Direct I3C NMR Evidence for Carbohydrate Moieties in Fulvic Acids R. L. Wershaw, US. Geological Survey, 5293 Ward Road, Arvada, Colorado 80002
M. A. Mikita and Cornelius Steelink Department of Chemistry, University of Arizona, Tucson, Arizona 8572 1
We have developed a method for facilitating the interpretation of the hydroxyl region in the 13C NMR spectra of fulvic acids. By treating the fulvic acid samples with diazomethane (CH2N2) followed by methylation with 1%-enriched methyl iodide (CH31) in NaH/dimethylformamide (DMF), we have obtained products in which phenolic and hydroxylic groups are 13C methylated. The 13C NMR spectra of these methyl fulvates show strong distinct OCH3 bands, as well as weak COOCH3 bands. Prior treatment with 13C-enriched CH2N2 enhances the ester and phenolic bands. Comparison of the spectra of these methyl fulvates with model phenolic glycosides methylated under identical conditions clearly reveals a significant carbohydrate character. A quantitative interpretation can be obtained under appropriate NMR conditions. Humic and fulvic acids are the most abundant organic compounds in soils and in natural waters. These groups of compounds are highly active, entering into a wide variety of reactions including ion exchange, sorption, complexation, and solubilization. In spite of their importance in soil and water chemistry, very little is known about their chemical structure. The preponderance of evidence indicates that both humic and fulvic acids have a phenolic acid skeleton with attached carbohydrate, aliphatic, and amino acid groups. Humic acids generally are of greater molecular weights and are thought to be more phenolic than fulvic acids (1).However, recent studies have shown that both humic and fulvic acids can be fractionated into components, some of which appear to be more phenolic than others (2).In a previous study (31, a generalized model for the chemical structure of humic acids was proposed in which humic acid molecules formed both homogeneous and heterogeneous aggregates. The homogeneous aggregates are composed of chemically similar molecules, while the heterogeneous aggregates are composed of different types of molecules or of different homogeneous aggregates. Recent unpublished studies indicate that a similar model applies to fulvic acid. These aggregates are held together by various weak-bonding mechanisms, the most important of which is hydrogen bonding between the carboxylic acid groups, phenolic groups, and other hydroxyl groups. The distribution and concentration of these functional groups may be determined by using the procedure reported here in which 13C-labeled methyl derivatives of these groups are prepared and analyzed by 13C NMR. A number of structural studies on humic and fulvic acids have used NMR (4-7),but the spectra obtained have yielded only general information. The proton NMR spectra showed line broadening and line shifting, apparently due to hydrogen bonding and the presence of exchangeable protons, and the 13C spectra consisted of a few broad, weak lines. Wilson (8) and Hatcher (9)were able to obtain more informative spectra
* Address correspondence to this author a t the foiow&;ddress: U.S. Geological Survey, Federal Center, Mail Stop 407, Denver, CO 80225. 0013-936X/81/0915-146 1$01.2510 @ 198 1 American Chemical Society
by using high-magnetic fields and a resolution-enhancement technique, or solid-state NMR. However, their results do not allow unambiguous differentiation between the various hydroxyl groups present in fulvic and humic acids; the most direct way to accomplish this is derivatization of the groups with 13C-enriched reagents. Derivatization allows labeling of the hydroxyl groups and elimination of hydrogen bonding. Experimental Procedure The methylation procedure used is a modification of the one developed by Wershaw and Pinckney (10). The fulvic acid was dissolved in NJJ-dimethylformamide(DMF) and methylated with diazomethane (CH2N2). The products from this reaction were recovered by vacuum drying and redissolved in DMF. The next methylation step was to add a 10-fold excess of SOdium hydride (NaH) and methyl iodide (CH3I) to this s o h tion. At the end of the reaction period, the solution was poured into water, acidified with hydrochloric acid, and extracted with methylene chloride. The methlene chloride solution was washed 5-6 times with water to remove the DMF and evaporated to dryness on a hot water bath. This procedure has been used for the methylation of both humic acid fractions and unfractionated fulvic acids; in all instances, infrared and lH NMR analyses has shown that complete methylation was obtained. Two variations of the methylation procedure were used to differentiate between carbohydrate hydroxyl groups and phenolic hydroxyl groups and phenolic hydroxyl and carboxylic acid groups. In one variation 90% 13C-enrichedCH2N2, prepared from N-methyl-13C-N-nitroso-p-toluenesulfonamide, was used in the first methylation step, and 90% 13Cenriched CH31 in the second step. In the