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Chemical characterization of size-fractionated humic and fulvic materials in aqueous samples
The Science of the Total Environment, 113 (1992) 159-177 Elsevier Scientific Publishers B.V., Amsterdam
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Chemical characterization of size-fractionated humic and fulvic materials in aqueous samples N . A . M a r l e y a, J.S. G a f f n e y a, K . A . O r l a n d i n i a, K . C . Picel a a n d G.R. Choppin b "Building 203, Environmental Research Division, Argonne National Laboratory, Argonne, IL 60439, USA bChemistry Department, Florida State University, Tallahassee, FL 32306, USA
ABSTRACT Humic and fulvic acids have been shown to be potentially important transport agents for inorganic and organic contaminants, including radionuclides, in surface and groundwaters. In this work, the possibility of characterizing humic and fulvic materials in the colloidal and macromolecular size ranges (i.e., between 3000 molecular weight (1 nm) and 0.45 #m) after size fractionation and collection with hollow-fiber ultrafilters has been investigated. Three surface waters have been examined as test cases. Using ultrafiltration, sized samples of humic and fulvic acids were chemically characterized with 13C-nuclear magnetic resonance spectroscopy (NMR), Fourier transform infrared spectroscopy (FTIR) using diffuse reflectance, and pyrolysis/gas chromatography (GC). These test cases demonstrate that sufficient material can be obtained by ultrafiltration with minimal or no alteration to the materials. In addition, inorganic analyses performed on the size fractions show that ultrafiltration can also allow the binding capacity of the humic materials in each fraction to be measured. Substantial variability among the different humic and fulvic fractions demonstrates the importance of sizing submicron materials in attempts to understand the mechanisms of pollutant transport by natural humic materials. Key words: humics; fulvics; ultrafiltration; chemical characterization; pollutant transport
INTRODUCTION N a t u r a l l y o c c u r r i n g h u m i c a n d fulvic acids are i m p o r t a n t c o m p l e x i n g agents for a v a r i e t y o f metals, r a d i o n u c l i d e s , a n d o t h e r c h e m i c a l species. T h e y c a n t h e r e f o r e act to e n h a n c e the m o b i l i t y o f c o n t a m i n a n t s in s u b s u r f a c e a n d surface waters. I n c r e a s i n g c o n c e r n s a b o u t g r o u n d w a t e r quality a n d c o n t a m i n a t i o n o f soils a n d w a t e r s b y h a z a r d o u s wastes h a v e e m p h a s i z e d the need to u n d e r s t a n d the role o f these m a t e r i a l s in ecological systems. H o w ever, d u e to their diverse a n d c o m p l e x n a t u r e , a t t e m p t s to describe the structure a n d b e h a v i o r o f h u m i c m a t e r i a l s h a v e o f t e n yielded c o n t r a d i c t o r y results
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[1]. A number of factors including source, method of extraction, or limitations of the characterization methods may be the cause of these differences. Traditionally soil humic materials have been studied by extraction into a base, followed by further separation into acid-insoluble (humic) and acidsoluble (fulvic) fractions which provided an operational distinction between humic and fulvic acids according to their acid solubilities. In an attempt to extend these studies to surface and subsurface waters, modifications of these same procedures have been applied to extract humic materials from water [2]. The methods most frequently used to characterize these substances include (i) infrared spectroscopy [3-9], (ii) nuclear magnetic resonance spectroscopy [3-7,10-13] and (iii) gas chromatography, following pyrolysis [13-18] or chemical degradation [19,20]. Aquatic humic materials occur in sizes ranging from colloidal particles (0.1 m~-10 A) to macromolecules (3000-500 mol. wt) [21]. It has been proposed that the larger, colloidal fractions are composed of humic acids and that the smaller macromolecules are the fulvic acids [22]. This suggests the possibility of separating and concentrating natural aquatic humic substances on the basis of molecular weight, thereby avoiding the harsh conditions traditionally used for the recovery of soil humics, and adapted for extraction from water [2]. In this study humic materials have been obtained from three surface waters by using hollow-fiber ultrafiltration techniques. The natural trace metal content of the size-fractionated humics and fulvics are reported as a measure of the binding capacity of each group. The structural characteristics and functional groups associated with these materials were investigated with FTIR, 13C-NMR, and pyrolysis GC in an attempt to compare the advantages of each method. These results are then compared with samples prepared by more conventional techniques [2]. MATERIALS AND METHODS
Sample sites Water samples were obtained from three surface locations, Volo Bog, Lake Bradford and Saganashkee Slough. At each site, a single 60-gallon sample was collected from just below the surface of the water, at - 20 feet from the shoreline. Volo Bog is located in an Illinois Nature Preserve northwest of Chicago. It is a small glaciated bog surrounded by sedge peat with no surface inlet or outlet. The water is of low nutrient content with a pH of 4-5. Saganashkee Slough, located in Cook County, Illinois, drains into the Calumet Sag Channel and finally into the Des Plaines River. It has a higher nutrient content
C H A R A C T E R I Z A T I O N O F H U M I C A N D F U L V I C M A T E R I A L S 1N A Q U E O U S S A M P L E S
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and is nearly at neutral pH. Lake Bradford is located near Tallahassee, Florida, is fed by both spring and surface run off, and has a pH of 5-6. Humic and fulvic acids isolated from this lake have previously been used as a source of humic materials for studies of radionuclide binding capacities [231.
Isolation of humic materials Water samples were first passed through a 35-/~m screen to remove large particulates, followed by prefiltering with a 0.45-/~m Millipore filter to remove the suspended solids. Humic materials were concentrated from the 60-gallon water samples using hollow-fiber filter cartridges obtained from the Amicon Division of W.R. Grace and Co. These filters operate using a cross-translational flow which avoids polarization on the filter due to pile up of the material on the filter surface. The cartridges are 60 cm in length and can be obtained with pore diameters of 0.1/~m and effective molecular weight filtration of 100 000, 30 000, 10 000 and 3000 mol. wt, based upon spherical model compounds. Flat disk membrane filters in a Vortex Mixing Stirred Cell were used to separate 1000 and 500 mol. wt species. The filters and stirred cells were obtained from Spectrum Medical Industries, Inc. and Amicon Corp., respectively. Lake Bradford humic and fulvic acids were also obtained using traditional separation and purification techniques [2,24]. Mass balances as determined by dissolved organic carbon determinations were observed to be within the errors of the DOC measurements (10%) for the natural humic and fulvic materials. Portions of the aqueous concentrates were dried, and the solid samples were used for the various spectral determinations. For the trace element and dissolved organic carbon measurements, the aqueous solutions were analyzed.
Trace element determinations Dissolved organic carbon (DOC) measurements were obtained on the sizefractionated concentrates with a Sybron PHOTOchem Organic Carbon Analyzer, Model E3500. Samples of 1-10 ml were injected into the analyzer with 0.05 N phosphoric acid. Trace metals were determined by inductively coupled plasma spectroscopy with an Instruments SA, Model JY 86 spectrometer equipped with an HF-compatible torch.
