chemical ionization (NCI, isobutane buffer gas), and low-resolution accurate mass measured E1 spectra (tetraiodoethylene internal reference) were collected as a complement, utilizing the manufacturer's recommended conditions. CuO Oxidation, CuO oxidation of Singletary Lake fulvic acid and the extraction and analysis of the resulting oxidation products were carried out by the procedures described by Hedges and Ertel (16) with only minor modification. Quantification of individual ether-extractable phenols was achieved by gas chromatographicanalysis (Perkin-Elmer Sigma 2; Perkin-Elmer, Norwalk, CT) with flame ionization detection (FID) of the corresponding trimethylsilyl derivatives in pyridine under appropriate GC conditions (16). Mass spectral analyses were carried out on the VG 7070F system. Sequential Chemical Degradation. The first degradation in the sequence was aqueous chlorination. Singletary Lake fulvic acid (301 mg) was reacted in 450 mL of deionized/distilled water at an initial chlorine-to-carbon molar ratio of 0.3:l and neutral pH for 5 h and 40 min in the dark. The final reaction mixture pH (before sodium arsenite quenching) was 5 . 792
Environ. Sci. Technol., Vol. 21, No. 8, 1987
Time (min)
Figure 2. NCI selected mass chromatogram ( m l z 35) of the etherextractable aqueous chlorination products of Singletary Lake fulvic acid.
Partially reacted fulvic acid was isolated from the reaction mixture by absorption onto a 200-mL bed volume column to solvent-extracted (acetone/hexane) XAD-8 resin (Rohm and Haas, Philadelphia, PA). The acidified (pH 1)reaction mixture was then eluted through the column at a flow rate of 5 mL/min. The column was then eluted with one column volume of distilled/deionized water to remove chloride, followed by one column volume of 0.1 N NaOH to desorb the fulvic acid. A brightly colored solution of 350-mL volume was collected and was immediately passed through a 130-mL bed volume column of methanol-extracted AG-MP-50 (Rohm and Haas) cation-exchange resin at a moderate flow rate for desalting. The resulting solution was lyophilized (Labconco Model 18, Fisher) to produce 150 mg of solid hydrogen exchanged fulvic acid. CuO oxidation was used as the second degradation in the sequence. Chromatographic analyses of oxidation products were performed on the Perkin-Elmer system and on a Carlo-Erba 5160 HRGC (Carlo-Erba, Milan, Italy) utilizing the previously indicated conditions.
Results and Discussion Elemental Analysis. The elemental composition of Singletary Lake fulvic acid (average of eight different batch determinations) was determined to be C 53.06% (S, = 1.21), H 4.57% (S, = 0.361, and N 1.22% (S, = 0.32) on an ash-free basis. The resulting H/C ratio of 1.03 is in the range reported for aquatic humic substances by Thurman (27) and indicates a relatively high degree of unsaturation and/or heteroatom substitution. The C/N ratio of 50.7 is also typical of aquatic humic substances (27). Aqueous Chlorination. A reconstructed total ion chromatogram (EI) of the methylated product ether extract is shown in Figure 1. The identification of individual chlorination products utilized the E1 fragmentation pattern, an element map provided by the accurate mass measured E1 spectrum, and the protonated molecular ion from the PCI spectrum (when available). Where authentic standards were available these were analyzed and their E1 spectra and retention indices (relative to methyl p chlorobenzoate internal reference) compared with the appropriate unknown data set. The complexity of the organohalide product mixture is indicated in Figure 2, which shows an NCI mass chromatogram of m / z 35 (Cl-). Each chromatographic peak represents an individual organohalide. Figure 1 also indicates two of the most prominent organohalides formed, dichloroacetic acid (DCAA) and trichloroacetic acid (TCAA). These and several other of the 20 major organohalide products are listed in Table I. Table I includes examples of possible phenolic ring rupture products (chloroform, dichloromaleic acid) as well as products that are not so easily rationalized by such mechanisms (dichlorosuccinic acid). The overall distribution of organohalides and other chlorination products
Table I. Major Organohalide Chlorination Products CHC13 HCC12COZH
chloroform dichloroethanoic acid (dichloroacetic acid, DCAA) CC13COZH trichloroethanoic acid (trichloroacetic acid, TCAA) HOzCCClzCOzH dichloropropanedioic acid (dichloromalonic acid) HOzCCClzCHzCOzH 2,2-dichlorobutanedioic acid (dichlorosuccinic acid) HO2CCCl=CC1CO2H cis-dichlorobutenedioic acid (dichloromaleic acid)
c-c
COOH
a
a
b
b a
Confirmed. E1 spectrum and GC retention index match those of an authentic standard; other spectral data agree. bConfident. Sufficient data are available to preclude all but the most closely related structures.