second variation unenriched CH2N2 and enriched CH31 were used. The 13C NMR spectrum obtained from the first variation will consist of absorption bands from all of the methyl esters and ethers produced by the methylation procedure, while in the second variation only the carbohydrate methyl esters and some of the more difficult to methylate phenols will be seen. The 13CNMR spectra were determined either on a Bruker Instruments WH-90 Fourier transform (FT) NMR spectrometer operating at a frequency of 22.62 MHz or on a Bruker WM 250 FT NMR spectrometer operating at 63.8 MHz with l H broad-band decoupling. Spectra were measured in solutions of chloroform-d which served as the internal deuterium lock. The spectra on the WH-90 were obtained by using 1000 data points over a 6000-Hz spectral’width with quadrature detection. The chemical shifts were assigned relative to internal tetramethylsilane (Me&). Because fulvic acid generally contains more carbohydrate groups than humic acids, only the results obtained in a typical soil fulvic acid (Contech, purified, “metal-free”, titrated fulvic acid) are reported. Contech fulvic acid was used because it has been extensively characterized and is readily available commercially for comparison (11).The phenolic glycoside rutin (Figure 1) was methylated by the same method in order to compare the fulvic acid results with those of a well-defined compound with similar structural elements. Volume 15, Number 12, December 1981
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Results and Discussion The spectrum of Contech fulvic acid methylated with both l3C-enriched CHzN2 and CHJ is given in Figure 2. Intense bands for methoxyl groups are seen in this fulvate spectrum between 50 and 62 ppm (relative to Me4Si). The chemical shifts in the region between 52 and 56 ppm are consistent with aromatic carboxylic acid methyl esters and phenolic esters. Thus, Scott (12) found that the methyl ester carbon atoms of methyl benzoate and methyl o -hydroxybenzoate have chemical shifts at 52.3 and 52.4 ppm, respectively. For the methyl carbon atoms of various methoxy ethers of benzoic acid derivatives, she reported chemical shifts of 55.3-56.3 ppm. Sundholm (13) has measured the 13Cspectrum of a symmetrical hexamethoxybenzophenone and found that the methoxy carbon atoms had chemical shifts between 55.3 and 56.2 ppm. Ludemann (14) has found that the 13Cchemical shift for the methoxy carbon atoms of ether groups of two lignins was 56.3 PPm. The relatively broad absorption band centered at 52.6 ppm is probably due to a mixture of aromatic and aliphatic ester groups. We have observed in another study of an aquatic fulvic acid (Mikita, Steelink, and Wershaw, in press) that the methyl ester band was resolved into two bands sharper than the one observed in this study; one was at -51 ppm and the other at -52 ppm. The first we have assigned to aliphatic methyl ester groups, and the second to aromatic ester groups. In a previous study (25) we have found by measuring the l3C NMR spectrum of humic and fulvic acids methylated with 13C-enriched CHzNzthat CH2N2 will methylate most of the carboxylic acid groups and some of the phenolic groups. Complete methylation of these groups required further
methylation with CHJ and dimethylsulfinyl carbanion. The carbohydrate hydroxyl groups, however, were not quite as efficiently methylated as in the procedure used here. The relative abundances of the various hydroxyl groups can be estimated from the peak areas in the full methylated fulvic acid in Figure 2. Thus, the ratio total carboxylhotal phenol is calculated from the integrated areas of the methyl ester band at 52.6 ppm and the methylaryl ether band at 56 ppm. This calculation yields a ratio of 2.5, which is in good agreement with the value of 2.3-2.4 based on titration data of Contech E.T.C., Ltd., who supplied us with the sample. Diazomethylation converts all phenolic groups to methyl ethers, with the exception of small amounts of phenolic groups adjacent to carbonyl functions. I t also converts carboxylic acids to methyl esters. This methylation behavior was deduced for the IH NMR spectra of model compounds. On the other hand, carbohydrate methyl esters, whose 13C NMR bands appear a t 58-62 ppm (16),are not formed by CH2N2. Thus, treatment of substrates with unenriched diazomethane does not enhance phenolic ether and carboxyl ester bands in the 13C NMR spectrum. The spectra in Figure 3 were obtained from samples that were methylated with unenriched CHzN2 and enriched CHsI. This procedure enhances aliphatic (or carbohydrate) methyl ether bands (58-62 ppm) at the expense of phenolic ether or acid ester bands (51-56 ppm). A comparison of the methyl fulvate spectra in Figure 3 with the spectra in Figure 2 dramatically illustrates this phenomenon. In another experiment, milder methylation conditions were used. Fulvic acid was treated with diazomethane; the product was then mixed with solid KOH and a 2-fold excess of 13C-
I
'I' Rutin NaH
I
Figure 1. Structure of rutin.