Pyrolysis gas chromatography Prior to analysis, dry samples were redissolved with high-purity water to
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obtain a concentration of 1 mg/ml. A Chemical Data Systems Model 122 pyrolysis unit with a platinum ribbon probe was used to pyrolyze samples. To evaporate the sample, 20/~1 of aqueous solution was applied slowly to the ribbon, which was held at 100°C. The probe was then inserted into the interface module and maintained at 220°C. After re-establishing the column flow, the sample was ramped at 20°C/ms to a temperature of 700°C for pyrolysis. The p y r o p r o b e interface was connected to a Hewlett-Packard HP5880A G C by means of a split/splitless capillary injector operated in the split mode. The G C was equipped with a 50 m (0.2 mm i.d.) Carbowax 20M column and a flame ionization detector. The G C temperature program was started at the same time as sample pyrolysis. It consisted of a 2-min hold at 50°C, followed by a linear temperature ramp, at 6°C/min to a final temperature of 220°C, which was held for 20 min. After analysis, the probe was cleaned by rapid heating to 1000°C for a short time period.
13C-Nuclear magnetic resonance ( N M R ) spectroscopy The N M R spectra were obtained on - 5 0 mg of solid sample using a Bruker 200 M H z ~3C spectrometer located at the Florida State University N M R laboratory and operated at 50.27 M H z for carbon. During the course of the experiment the sample was spun at - 3 . 7 kHz at the magic angle [10]. Pulse time was 6 #s, and cross polarization time was 1 ms. Recycle time was 3 s. Approximately 20 000 sample scans were obtained, with the number increasing for smaller sample sizes. Chemical shifts were referenced to tetramethylsilane (TMS). Dipolar dephased spectra were obtained with a dephasing time of 50 gs.
Infrared spectroscopy Infrared spectra were obtained using a Mattson Polaris Fourier transform spectrometer. The samples were mixed with dried, reagent grade KBr, and diffuse reflectance spectra were taken from 400 to 4000 wavenumbers with a Spectra Tech D R I F T accessory. Diffuse reflectance was automatically corrected for specular reflectance, and the data are reported in Kubelka-Munk units [25]. Spectra were taken at 2 cm-~ resolution and averaged over 64 scans. Reported results were baseline corrected and smoothed using a sevenpoint boxcar function. RESULTS AND DISCUSSION The purpose of this study was to determine if hollow-fiber ultrafiltration could be effective in obtaining useful samples of size-fractionated humic and
C H A R A C T E R I Z A T I O N O F H U M I C A N D F U L V I C M A T E R I A L S IN A Q U E O U S S A M P L E S
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TABLE 1 Dissolved organic carbon (DOC) and trace element levels determined in the total water samples taken at Volo Bog, Lake Bradford and Saganashkee Slough. Analyses were made after filtration through a 0.45-#m filter to remove large particulate matter. D O C is reported in ppm, and all trace elements are in ppb
DOC AI Ba Ca Cu Fe Mg Mn Na Si Sr Zn
Volo Bog
Lake Bradford
Saganashkee Slough
24.7 < 50 13 6740 < 10 340 3900 13 990 590 7 < 10
11.0 200 < 10 1190 < 10 340 460 26 1840 < 100 <5 21
13.3 63 33 50200 9 11 23900 13 4600 < 100 80 11
fulvic materials from water. For all three test cases, sufficient materials were collected to obtain spectral characterization and trace element distributions for the sized samples. The following results highlight the potential applications for size-fractionated humic and fulvic acid characterization using these techniques. The natural trace metal concentrations are listed in Table 1 for all three sample sites. Size-fractionated materials ranging from colloidal to macromolecular were separated from the samples by hollow-fiber ultrafiltration techniques and analyzed for organic and trace metal content. The results for each size fraction were compared to those obtained for the whole water samples and are shown in Figs. 1-3 for Volo Bog, Lake Bradford, and Saganashkee Slough, respectively. Sizes ranging between 0.45/~m and 3000 molecular weight represent the humics, while those less than 3000 molecular weight are the fulvic acids. Of the three sites studied, Lake Bradford has the highest concentration of larger particulates (0.45-0.1 /~m). These materials are of relatively low organic content, however, and are most likely of clay origin. In each case, at least 50% of the dissolved organic material is in size fractions < 30 000 molecular weight. These are also the species that are the most active in complexing the major trace metals.
164
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CHARACTERIZATION OF HUMIC AND FULVIC MATERIALS IN AQUEOUS SAMPLES
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Pyrolysis GC was performed on the size-fractionated samples. A typical pyrogram is shown in Fig. 4. The peak numbers in the figure correspond to the signature compounds listed in Table 2. Due to the great complexity of the pyrograms, only the major pyrolysis products have been identified and quantified. These have been grouped according to organic functionality for comparison to N M R results. The relative abundances are given in Tables 3 and 4 for Volo Bog and Lake Bradford, respectively. The pyrograms for the Lake Bradford samples generally contain larger amounts of phenol and thiophene than those from Volo Bog. Phenol is a pyrolysis product of lignin type structures and indicates the aromatic content of the parent molecules [14]. The organics contained in the larger particulates (0.45-0.1 #m) from Lake Bradford yielded the highest amount of phenolic products and therefore are most likely composed of large aromatic polymers. The two intermediate fractions have an aromatic content between those of the large and small sizes. However, in general, the signature products are most similar to the large particulates. The small macromolecular
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(min.) Fig. 4. Typical pyrolysis/gas chromatographic pyrogram obtained for a size-fractionated humic acid sample (Volo Bog, 0.45-0.1 /zm). Numbered peaks correspond to the signature compounds listed in Table 2.
fraction (less than 3000 mol. wt) is extremely high in thiophene, acetonitrile and acetic acid indicating a high proportion of amino acid groups in the parent molecules [15]. This is in agreement with the previous generalization that, humic acids are higher in phenolate content while fulvic acids are higher in carboxylates [26]. The pyrograms for Volo Bog yielded more acetonitrile, a pyrolysis product of proteins and nucleic acids [14]. In addition, although they have low levels of the phenols, the pyrolysis produced significant amounts of benzene and its derivatives. These arise from decarboxylation of aromatic acids such as phthalates [17]. These observations indicate that, although they do not possess large amounts of lignin type structures like the Lake Bradford humics, Volo Bog humics do contain smaller aromatic carboxylate units. The small fraction from the Volo Bog showed the highest carboxylate content, as evaluated from the acetic acid GC/pyrolysis data. The large fraction, however is highest in amino acid type groups. Analysis of the pyrograms for aliphatic carboxylate structures yields very low values in all cases. This is because the major product from the pyrolysis of these groups is carbon dioxide. A more accurate measure of the aliphatic
CHARACTERIZATIONOF HUMICANDEULVICMATERIALSIN AQUEOUSSAMPLES
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TABLE 2 Identified products from humic sample pyrolysis/gas chromatography analyses (see Fig. 4). Retention times are in minutes Peak no.
