Anal. Chem. 1983, 55, 862-866
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Cross Polarization Carbon- 13 Nuclear Magnetic Resonance and Fast Atom Bombardment Mass Spectrometry of Fractionated Fulvic Acid Farlda Y. Saleh” and David Y. Chang Institute of Applied Sciences and Department of Chemistty, North Texas State University, Denton, Texas 76203-3078
James S. Frye Department of Chemistty, Colorado State Unlverslty, Fort Collins, Colorado 80523
A major component of aquatlc fulvlc acld was lnvestlgated by cross polarlzatlon magic angle splnnlng carbon-I3 nuclear magnetlc resonance (CP-MAS 13C NMR) and fast atom bombardment mass spectrometry (FAB-MS). Both technlques provided complementary Informatlon. The lnvestlgated fuivlc acld contalned more aliphatic than aromatlc moletles. The ailphatlc moletles are represented by branched allphatlcs wlth methylene carbons a,(3, and y from the end of an alkyl chaln or an aromatlc ring. The aromatic moietles Included substltuted aromatlcs wlth structures that contaln metal complexlng sites and are potential precursors of trihalomethanes upon chlorlnatlon. Methoxyl, carboxylic acids, and esters are well-deflned moleties of the macromolecule. Phenoilc compounds were detected only In the FAB-MS and were not obNMR, possibly due to the presence of stable served In the free radicals.
Aquatic fulvic acid (FA) plays an important role in many environmental reactions such as the formation of trihalomethanes (THMs) upon chlorination ( I ) , complexation with heavy metals (2),and solubilization of organic pesticides (3). To date, the structure of fulvic acid is unknown, even though it has been extensively investigated by chemical and spectroscopic methods in the past few decades (4,5). The preponderance of evidence indicates that FA is a polymeric material of molecular weight ranging from a few hundred to several thousand. It is also known that FA behaves like linear flexible polyelectrolytes that are readily aggregated at low pH with the aid of hydrogen bonding, van der Waals interactions, and interactions between the a-electron system of adjacent molecules. The carbon skeleton of FA consists of a broken network of poorly condensed aromatic rings with an appreciable number of disordered aliphatic chains and alycyclic structures. Fulvic acid contains several oxygen functional groups such as carboxyl, carbonyl, phenolic, and methoxy groups. Chemical degradation followed by GC/MS and IH and 13C NMR studies have established the presence of aliphatic structures in addition to the aromatic moieties in FA macromolecules (6-9). One of the major difficulties in humic material research is the limitations of spectroscopic instruments to identify the molecular structure of high molecular weight complex molecules. Recently, there has been a major breakthrough in spectroscopic instrumentation which allowed the establishment of the structures of several complex macromolecules of biochemical and medical importance (10, 11). This paper presents the results of application of cross polarization magic angle spinning (CP-MAS) carbon-13 nuclear magnetic resonance and fast atom bombardment (FAB) mass spectrometry to aquatic FA that has been fractionated on
macroreticular resin (Chromasorb 108), under acidic and basic conditions. Carbon-13 NMR spectrometry is one of the most powerful tools that provides information on the carbon skeleton of organic molecules. The recent development of CP-MAS technique has overcome inherent problems due to dipoledipole interactions and length of spin-lattice relaxation times. In the CP-MAS technique protons are decoupled from 13C nuclei and then used to enhance the relaxation of the 13C nuclei which result in improved resolution and sensitivity. Principles of CP-MAS 13CNMR can be found in publications by Pines, Gibby, and Waugh (22),Schaefer and Stejskal (13), Wilson (14), and Miknis, Bartuska, and Maciel(15). Several workers have applied CP-MAS 13C NMR to study the structural features of coal and shale oil (16),legnin and pine wood (17,18), and coal and soil organic matter (19,20). Soil humic acids from different climatic zones and soil FA from Armadale soil, Prince Edward, Canada, have been examined by CP-MAS 13CNMR (21). Aromaticities and carboxylic acid content were calculated from the NMR spectra and the results were compared with those previously obtained on the same samples by chemical methods. It was reported that chemical techniques have overestimated the degree of aromaticity and underestimated the carboxylic acid content of humic substances. Fast atom bombardment (FAB) mass spectroscopy has recently been developed by Barber and his colleagues (22) to study organic salts, polar antibiotics, nucleoside phosphates, and underivatized peptides. The technique is evolving as one of the most powerful tools to study polar high molecular weight compounds. Reviews on the technique, its performance, and application have been recently published (23, 24). EXPERIMENTAL SECTION Samples Origin and Preparation. Fulvic acid used in this study was extracted from aquatic sediments collected from Cross Lake, Louisiana. This was one of three sampling sites, in which water and sediment FA were fractionated by different HPLC models. Details of the experimental procedures and the HPLC results have been published (25). High-Performance Liquid Chromatography. HPLC was carried out on Waters ALC-201 instrument with a Model 6000 pump. Both UV detectors (Model ISCO Au-5) and fluorescence detector (Schoeffel Model 970-A) were simultaneously used to monitor the HPLC fractionation. A preparative stainless steel column (50 cm length X 8 mm i.d.) was packed with methyl methacrylate resin (Johns Manville, Chromosorb 108 of 110-120 mesh). A 200-mL sample of Cross Lake sediment FA (corresponding to 20 g dry weight of the sediment) was fractionated into two fractions, using acetic acid at pH 3.1 and ammonium hydroxide at pH 11.70 under stepwise gradient conditions. The eluant was simultaneously monitored by the UV detector set at 254 nm and the fluorescence detector was set at A,, 273 nm and A, 387 nm. The solvents were removed by freeze-drying. Acidic solutions (HN03,pH 2.2) of the fractions were reinjected on the
0003-2700/83/0355-0862$01.50/0 0 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 6, MAY 1983
863
Table I. Molecular Weight Data“ of Total and Fractionated Aquatic Fulvic Acid wt BV mol wt “Mi?’ no. av mol wt “ME” range of mol wt ~
a
total sample
fraction 1
fraction I1
3.93 x 103 1.93 X l o 3 31.62 x 103 to 0.18 x 103
0.8 x 103 7.08 x 103 to 0.09 x 103
1.8 x 103
6.05 x 103 2.36 x 103 28.18 x 103 to 0.14 x 103
Determined by size exclusion HPLC ( 2 6 ) . -
.