methylatlon
' i ,
J
NaH m e t h y l a t i o n
F u l v l c acld KOH methylation
6s ,
65
I
55
45
ppm(TMS)
Figure 2. I3C NMR spectrum of Contech fulvic acid methylated with I3C-enrichedCH2N2and I3C-enrichedCH31.Spectrum was measured on a Bruker WM 250 NMR spectrometer. 1462
Environmental Science & Technology
'
55
d5 p p r n ( 1 M S )
Flgure 3. I3C NMR spectra of rutin and Contech fulvic acid methylated with unenriched CH2N2and I3C-enrichedCH3L Spectra were measured on a Bruker WH-90 NMR spectrometer. Peak assignments for permethylated rutin: 6(CDC13)55.1,56.7 (methyl ethers at aromatic positions 3', 4',5, and 7), 58.0, 59.0, 60.0(methyl ethers at carbohydrate positions 2, 3, 4,and 6).
enriched CHsI and Me2SO and stirred for 1h a t room temperature ( 1 7 ) .The enriched compounds were examined by NMR. Somewhat less intense bands in the 56-62-ppm region were observed at the same chemical shifts as in the rigorous methylation procedure (Figure 3). The ratio of carbohydrate hydroxyl groups of carboxyl groups was calculated in the following manner: methyl ether band areas in the 57-62-ppm region were compared to methyl ester band areas in the 51-53-ppm region. A ratio of 0.46 was obtained, which is very close to the ratio of 0.47, which was calculated from the data of Gamble and Schnitzer (18) pertaining to aliphatic hydroxyl groups and acid groups in this fulvic acid. These results demonstrate that Contech fulvic acid contain carbohydrate-like hydroxyl groups, in addition to carboxyl and phenolic groups. The relative abundances of these three groups COOH/carbohydrate/phenolic are 2.5/1,1/1.0, in close agreement with literature values (18). The methylation method described in this study enables one to distinguish between various OH groups in complex macromolecules, by selectively enhancing the I3C concentration of the derivatized OH groups, and to calculate their relative concentrations. Preliminary results suggest that the relative concentration measurements are reliable; however, further work is necessary in order to verify the results and to determine those factors which limit the accuracy of the results. There is some overlap of the carbohydrate methyl ether region by the methyl ethers of other alcohols. Acid hydrolysis of humic and fulvic acids yields identified monosaccharides. Comparison of the saccharide ion concentration obtained by hydrolysis with that calculated from the NMR spectrum should allow us to determine whether other hydroxylated species are contributing to the observed NMR spectrum in the carbohydrate methyl ether region.
Literature Cited (1) Steelink, Cornelius. J. Chem. Educ., 1977,54, 599. ( 2 ) Thurman, E. M.; Malcolm, R. L. U S . Geol. Suru., Water-Supply Pap. 1979,1817-G, G-1. (3) Wershaw, R. L.; Pinckney, D. J.; Booker, S. E. U.S. Geol. Surv., J . Res. 1977,5,565. (4) Ludemann, H. D.: Lentz, Harro: Ziechmann, Wolfgang. Erdoel Kohle, Erdgas, Petrochem. Brennst.-Chem. 1973,26,566. ( 5 ) Oka, Hiroshi; Susaki, Mitsuo; Itoh, Mitsuomi; Suzuki, Akira. Nenryo Kyokaishi 1969,48,295. (6) Gonzalez Vila, F. J. Biochem. Biophys. Res. Commun. 1976,72, 1063. ( 7 ) Wilson, M. A.; Goh, K. M. Plant Soil 1977,46,287, (8)Wilson. M. A.: Jones. A. J.: Williamson. Bruce. Nature (London) ‘ 1978,276,487.’ (9) Hatcher, P. G.; Rowan, R.; Mattingly, M. A. Org. Geochem. 1980, 2.77.