Compound
Retention time
1 2 3 4 5 6 7 8 9 10 11
Benzene Acetonitrile Thiophene Toluene Anisole Acetic Acid Pyrrole Propionic Acid Acetamide Phenol, o-Cresol m-Cresol, p-Cresol
5.30 5.97 6.10 6.33 11.90 13.82 15.64 15.96 20.34 25.18 26.67
n-Alkyls
CII saturated Cll unsaturated Ci2 saturated C~2 unsaturated CI3 saturated C13 unsaturated CI4 saturated C 14 unsaturated CI5 saturated CI5 unsaturated
7.62 8.27 9.69 10.44 11.98 12.78 14.31 15.12 16.58 17.38
TABLE 3 Relative abundances of signature compounds determined from humic pyrolysis/gas chromatography for size-fractionated samples from Volo Bog. Areas are normalized for 45/~g equivalent samples. K indicates 1000 molecular weight units Compounds
Benzene, toluene Phenol, cresols Thiophene Pyrrole Acetonitrile Acetamide Acetic acid
Relative abundances of size fractions 0.45-0.1 /zm
0.1tzm-100 K
100 K - 3 0 K
30 K - 3 K
33.7 8.3 3.0 6.7 60.7 1.9 1.3
24.4 2.8 1.3 2.7 18.0 1.2 0.3
22.4 10.3 3.3 3.9 29.4 2.3 0.3
62.8 8.5 0.7 3.8 25.9 3.4 0.1
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TABLE 4 Relative abundances of signature compounds determined from humic pyrolysis/gas chromatography for size-fractionated samples from Lake Bradford. Areas are normalized for 45 p.g equivalent samples. K indicates 1000 molecular weight units Compounds
Benzene, toluene Phenol, cresols Thiophene Pyrrole Acetonitrile Acetamide Acetic acid Anisole
Relative abundances of size fractions 0.45-0.1/~m
0.1/~m-100 K
100 K-30 K
30 K - 3 K
23.6 42.3 19.9 7.1 22.6 16.2 7.4 8.9
23.8 35.2 24.4 5.0 25.6 14.0 4.2 7.0
17.4 37.0 14.1 4.3 19.1 8.2 1.1 7.2
19.9 23.3 85.5 3.5 47.5 11.1 0.7 4.0
carboxylate content can be obtained from the analysis if an infrared detector is used to determine the amount of carbon dioxide produced during pyrolysis. Plans have been made in this laboratory to include this additional feature. The ~3C-NMR spectra were obtained on the large (0.45-0.1 ~m) and small (30 000-3000 mol. wt) sample fractions from Volo Bog. They are shown in Fig. 5 (B and C). These size-fractionated samples are compared with humic materials collected from the total sample before fractionation (Fig. 5A). The total sample is the humic matter contained in the water that is less than 0.45 ~m in size. The major carbon signals are listed in Table 5 along with the relative intensities for each sample. Results for the total sample of humic materials most resemble those for the 30 000-3000 molecular weight fraction. This is because this size range comprises more than 40% of the humics present in the sample. Therefore, analysis of total humics removed from a water sample without further size fractionation can lead to biased results. The large fraction (Fig. 5B) possesses the most intense signal in the 50-100 ppm range. The signals in this range of chemical shift arise from carbons that are singly bonded to either oxygen or nitrogen (alcohol, ether, or amine derivatives). These are usually termed the carbohydrate carbons because it has been suspected that for most humic materials, they consist of polysaccharide units [12]. Therefore, the major assignment for this range has been
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(pp~) Fig. 5. The ]3C-NMR spectra of organic materials obtained from Volo Bog for (A) the total sample, (B) 0.45-0.1 /xm and (C) 30 000-3000 mol. wt size fractions.
O-alkyl carbons. Comparison with the pyrolysis GC results for this sample, however, leads to the conclusion that a large percentage of this signal may be due to amino acid groups. The dipolar dephased spectrum is shown in Fig. 6 for the 0.45-0.1/xm sample. A decrease in all signals from 0-100 ppm with a dephasing time o f 50 its demonstrates that these carbons are protonated [11]. This is consistent with either ether or amine assignments.
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N.A. M A R L E Y ET AL.
TABLE 5 Results for ]3C-NMR analyses of size-fractionated humics collected from Volo Bog. Functional groups contributions are given as the relative areas of the carbon signals observed in the solid magic angle spectra. The results for the total sample are the abundances for the whole water sample after filtration through a 0.45-/~m filter to remove particulate matter. Functional group chemical shift ranges are given in parentheses and are referenced to tetramethylsilane as the standard Functional group (Shift, ppm)
Relative carbon abundances Size fractions
Alkyl (25-50) O-Alkyl (50-100) Acetal (100-107) Aromatic (110-140) Phenolic (140-160); Carboxyl (160-200) Carbonyl (200-220)
=
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0.45-0.1 ~m
30 K - 3 K
25 43 6 8 2 14 2
35 30
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100
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41 23
1
1
11 4 15 4
8 3 19 5
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(ppm) Fig. 6. The ]3C-NMR spectra of humic materials from Volo Bog (0.45-0.1 t~m) obtained with (lower) and without (upper) dipolar dephasing.
171
CHARACTERIZATION OF HUMIC AND FULVIC MATERIALS IN AQUEOUS SAMPLES
TABLE 6 Comparison of functional group relative abundances in percent as determined by 13Cnuclear magnetic resonance spectroscopy and pyrolysis/gas chromatography analyses of sizefractionated humics collected from Volo Bog (Volo) and Saganashkee Slough (Sag) Functional group
Alkyl O-Alkyl Acetal Aromatic Phenolic Carboxyl Carbonyl
Relative carbon NMR abundances Sag < 3 K
Volo 0.45-0.1 #m
Volo 30 K - 3 K
37 24 4 10 3 18 2
21 40 11 6 4 15 4
35 24 6 9 4 15 6
Product (parent) pyrolysis~gas chromatography Alkyls Nitriles (amino acids) Aromatics (Ar-COOH) Phenols (aromatics) Carboxylates (R-COOH)
13 28
11 50
13 22
16
28
52
22
7
7
19
3
3
Table 6 compares results obtained from ~3C-NMR and pyrolysis GC for the Volo Bog humics and Saganashkee Slough fulvic acids. Results compare favorably for the aromatic content of the Volo Bog humics. For the Saganashkee fulvics, pyrolysis results are somewhat higher than NMR results. Again, comparison of amino acid content determined by pyrolysis with the carbon signals in the 50-100 ppm region shows good agreement, which may be an indication that a large portion of this N M R signal is from nitrogen-bonded carbon. Results for alkyl content obtained by pyrolysis GC include only long chain units (more than 11 carbon atoms) and are therefore lower than N M R results, which reflect more accurately the total alkyl content. Carboxylate results do not agree because decarboxylation reactions occur upon pyrolysis. There is also the possibility of the formation of benzene derivatives by elimination and cyclization reactions. It has been estimated
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Fig. 7. The FTIR spectra of size-fractionated samples from Volo Bog: (A) 0.45-0.1 tim, (B) 0.1 #m to 100 000 mol. wt, (C) 100 000-30 000 mol. wt, (D) 30 000-3000 mol. wt, (E) less than 3000 mol. wt and (F) 1000-500 mol. wt.