,
,
1
1
,
I1 (50-110 ppm) I11 (110-160 ppm)
IV (160-220 ppm)
1
1
,
1
1
1
1
1
0
‘ 1 1 H2°
% contri-
I (0-50 ppm)
1
100
Table 11. 13C (A) and ‘H(B) Chemical Shift Regions region
,
200
carbon type
bution
methyl, methylene, methine (etc.) alcohol, amine, carbohydrate, ether, methoxyl, acetal (etc.) olefinic, aromatic, phenolic carboxyl, ester, amide, aldehyde, ketone
29
l
22 Ar-C-H H-C-COOH
!i
21
H-C-C
24 % contri-
region
proton type
bution
I (0.4 - 1.7 ppm)
methyl, methylene, methine (etc.) methyl, methylene ( a to aromatic rings or carboxyl groups), a and p groups of indanes and tetralins protons on carbons, a to oxygen aromatic
38
I1 (1.7-3.3 ppm)
I11 (3.3-4.6 ppm) IV (6.5-8.1 m m )
I
I
I
I
I
I
10
9
8
7
6
5
4
I
’
l
l
3
2
1
0 PPm
36
16 9
is qualitatively and semiquantitatively identical with those observed in previous studies (8-1 I) from other aquatic humic substances. I3C and ‘H NMR Spectral Interpretations. CP/ MAS 13Cand lH NMR spectra of Singletary Lake fulvic acid are shown in Figure 3. The CP/MAS spectrum (Figure 3A) resulted from the collection and averaging of 95 000 individual free-induction decays and is striking similar to spectra reported for other aquatic fulvic acids (27). Our treatment of these data is patterned after the work of Hatcher et al. (20) and divides the spectrum into four regions as outlined in part A of Table 11. These include region I, paraffinic carbons; region 11, aliphatic carbons substituted with oxygen; region 111, aromatic/ olefinic carbons; and region IV, carbonyl carbons. The most interesting piece of information contained in this spectrum is the relative importance of aliphatic structures in this aquatic fulvic acid (regions I and 11). The anisotropic peak in region I with resonance maxima at 23.5 and 40.8 ppm is probably due to highly cross-linked methylene and methine carbon chains. Region I1 shows an intense resonance centered at 76 ppm, which is usually assigned to a polyhdyroxy aliphatic carbon such as carbohydrate carbon. The presence of carbohydrates cannot, however, be confirmed since a distinct peak for the anomeric carbon of polysaccharides (105 ppm) is not present. The aliphatic carbon in these two spectral regions accounts for 51% of the total carbon.
Figure 3. CP/MAS 13C NMR (A) and ‘H NMR (E) (D,O/NaOD solution) spectra of Singletary Lake fulvic acid.
The aromatic/olefinic region (111) has a maximum centered at 129 ppm, most likely due to aromatic carbons not substituted with heteroatoms. Oxygen-substituted aromatic carbons of phenols and aryl ethers should exhibit signals at approximately 150 ppm. Note that a reasonably intense shoulder but no distinct peak is apparent at 150 ppm in this spectrum. The aromatic region accounts for 21% of the total fulvic acid carbon, which translates to three aromatic rings per 100 carbon atoms in the macromolecule. These results are surprising with regard to the traditional view of humic chlorination chemistry that emphasizes the reactivity of aromatic organohalide precursors. The carbonyl region (IV) has a maximum at 173 ppm (carbonyl of carboxy groups) and a more diffuse resonance centered at approximately 205 ppm (carbonyl of aldehydes and ketolres). A typical proton spectrum of Singletary Lake fulvic acid appears in Figure 3B. Our interpretations have been influenced by Wilson’s (17)summary of chemical shift assignments for humic substances and are summarized in part B of Table 11. Like the CP/MAS spectrum, the proton spectrum can be divided into four regions. These include region I, protons on aliphatic carbons at least two carbons removed from aromatic rings or other polar functional groups; region 11,protons on carbons a to aromatic rings or carboxyl groups; region 111, protons on carbons attached to oxygen functions; and region IV, protons bound to aromatic rings. Note the excellent correlation between this proton spectrum and the CP/ MAS spectrum. An interesting observation is that the aromatic protons account for only 9.2% of the total nonexchangeable proton whereas 21% of the carbon is present in aromatic rings. This result implies that aromatic rings Environ. Sci. Technol., Vol. 21, No. 8, 1987
793
I Flgure 4. CP/MAS I3C NMR spectrum of a lignin extracted from Douglas fir.
n
1 ~ ' ~ ' 1 " " 1 ' ~ ~ ' I " " 1 " ~ ~ I ' " ' / ' ' ' ' I ' ' '
200
100
0
ppm
Flgure 6. CP/MAS 13C NMR spectra of three humic acids extracted from (A) Everglades peat, (B)histosol soil, and (C) mollisol soil.
1 ' " ' 1 " ' ' I " " I
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200
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