(16j Wershaw, R. L.; Pinckney, D. J. Science 1978,199,906. (11) Burch, R. D.; Langford, C. H.; Gamble, D. S. Can J. Chem. 1978, 56,1196. (12) Scott, K. N., Jr. J. Am. Chem. Soc. 1972,94,8564. (13) Sundholm, E. G. Tetrahedron 1977,33,991. (14) Ludemann, H. D. Biochem. Biophys. Res. Commun. 1973,52, 1162. (15) Wershaw, R. L.; Pinckney, D. J.; Cary, Lewis, presented at the 20th Annual Rocky Mountain Conference on Analytical Chemistry, Denver, CO, 1978. (16) Blunt, J. W.; Munro, M. H. G.; Paterson, A. J. Aust. J. Chem. 1976,29,1115. (17) Johnstone, R. A. W.; Rose, M. E. Tetrahedron 1979,35,2169. (18) Gamble, D. S.; Schnitzer, Morris. In “Trace Metals and Metal Organic Interactions in Natural Waters”; Singer, P. C., Ed.; Ann Arbor Science: Ann Arbor, MI, 1973; p 265. Received for review September 22, 1980. Revised Manuscript Received April 20, 1981. Accepted July 6, 1981. This work was supported in part by funds provided by the officeof Water Research and Technology, A-094, US.Department of Interior, Washington, DC, as authorized by the Water Research and Development Act of 1978.
Gas-Chromatographic Technique for the On-Line Evaluation of Solvent Emission Abatement Devices Larry P. Haack,* Rick S. Marano, Tim t. Riley, and Steve P. Levine Ford Motor Company, Engineering and Research Staff, P.O.Box 2053, Rm S-3061,Dearborn, Michigan 48121
Government regulations have been enacted which limit the amount of volatile organic compounds (VOCs) that can be emitted into the air from painting facilities (1-3). A number of hardware abatement devices are being evaluated to achieve compliance with these regulations ( 4 ) . Continuous totalhydrocarbon (THC) analyzers employing flame ionization detection (FID) are most frequently used to measure VOC emissions ( 5 ) .However, when monitoring an abatement device, the THC analyzer cannot provide information about the abatement efficiency for specific solvents. This type of information is especially useful in the evaluation of a carbon adsorber which adsorbs certain solvent vapors with high efficiency while being relatively ineffective with others (6, 7). Furthermore, the THC analyzer is calibrated by using a single-component standard, usually propane in air or nitrogen. As a result, the analyzer cannot be used to determine accurately the mass emission rate of an abatement device unless the composition of the inlet and outlet vapor streams is known. This is because the FID response (i.e., relative to 0013-936X/81/0915-1463$01.25/0
@ 1981 American Chemical Society
propane) to each particular compound depends on its molecular structure. Gas-chromatography (GC) compositional results can be used to transform the THC-FID values in terms of equivalent propane to an actual solvent basis through the use of effective carbon number (ECN) response values which represent an apparent number of carbon atoms as seen by the FID (4,8,9). Thus, a more accurate measure of mass emission rate and abatement efficiency can be determined. Therefore, in order to correctly assess the operational performance of an abatement device, instrumentation has been developed based on an on-line gas chromatograph equipped with an automated inlet system. The utility of this instrument is demonstrated with data gathered through its application to a carbon adsorber used for automobile paint spraying emission control. This type of application may be subject to a number of difficulties including the following: (1)a rapidly varying inlet vapor concentration and composition, (2) high inlet vapor concentration (150-1000 ppm THC as propane) and low outlet vapor concentration (as low as 10 ppm or less) Volume 15, Number 12, December 1981
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