that, during sample pyrolysis, 50% of the original sample weight is lost as carbon dioxide, whereas the formation of aromatic products by cyclization is less than 0.1 percent [12]. These questions could be answered and agreement with N M R results strengthened with an added infrared detector. The F T I R spectra obtained for Volo Bog and Lake Bradford samples are shown in Figs 7 and 8. The major bands are listed in Table 7 along with the most probable band assignments [6-9,21]. The broad band centered at - 3 4 0 0 cm -1 arises primarily from hydrogen-bonded O H stretches of phenols, alcohols and carboxylic acid groups. This broad band is present in all humic and fulvic acid spectra and therefore contributes little information regarding structural differences. However, a shoulder that appears near 2900 cm-1 is due to aliphatic C - H stretch. This band has been reported to vary considerably among different humics [21]. In all spectra shown in Figs. 7 and 8 this band is very weak, indicating a low aliphatic content. Aromatic C - H stretch appears as a shoulder slightly higher than 3000 cm-1. This band can easily be masked by the broad O H stretch and interpretation is sometimes
173
CHARACTERIZATION OF HUMIC AND FULV1C MATERIALS IN AQUEOUS SAMPLES
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B
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lobo
30'00
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3600
2000
1000
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Fig. 8. The FTIR spectra of size-fractionated samples from Lake Bradford: (A) 0.45-0.1 #m, (B) 100 000-30 000 mol. wt, (C) 30 000-3000 mol. wt and (D) <3000 mol. wt.
TABLE 7 Infrared band assignments for diffuse reflectance spectra taken on the size-fractionated humic samples. Band shoulders are designated by (sh) Frequency (cm-I)
Band assignments
3400 3250 (sh) 2950 (sh) 2600 (sh) 1720 1560 1520 (sh) 1450 (sh) 1400 1384 1200-1000 620
H-bonded OH (phenol, alcohol, carboxyl) Aromatic C - H stretch Aliphatic C - H stretch H bonded carboxyl, OH stretch COOH stretch C O O - asymmetric stretch N - H deformation of peptides C - H deformation (CH2-, CH3- ) C O O - symmetric stretch Si-O-C stretch C - O stretch of polysaccharides SO3Hstretch
NA .M .ARLEY T AL,
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difficult. However, for the Lake Bradford samples (Fig. 8), it reveals a larger aromatic content for the larger fraction and the smallest aromatic content for the less than 1000 molecular weight species, in agreement with pyrolysis GC results. The aliphatic content, appears largest for the two intermediate fractions. Volo Bog samples (Fig. 7) yielded similar results for all fractions. Examination of the carboxyl stretching regions can yield information concerning complexation. The symmetric and asymmetric stretches of the carboxylate ion appear at 1560 and 1400 cm -~, respectively. The asymmetric stretch at 1400 cm -] is overlapped by a sharp peak at 1384 cm -J from carboxylate binding to silicate [27]. The a m o u n t of silica bound to the humics can be determined from the height of this peak. This sharp peak can also be used to differentiate between organically bound and inorganic colloidal silica. The band that appears at 1720 cm-~ is due to the stretching vibrations of the protonated carboxylic acid and is a measure of uncomplexed carboxyl. Comparing spectra of Fig. 7 with those of Fig. 8 shows that more uncomplexed carboxyl groups are present in the Lake Bradford samples than in those of Volo Bog. The sample with the least amount of free carboxyls be-
A
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0
_ s6oo
~ 2o'oo
16oo
WAVENUMBERS
Fig. 9. The FTIR spectra of purified humics and fulvics obtained by acid-base extraction procedures for Lake Bradford samples. Spectra are for (A) humics and (B) fulvics.
CHARACTERIZATION OF HUM1C AND FULVIC MATERIALS IN AQUEOUS SAMPLES
175
ing the large (0.45-0.1/~m) particulates, due to the large amount of inorganic matrix present. The Volo Bog samples also have a prominent band centered at 1200-1000 cm-~, which is the C - O stretch of the polysaccharide units. The N - H band, due to the peptides, appears as a weak shoulder at 1520 cm-~; although it is present in the spectra, it cannot be reliably used to determine the amount of amino acid groups. The symmetric carboxylate stretch can occur between 1610 and 1550 cm -~. This band can be complicated by overlap from unsaturated CC functional groups, which lead to bands at 1640-1675 cm -~. The effect of these contributions is seen in the shift of the peak maximum toward higher frequencies. Figure 9 shows FTIR spectra of humic and fulvic acids extracted from Lake Bradford by acid-base precipitation methods, with subsequent purification with HC1 and HF to remove metals and silica [24]. Humic acids, after purification, show a much higher aliphatic content (2925-2825 cm-~). This is most likely due to decarboxylation during purification. Comparison of the carboxyl bands at 1720 cm -~ and 1400 cm -~ reveals that all measurable groups are uncomplexed and in the protonated form. The silica band at 1384 cm-~ is also absent. The band at 1560 cm-~ is shifted in frequency to 1620 cm -~, indicating that a larger percentage of unsaturated groups in the purified humics. Comparison of Fig. 9B with Fig. 8D clearly demonstrates that the purified fulvic acids contain mostly aliphatic character and that the broad band from hydrogen-bonded OH is greatly decreased. These observations are consistent with the humics undergoing substantial chemical change during the acid-base precipitation, as compared to the much milder ultrafiltration separations. CONCLUSIONS
In this work we have demonstrated the potential for ultrafiltration to separate colloidal and macromolecular sized humic and fulvics in natural waters. We have also shown that with hollow-fiber ultrafiltration, sufficient materials can be obtained for chemical characterization of both the natural organic materials and the associated inorganic substituents in the various size fractions. These methods offer a much milder approach to the collection and characterization of natural humic and fulvic acids than previous procedures and allow a much more detailed picture of the importance of the various sizes of materials in the transport of pollutants in surface and subsurface waters. ACKNOWLEDGEMENTS
The authors wish to acknowledge the help of Dr. Edmund A. Huff of