-.
column to check the separation efficiency. Total sample and fractions were subjected to size exclusion chromatography according to the published procedure (26). The freeze-dried residues of the two HPIL! separated fractions and the unfractionated residue were subjected to CP-MAS NMR and FAB mass spectroscopy. Cross Polarization Magic Angle Spinning lac,13C NMR spectra were obtained with a Nicolet NT-150 spectrometer at 37.7 MHz using a home built CP-MAS modification including the probe. The cross polarization or contact time was 1ms and the pulse repetition time was 1 s. The ‘H irradiation field was 11 G and 1K data points were zero filled to 2K points in the spectra. Chemical shifts were measured with respect to tetramethylsilane via hexamethylbenzene as a secondary substitution reference (aromatic peak a k 132.3 ppm). Usually 15000-50000 scans were accumulated. Bullet-shaped spinners (27)were used with sample volume of 0.4 cm3 and were spun at about 3.8 kHz. Fast Atom Bombardment Mass Spectroscopy. FAB mass spectra were obtained on a Kratos MS-50 instrument equipped with a 23 kG magnet which extends the mass range to 3000 amu at the full accelerating voltage of 8 kV. Spectra were recroded oscillographicallyat low resolution with an accelerating potential of 4 kV and scan rate of 100 s/decade. The pressure in the ion source housing vvas maintained within the lo4 torr region. An argon beam of 4-6 keV produced from an FAB source was impacted into the sample dissolved in glycerol. FAB spectra were obtained in the positive ion mode. The method of sample Entroduction was similar to that described by Grigsby et al. (28). RESULT8 AND DISCUSSION Figure 1 shows a typical HPLC chromatogram of FA extracted from Cross Lake sediment. It is noted that the area under peak I1 represents -80% of the UV absorption signals and =90% of the fluorescence siganls. Table I shows molecular size distribution data of the total sample and fractions. Results of the first phase of this study (25) have shown that aquatic FA extracted from different locations have common characteristic features under several HPLC modes with differences only in the magnitude of the UV and fluorescence responses. CP-MAS 13C NMR. Figure 2 shows the CP-MAS 13C NMR spectrum of the freeze-dried residue (0.220 g) of peak I1 in Figure 1. Three resonance envelops are characteristic of the spectrum. The chemical shift in the region between 6 0 and 70 ppm, corresponds to aliphatic carbons. The chemical shift in the region between b 100 and 160 pprn corresponds to aromatic, heteroaromatic, and olefinic carbons. The region between 6 170 and 190 ppm corresponds to carbons in the carboxyl or amide groups. To interpret the CP-MAS 13C NMR spectra, two questions must be addressed. First, are all the carbons in the sample cross polarized equally so that all the carbon types contribute proportionately l o the integrated intensities? Second, what types of interferences, if any, may arise from other components of the sample? The answer to these questions is partly dependent on factors related to the structure of the organic material (e.g., aromaticity, ring size, tertiary and quaternary carbons) and experimental factors (contact time and pulse repetition rates). Several workers have discussed provisions in interpretation of CP-MAS 13C NMR spectra of complex organic materials) (16-20). In spectra of such material, there is a considerable overlap between signals from different types of carbons. Also, uncertainty may result from effects of
UV Response h 254nm
F uorescence Response
h e x 273nm 387nm
, ,A
f-
-- Retention (mi;
-
1
Retention
(mL)
Flgure 1. C-108 chromatogram of fulvic acid extracted from Cross
Lake sedlment (solid lines)and procedure blank (dashed lines): solvent (l), acetic acid at pH 3.1; solvent (2), NH,OH at pH 11.7.