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Argonne National Laboratory and Mr. Lin Feng Rao of Florida State University in obtaining the inorganic and N M R data, respectively. We also wish to thank Ms. J. Jastrow and Dr. R.M. Miller of Argonne National Laboratory for reviewing the manuscript. This work was performed at Argonne National Laboratory and at Florida State University and was supported by the United States Department of Energy, Assistant Secretary for Energy Research, Office of Health and Environmental Research, under contract W-31-109-ENG-38. The authors also wish to acknowledge the continuing support and encouragement of Dr. Frank Wobber of the U.S. Department of Energy's Subsurface Science Program. REFERENCES 1 F.H. Frimmel and R.F. Christman, Humic Substances and their Role in the Environment, Wiley-lnterscience, New York, 1988. 2 E.M. Thurman and R.L. Malcolm, Preparative isolation of aquatic humic substances. Environ. Sci. Technol., 15 (1981) 463-466. 3 J.C. Lobartini, K.H. Tan, L.E. Asmussen, R.A. Leonard, D. Himmelsbach and A.R. Gingle, Humic matter isolated from soils and water by the XAD-8 resin and conventional NaOH methods. Commun. Soil Sci. Plant Anal., 20 (1989) 1453-1477. 4 J.J. Alberts and Z. Filip, Sources and characteristics of fulvic and humic acids from a salt marsh estuary. Sci. Total Environ., 81/82 (1989) 353-361. 5 M.A. Arshad and M. Schnitzer, Chemical characteristics of humic acids from five soils in Kenya, Z. Pflanzenernahr. Bodenk., 152 (1989) 11-16. 6 K. Yonebayashi and T. Hattori, Chemical and biological studies on environmental humic acids. II. IH-NMR and IR spectra of humic acids. Soil Sci. Plant Nutr., 35 (1989) 383-392. 7 R. Candler, W. Zech and H.G. Alt, Characterization of water-soluble organic substances from a typic dystrochrept under spruce using GPC, IR, tH NMR, and 13NMR spectroscopy. Soil Science, 146 (1988) 445-452. 8 A.U. Baes and P.R. Bloom, Diffuse reflectance and transmission fourier transform infrared (DRIFT) spectroscopy of humic and fulvic acids. Soil Sci. Soc. Am. J., 53 (1989) 695-700. 9 A. Piccolo, Characteristics of soil humic extracts obtained by some organic and Inorganic solvents and purified by HC1-HF treatment. Soil Science, 146 (1988) 418-426. 10 P.G. Hatcher, M. Schnitzer, L.W. Dennis and G.E. Maciel, Aromaticity of humic substances in soils, Soil Sci. Soc. Am. J., 45 (1981) 1089-1094. 11 I. Kogel-Knabner and P.G. Hatcher, Characterization of alkyl 13C NMR spectroscopy and dipolar dephasing. Sci. Total Environ., 81/82 (1989) 169-177. 12 R. Frund and H.-D. Ludeman, The quantitative analysis of solution and CPMAS-C-13 NMR spectra of humic material. Sci. Total Environ., 81/82 (1989) 157-168. 13 G.K. Stearman, R.J. Lewis, L.J. Tortorelli and D.D. Tyler, Characterization of humic acid from no-tilled and tilled soils using carbon-13 nuclear magnetic resonance. Soil Sci. Soc. Am. J., 53 (1989) 744-749. 14 M.A. Wilson, R.P. Philp, A.H. Gillam, T.D. Gilbert and K.R. Tate, Comparison of the structures of humic substances from aquatic and terrestrial sources by pyrolysis gas chromatography mass spectrometry. Geochim. Cosmochim. Acta, 47 (1983) 497-502.
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15 A.C. Sigleo, T.C. Hoering and G.R. Helz, Composition of estuarine colloidal material: organic components. Geochim. Cosmochim. Acta, 46 (1982) 1619-1626. 16 C. Saiz-Jimenez and J.W. De Leeuw, Pyrolysis-gas chromatography-mass spectrometry of soil polysaccharides, soil fulvic acids and polymaleic acid. Org. Geochem., 6 (1984) 287-293. 17 J.M. Bracewell, G.W. Robertson and D.I. Welch, Polycarboxylic acids as the origin of some pyrolysis products characteristic of soil organic matter. J. Anal. Appl. Pyrolysis, 2 (1980) 239-248. 18 J. Alcaniz, J. Romera, L. Comellas, R. Munne and A. Puigbo, Effects of some mineral matrices on flash pyrolysis-GC of soil humic substances. Sci. Total Environ., 81/82 (1989) 81-90. 19 W. Michaelis, H.H. Richnow and A. Jenisch, Structural studies of marine and riverine humic matter by chemical degradation. Sci. Total Environ., 81/82 (1989) 41-50. 20 S. Morinaga and R. Ishiwatari, Gas chromatographic determination of CI-C 5 lowmolecular-weight organic acids in alkaline permanganate oxidation products of humic substances. J. Chromatogr., 403 (1987) 225-231. 21 F.J. Stevenson, Colloidal properties of humic substances, in Humus Chemistry, Wiley, New York, 1982, Ch. 12. 22 E.M. Thurman, R.L. Wershaw, R.L. Malcolm and D.J. Pinkney, Molecular size of aquatic humic substances. Org. Geochem., 4 (1982) 27-35. 23 J.I. Kim, G. Buckau, E. Bryant and R. Klenze, Complexation of Americium (III) with humic acid. Radiochim. Acta, 48 (1989) 135-143. 24 K.L. Nash and G.R. Choppin, Interaction of humic and fulvic acids with Th(IV), J. Inorg. Nucl. Chem., 42 (1980) 1045-1050. 25 R.G. Messerschmidt, Complete elimination of specular reflectance in infrared diffuse reflectance measurements. Appl. Spectrosc., 39 (1985) 737-739. 26 G.R. Choppin and B. Allard, Complexes of actinides with naturally occurring organic compounds, in A.J. Freeman, C. Keller, (Ed), Handbook on the Physics and Chemistry of the Actinides, North Holland, New York, 1985, pp. 407-429. 27 N.A. Marley, P. Bennett, D.R. Janecky and J.S. Gaffney, Spectroscopic evidence for organic diacid complexation with dissolved silica in aqueous systems I. Oxalic Acid, Org. Geochem., 14 (1989) 525-528.
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Chemical characterization of size-fractionated humic and fulvic materials in aqueous samples N . A . M a r l e y a, J.S. G a f f n e y a, K . A . O r l a n d i n i a, K . C . Picel a a n d G.R. Choppin b "Building 203, Environmental Research Division, Argonne National Laboratory, Argonne, IL 60439, USA bChemistry Department, Florida State University, Tallahassee, FL 32306, USA
ABSTRACT Humic and fulvic acids have been shown to be potentially important transport agents for inorganic and organic contaminants, including radionuclides, in surface and groundwaters. In this work, the possibility of characterizing humic and fulvic materials in the colloidal and macromolecular size ranges (i.e., between 3000 molecular weight (1 nm) and 0.45 #m) after size fractionation and collection with hollow-fiber ultrafilters has been investigated. Three surface waters have been examined as test cases. Using ultrafiltration, sized samples of humic and fulvic acids were chemically characterized with 13C-nuclear magnetic resonance spectroscopy (NMR), Fourier transform infrared spectroscopy (FTIR) using diffuse reflectance, and pyrolysis/gas chromatography (GC). These test cases demonstrate that sufficient material can be obtained by ultrafiltration with minimal or no alteration to the materials. In addition, inorganic analyses performed on the size fractions show that ultrafiltration can also allow the binding capacity of the humic materials in each fraction to be measured. Substantial variability among the different humic and fulvic fractions demonstrates the importance of sizing submicron materials in attempts to understand the mechanisms of pollutant transport by natural humic materials. Key words: humics; fulvics; ultrafiltration; chemical characterization; pollutant transport
INTRODUCTION N a t u r a l l y o c c u r r i n g h u m i c a n d fulvic acids are i m p o r t a n t c o m p l e x i n g agents for a v a r i e t y o f metals, r a d i o n u c l i d e s , a n d o t h e r c h e m i c a l species. T h e y c a n t h e r e f o r e act to e n h a n c e the m o b i l i t y o f c o n t a m i n a n t s in s u b s u r f a c e a n d surface waters. I n c r e a s i n g c o n c e r n s a b o u t g r o u n d w a t e r quality a n d c o n t a m i n a t i o n o f soils a n d w a t e r s b y h a z a r d o u s wastes h a v e e m p h a s i z e d the need to u n d e r s t a n d the role o f these m a t e r i a l s in ecological systems. H o w ever, d u e to their diverse a n d c o m p l e x n a t u r e , a t t e m p t s to describe the structure a n d b e h a v i o r o f h u m i c m a t e r i a l s h a v e o f t e n yielded c o n t r a d i c t o r y results
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[1]. A number of factors including source, method of extraction, or limitations of the characterization methods may be the cause of these differences. Traditionally soil humic materials have been studied by extraction into a base, followed by further separation into acid-insoluble (humic) and acidsoluble (fulvic) fractions which provided an operational distinction between humic and fulvic acids according to their acid solubilities. In an attempt to extend these studies to surface and subsurface waters, modifications of these same procedures have been applied to extract humic materials from water [2]. The methods most frequently used to characterize these substances include (i) infrared spectroscopy [3-9], (ii) nuclear magnetic resonance spectroscopy [3-7,10-13] and (iii) gas chromatography, following pyrolysis [13-18] or chemical degradation [19,20]. Aquatic humic materials occur in sizes ranging from colloidal particles (0.1 m~-10 A) to macromolecules (3000-500 mol. wt) [21]. It has been proposed that the larger, colloidal fractions are composed of humic acids and that the smaller macromolecules are the fulvic acids [22]. This suggests the possibility of separating and concentrating natural aquatic humic substances on the basis of molecular weight, thereby avoiding the harsh conditions traditionally used for the recovery of soil humics, and adapted for extraction from water [2]. In this study humic materials have been obtained from three surface waters by using hollow-fiber ultrafiltration techniques. The natural trace metal content of the size-fractionated humics and fulvics are reported as a measure of the binding capacity of each group. The structural characteristics and functional groups associated with these materials were investigated with FTIR, 13C-NMR, and pyrolysis GC in an attempt to compare the advantages of each method. These results are then compared with samples prepared by more conventional techniques [2]. MATERIALS AND METHODS
Sample sites Water samples were obtained from three surface locations, Volo Bog, Lake Bradford and Saganashkee Slough. At each site, a single 60-gallon sample was collected from just below the surface of the water, at - 20 feet from the shoreline. Volo Bog is located in an Illinois Nature Preserve northwest of Chicago. It is a small glaciated bog surrounded by sedge peat with no surface inlet or outlet. The water is of low nutrient content with a pH of 4-5. Saganashkee Slough, located in Cook County, Illinois, drains into the Calumet Sag Channel and finally into the Des Plaines River. It has a higher nutrient content
C H A R A C T E R I Z A T I O N O F H U M I C A N D F U L V I C M A T E R I A L S 1N A Q U E O U S S A M P L E S
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and is nearly at neutral pH. Lake Bradford is located near Tallahassee, Florida, is fed by both spring and surface run off, and has a pH of 5-6. Humic and fulvic acids isolated from this lake have previously been used as a source of humic materials for studies of radionuclide binding capacities [231.
Isolation of humic materials Water samples were first passed through a 35-/~m screen to remove large particulates, followed by prefiltering with a 0.45-/~m Millipore filter to remove the suspended solids. Humic materials were concentrated from the 60-gallon water samples using hollow-fiber filter cartridges obtained from the Amicon Division of W.R. Grace and Co. These filters operate using a cross-translational flow which avoids polarization on the filter due to pile up of the material on the filter surface. The cartridges are 60 cm in length and can be obtained with pore diameters of 0.1/~m and effective molecular weight filtration of 100 000, 30 000, 10 000 and 3000 mol. wt, based upon spherical model compounds. Flat disk membrane filters in a Vortex Mixing Stirred Cell were used to separate 1000 and 500 mol. wt species. The filters and stirred cells were obtained from Spectrum Medical Industries, Inc. and Amicon Corp., respectively. Lake Bradford humic and fulvic acids were also obtained using traditional separation and purification techniques [2,24]. Mass balances as determined by dissolved organic carbon determinations were observed to be within the errors of the DOC measurements (10%) for the natural humic and fulvic materials. Portions of the aqueous concentrates were dried, and the solid samples were used for the various spectral determinations. For the trace element and dissolved organic carbon measurements, the aqueous solutions were analyzed.
Trace element determinations Dissolved organic carbon (DOC) measurements were obtained on the sizefractionated concentrates with a Sybron PHOTOchem Organic Carbon Analyzer, Model E3500. Samples of 1-10 ml were injected into the analyzer with 0.05 N phosphoric acid. Trace metals were determined by inductively coupled plasma spectroscopy with an Instruments SA, Model JY 86 spectrometer equipped with an HF-compatible torch.
Pyrolysis gas chromatography Prior to analysis, dry samples were redissolved with high-purity water to
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obtain a concentration of 1 mg/ml. A Chemical Data Systems Model 122 pyrolysis unit with a platinum ribbon probe was used to pyrolyze samples. To evaporate the sample, 20/~1 of aqueous solution was applied slowly to the ribbon, which was held at 100°C. The probe was then inserted into the interface module and maintained at 220°C. After re-establishing the column flow, the sample was ramped at 20°C/ms to a temperature of 700°C for pyrolysis. The p y r o p r o b e interface was connected to a Hewlett-Packard HP5880A G C by means of a split/splitless capillary injector operated in the split mode. The G C was equipped with a 50 m (0.2 mm i.d.) Carbowax 20M column and a flame ionization detector. The G C temperature program was started at the same time as sample pyrolysis. It consisted of a 2-min hold at 50°C, followed by a linear temperature ramp, at 6°C/min to a final temperature of 220°C, which was held for 20 min. After analysis, the probe was cleaned by rapid heating to 1000°C for a short time period.
13C-Nuclear magnetic resonance ( N M R ) spectroscopy The N M R spectra were obtained on - 5 0 mg of solid sample using a Bruker 200 M H z ~3C spectrometer located at the Florida State University N M R laboratory and operated at 50.27 M H z for carbon. During the course of the experiment the sample was spun at - 3 . 7 kHz at the magic angle [10]. Pulse time was 6 #s, and cross polarization time was 1 ms. Recycle time was 3 s. Approximately 20 000 sample scans were obtained, with the number increasing for smaller sample sizes. Chemical shifts were referenced to tetramethylsilane (TMS). Dipolar dephased spectra were obtained with a dephasing time of 50 gs.