-
- I ~ I I 250
200
~~~~~I~~~
I50
I00 50 Chemical S h i f t 8
0
1
-50
J
-100 PPM
Carboxyl c
A m de Carbon.
0 Alkyl
Corbons
AIken c Carbons
Flgure 2. CP-MAS I3C NMR spectrum of fractionated FA, 2 5 0 0 0 scans. Chemical shifts are measured with tetramethylsilane via hetx-
amethylbenzeneas a secondary substiiution resonance (aromaticpeak at 132.3 ppm). paramagnetic material on signal intensities and line widths. Nevertheless, qualitative information on the different types of carbon can be derived from the spectra. With semiquantitative estimates, it may be possible to draw conclusioiis regarding the relative abundance of the various carbon types. Considering the forementioned discussion, additional information can be derived from the spectrum in Figure 2. Table I1 summarizes the chemical shifts and structural assignments. The spectrum reveals prominent signals in the alkyl region extending from 6 10 to 50 ppm. Intensities aire
864
ANALYTICAL CHEMISTRY, VOL. 55, NO. 6, MAY 1983
Table 11. Chemical Shifts and Structural Assignmentsa in the CP-MAS I3C NMR Spectrum of FA (Figure 2)
chemical shift region aliphatic carbons (0-50 ppm)
obsd resonance peaks, ppm 12-1 6
assignment terminal methyl carbon or methyl carbon y or further aromatic ring methyl carbon a to aromatic ring or methylene carbon p , y , or 6 from terminal methyl group methylene carbons of branched alkyls methoxy carbons carbon atoms in polyhydroxy compounds and certain amino acids aromatic carbon ortho to ether oxygen or hydroxy group aromatic and substituted aromatic carbons carbon in carboxylic acids, esters, or amides
13-1 7
27-34
19-35
oxygen alkyl carbons (50-110 ppm)
41-45 54,55 58, 67,72, 77,82
37-53 54-56 60-101
aromatic and alkenic carbons (100-1 60 ppm)
117
108-118
131,139 176, 180
118-148 150-196
carboxylic, carbonyl, or amide carbons (160-220 ppm) a
range of chemical shift, ppm
Some overlap of shift ranges is likely.
90
-
80
-
!
70 -
70
I
60
I00
Flgure 3. FAB mass spectrum of fractionated FA dissolved in glycerol.
defined by peaks at 6 27,34,41, and 45 ppm. These signals typify aliphatic side chain carbons and carbons in a,(3, or y position from terminal methyl group or an aromatic ring. The shoulders at 6 10-16 ppm arise from terminal methyl groups. The 0-alkyl region extends from 6 50 to 100 ppm. The rather strong signals at 54 and 55 ppm can be assigned to methoxy carbons. Ether and carbohydrate carbons resonate at 6 60-72 ppm and a t 101 ppm and small amounts of these materials may be present. The aromatic carbons extend over the region from 6 100 to 160 ppm. The three peaks a t 6 117,131, and 139 in this region suggest the presence of different types of substituted aromatic structures. The general configuration of the 13C NMR spectrum is similar to the one reported by Hatcher et al. (21) for soil FA, except in the resonance region between 6 60 and 100 ppm which indicates higher alcoholic and carbohydrate carbons in soil FA. Apparently, aquatic FA contains relatively few of these carbons, It is also possible that these components were not eluted with this fraction of FA. Peaks corresponding to methoxy carbons are more pronounced in the fractionated aquatic FA than in soil FA (21). Similarity between the Hatcher et al. (21)FA spectrum and the current spectrum lies essentially in the predominance of aliphatic moieties over the aromatic ones. Also, as noted by Hatcher et al. (21), phenolic
120
140
Figure 4. FAB mass spectrum of fractionated FA dissolved in glycerol, NaCl added.
carbons which resonate between 6 148 and 153 ppm are not detected in the 13CNMR spectra. Hatcher et al. (21) offered two possible explanations to this observation. The first is the possibility of overestimation of phenolic compounds in FA, using chemical methods. The second is the possible masking of the phenolic carbons due to the stable free radicals in the sample. We believe that the second explanation is more likely. Presence of stable free radicals in FA and humic material have been reported by several workers using electron spin resonance (ESR) spectrometry (29-31). Preliminary ESR work on the fractionated FA sample confirmed the presence of free radicals. The CP-MAS 13CNMR spectra on the freeze-dried residue of the total sample and the fraction corresponding to peak I in Figure 1did not reveal measurable signals above the noise level possibly because of the relatively low carbon content of the sample and the presence of additional paramagnetic ions. Fast Atom Bombardment Spectra. The same sample subjected to CP-MAS 13C NMR was subjected to FAB mass spectrometry. Figures 3 and 4 show the FAB spectra of the sample dissolved in glycerol and in glycerol plus sodium chloride. Addition of Na+ is known (23) to enhance the production of cationized species (M + Na)+ and reduce, to
ANALYTICAL CHEMISTRY, VOL. 55, NO. 6, MAY 1983
OH
I
M/2
w/