Infrared spectroscopy Infrared spectra were obtained using a Mattson Polaris Fourier transform spectrometer. The samples were mixed with dried, reagent grade KBr, and diffuse reflectance spectra were taken from 400 to 4000 wavenumbers with a Spectra Tech D R I F T accessory. Diffuse reflectance was automatically corrected for specular reflectance, and the data are reported in Kubelka-Munk units [25]. Spectra were taken at 2 cm-~ resolution and averaged over 64 scans. Reported results were baseline corrected and smoothed using a sevenpoint boxcar function. RESULTS AND DISCUSSION The purpose of this study was to determine if hollow-fiber ultrafiltration could be effective in obtaining useful samples of size-fractionated humic and
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TABLE 1 Dissolved organic carbon (DOC) and trace element levels determined in the total water samples taken at Volo Bog, Lake Bradford and Saganashkee Slough. Analyses were made after filtration through a 0.45-#m filter to remove large particulate matter. D O C is reported in ppm, and all trace elements are in ppb
DOC AI Ba Ca Cu Fe Mg Mn Na Si Sr Zn
Volo Bog
Lake Bradford
Saganashkee Slough
24.7 < 50 13 6740 < 10 340 3900 13 990 590 7 < 10
11.0 200 < 10 1190 < 10 340 460 26 1840 < 100 <5 21
13.3 63 33 50200 9 11 23900 13 4600 < 100 80 11
fulvic materials from water. For all three test cases, sufficient materials were collected to obtain spectral characterization and trace element distributions for the sized samples. The following results highlight the potential applications for size-fractionated humic and fulvic acid characterization using these techniques. The natural trace metal concentrations are listed in Table 1 for all three sample sites. Size-fractionated materials ranging from colloidal to macromolecular were separated from the samples by hollow-fiber ultrafiltration techniques and analyzed for organic and trace metal content. The results for each size fraction were compared to those obtained for the whole water samples and are shown in Figs. 1-3 for Volo Bog, Lake Bradford, and Saganashkee Slough, respectively. Sizes ranging between 0.45/~m and 3000 molecular weight represent the humics, while those less than 3000 molecular weight are the fulvic acids. Of the three sites studied, Lake Bradford has the highest concentration of larger particulates (0.45-0.1 /~m). These materials are of relatively low organic content, however, and are most likely of clay origin. In each case, at least 50% of the dissolved organic material is in size fractions < 30 000 molecular weight. These are also the species that are the most active in complexing the major trace metals.
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CHARACTERIZATION OF HUMIC AND FULVIC MATERIALS IN AQUEOUS SAMPLES
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Pyrolysis GC was performed on the size-fractionated samples. A typical pyrogram is shown in Fig. 4. The peak numbers in the figure correspond to the signature compounds listed in Table 2. Due to the great complexity of the pyrograms, only the major pyrolysis products have been identified and quantified. These have been grouped according to organic functionality for comparison to N M R results. The relative abundances are given in Tables 3 and 4 for Volo Bog and Lake Bradford, respectively. The pyrograms for the Lake Bradford samples generally contain larger amounts of phenol and thiophene than those from Volo Bog. Phenol is a pyrolysis product of lignin type structures and indicates the aromatic content of the parent molecules [14]. The organics contained in the larger particulates (0.45-0.1 #m) from Lake Bradford yielded the highest amount of phenolic products and therefore are most likely composed of large aromatic polymers. The two intermediate fractions have an aromatic content between those of the large and small sizes. However, in general, the signature products are most similar to the large particulates. The small macromolecular
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fraction (less than 3000 mol. wt) is extremely high in thiophene, acetonitrile and acetic acid indicating a high proportion of amino acid groups in the parent molecules [15]. This is in agreement with the previous generalization that, humic acids are higher in phenolate content while fulvic acids are higher in carboxylates [26]. The pyrograms for Volo Bog yielded more acetonitrile, a pyrolysis product of proteins and nucleic acids [14]. In addition, although they have low levels of the phenols, the pyrolysis produced significant amounts of benzene and its derivatives. These arise from decarboxylation of aromatic acids such as phthalates [17]. These observations indicate that, although they do not possess large amounts of lignin type structures like the Lake Bradford humics, Volo Bog humics do contain smaller aromatic carboxylate units. The small fraction from the Volo Bog showed the highest carboxylate content, as evaluated from the acetic acid GC/pyrolysis data. The large fraction, however is highest in amino acid type groups. Analysis of the pyrograms for aliphatic carboxylate structures yields very low values in all cases. This is because the major product from the pyrolysis of these groups is carbon dioxide. A more accurate measure of the aliphatic
CHARACTERIZATIONOF HUMICANDEULVICMATERIALSIN AQUEOUSSAMPLES
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TABLE 2 Identified products from humic sample pyrolysis/gas chromatography analyses (see Fig. 4). Retention times are in minutes Peak no.
Compound
Retention time
1 2 3 4 5 6 7 8 9 10 11
Benzene Acetonitrile Thiophene Toluene Anisole Acetic Acid Pyrrole Propionic Acid Acetamide Phenol, o-Cresol m-Cresol, p-Cresol
5.30 5.97 6.10 6.33 11.90 13.82 15.64 15.96 20.34 25.18 26.67
n-Alkyls
CII saturated Cll unsaturated Ci2 saturated C~2 unsaturated CI3 saturated C13 unsaturated CI4 saturated C 14 unsaturated CI5 saturated CI5 unsaturated
7.62 8.27 9.69 10.44 11.98 12.78 14.31 15.12 16.58 17.38
TABLE 3 Relative abundances of signature compounds determined from humic pyrolysis/gas chromatography for size-fractionated samples from Volo Bog. Areas are normalized for 45/~g equivalent samples. K indicates 1000 molecular weight units Compounds
Benzene, toluene Phenol, cresols Thiophene Pyrrole Acetonitrile Acetamide Acetic acid
Relative abundances of size fractions 0.45-0.1 /zm
0.1tzm-100 K
100 K - 3 0 K
30 K - 3 K
33.7 8.3 3.0 6.7 60.7 1.9 1.3
24.4 2.8 1.3 2.7 18.0 1.2 0.3
22.4 10.3 3.3 3.9 29.4 2.3 0.3
62.8 8.5 0.7 3.8 25.9 3.4 0.1
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TABLE 4 Relative abundances of signature compounds determined from humic pyrolysis/gas chromatography for size-fractionated samples from Lake Bradford. Areas are normalized for 45 p.g equivalent samples. K indicates 1000 molecular weight units Compounds
Benzene, toluene Phenol, cresols Thiophene Pyrrole Acetonitrile Acetamide Acetic acid Anisole
Relative abundances of size fractions 0.45-0.1/~m
0.1/~m-100 K
100 K-30 K
30 K - 3 K
23.6 42.3 19.9 7.1 22.6 16.2 7.4 8.9
23.8 35.2 24.4 5.0 25.6 14.0 4.2 7.0
17.4 37.0 14.1 4.3 19.1 8.2 1.1 7.2
19.9 23.3 85.5 3.5 47.5 11.1 0.7 4.0
carboxylate content can be obtained from the analysis if an infrared detector is used to determine the amount of carbon dioxide produced during pyrolysis. Plans have been made in this laboratory to include this additional feature. The ~3C-NMR spectra were obtained on the large (0.45-0.1 ~m) and small (30 000-3000 mol. wt) sample fractions from Volo Bog. They are shown in Fig. 5 (B and C). These size-fractionated samples are compared with humic materials collected from the total sample before fractionation (Fig. 5A). The total sample is the humic matter contained in the water that is less than 0.45 ~m in size. The major carbon signals are listed in Table 5 along with the relative intensities for each sample. Results for the total sample of humic materials most resemble those for the 30 000-3000 molecular weight fraction. This is because this size range comprises more than 40% of the humics present in the sample. Therefore, analysis of total humics removed from a water sample without further size fractionation can lead to biased results. The large fraction (Fig. 5B) possesses the most intense signal in the 50-100 ppm range. The signals in this range of chemical shift arise from carbons that are singly bonded to either oxygen or nitrogen (alcohol, ether, or amine derivatives). These are usually termed the carbohydrate carbons because it has been suspected that for most humic materials, they consist of polysaccharide units [12]. Therefore, the major assignment for this range has been
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O-alkyl carbons. Comparison with the pyrolysis GC results for this sample, however, leads to the conclusion that a large percentage of this signal may be due to amino acid groups. The dipolar dephased spectrum is shown in Fig. 6 for the 0.45-0.1/xm sample. A decrease in all signals from 0-100 ppm with a dephasing time o f 50 its demonstrates that these carbons are protonated [11]. This is consistent with either ether or amine assignments.
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TABLE 5 Results for ]3C-NMR analyses of size-fractionated humics collected from Volo Bog. Functional groups contributions are given as the relative areas of the carbon signals observed in the solid magic angle spectra. The results for the total sample are the abundances for the whole water sample after filtration through a 0.45-/~m filter to remove particulate matter. Functional group chemical shift ranges are given in parentheses and are referenced to tetramethylsilane as the standard Functional group (Shift, ppm)
Relative carbon abundances Size fractions
Alkyl (25-50) O-Alkyl (50-100) Acetal (100-107) Aromatic (110-140) Phenolic (140-160); Carboxyl (160-200) Carbonyl (200-220)
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CHARACTERIZATION OF HUMIC AND FULVIC MATERIALS IN AQUEOUS SAMPLES
TABLE 6 Comparison of functional group relative abundances in percent as determined by 13Cnuclear magnetic resonance spectroscopy and pyrolysis/gas chromatography analyses of sizefractionated humics collected from Volo Bog (Volo) and Saganashkee Slough (Sag) Functional group
Alkyl O-Alkyl Acetal Aromatic Phenolic Carboxyl Carbonyl
Relative carbon NMR abundances Sag < 3 K
Volo 0.45-0.1 #m
Volo 30 K - 3 K
37 24 4 10 3 18 2
21 40 11 6 4 15 4
35 24 6 9 4 15 6
Product (parent) pyrolysis~gas chromatography Alkyls Nitriles (amino acids) Aromatics (Ar-COOH) Phenols (aromatics) Carboxylates (R-COOH)
13 28
11 50
13 22
16
28
52
22
7
7
19
3
3
Table 6 compares results obtained from ~3C-NMR and pyrolysis GC for the Volo Bog humics and Saganashkee Slough fulvic acids. Results compare favorably for the aromatic content of the Volo Bog humics. For the Saganashkee fulvics, pyrolysis results are somewhat higher than NMR results. Again, comparison of amino acid content determined by pyrolysis with the carbon signals in the 50-100 ppm region shows good agreement, which may be an indication that a large portion of this N M R signal is from nitrogen-bonded carbon. Results for alkyl content obtained by pyrolysis GC include only long chain units (more than 11 carbon atoms) and are therefore lower than N M R results, which reflect more accurately the total alkyl content. Carboxylate results do not agree because decarboxylation reactions occur upon pyrolysis. There is also the possibility of the formation of benzene derivatives by elimination and cyclization reactions. It has been estimated
172
N.A. MARLEY ET AL.
B
C
.1
0 4000
3600
20'00
10'00 4-0'00
30'00
20'00
10'00
E
40'00
30'00
20'00
1 (3'00
30'00
20'00
l doo
F
.2 ,1 ,,%
O
4000
30bo
20bo
10'00 40bo
'~ 30'00
20'00
10'OO
4600
WAVENUMBERS
Fig. 7. The FTIR spectra of size-fractionated samples from Volo Bog: (A) 0.45-0.1 tim, (B) 0.1 #m to 100 000 mol. wt, (C) 100 000-30 000 mol. wt, (D) 30 000-3000 mol. wt, (E) less than 3000 mol. wt and (F) 1000-500 mol. wt.
that, during sample pyrolysis, 50% of the original sample weight is lost as carbon dioxide, whereas the formation of aromatic products by cyclization is less than 0.1 percent [12]. These questions could be answered and agreement with N M R results strengthened with an added infrared detector. The F T I R spectra obtained for Volo Bog and Lake Bradford samples are shown in Figs 7 and 8. The major bands are listed in Table 7 along with the most probable band assignments [6-9,21]. The broad band centered at - 3 4 0 0 cm -1 arises primarily from hydrogen-bonded O H stretches of phenols, alcohols and carboxylic acid groups. This broad band is present in all humic and fulvic acid spectra and therefore contributes little information regarding structural differences. However, a shoulder that appears near 2900 cm-1 is due to aliphatic C - H stretch. This band has been reported to vary considerably among different humics [21]. In all spectra shown in Figs. 7 and 8 this band is very weak, indicating a low aliphatic content. Aromatic C - H stretch appears as a shoulder slightly higher than 3000 cm-1. This band can easily be masked by the broad O H stretch and interpretation is sometimes
173
CHARACTERIZATION OF HUMIC AND FULV1C MATERIALS IN AQUEOUS SAMPLES
A
B
.4-
.2
0
~o'oo
2obo
lobo
30'00
20'00
10'00
3600
2000
1000
30'00
20'00
10'00
.4-
.2
WAVENUMBERS
Fig. 8. The FTIR spectra of size-fractionated samples from Lake Bradford: (A) 0.45-0.1 #m, (B) 100 000-30 000 mol. wt, (C) 30 000-3000 mol. wt and (D) <3000 mol. wt.
TABLE 7 Infrared band assignments for diffuse reflectance spectra taken on the size-fractionated humic samples. Band shoulders are designated by (sh) Frequency (cm-I)
Band assignments
3400 3250 (sh) 2950 (sh) 2600 (sh) 1720 1560 1520 (sh) 1450 (sh) 1400 1384 1200-1000 620
H-bonded OH (phenol, alcohol, carboxyl) Aromatic C - H stretch Aliphatic C - H stretch H bonded carboxyl, OH stretch COOH stretch C O O - asymmetric stretch N - H deformation of peptides C - H deformation (CH2-, CH3- ) C O O - symmetric stretch Si-O-C stretch C - O stretch of polysaccharides SO3Hstretch
NA .M .ARLEY T AL,
174
difficult. However, for the Lake Bradford samples (Fig. 8), it reveals a larger aromatic content for the larger fraction and the smallest aromatic content for the less than 1000 molecular weight species, in agreement with pyrolysis GC results. The aliphatic content, appears largest for the two intermediate fractions. Volo Bog samples (Fig. 7) yielded similar results for all fractions. Examination of the carboxyl stretching regions can yield information concerning complexation. The symmetric and asymmetric stretches of the carboxylate ion appear at 1560 and 1400 cm -~, respectively. The asymmetric stretch at 1400 cm -] is overlapped by a sharp peak at 1384 cm -J from carboxylate binding to silicate [27]. The a m o u n t of silica bound to the humics can be determined from the height of this peak. This sharp peak can also be used to differentiate between organically bound and inorganic colloidal silica. The band that appears at 1720 cm-~ is due to the stretching vibrations of the protonated carboxylic acid and is a measure of uncomplexed carboxyl. Comparing spectra of Fig. 7 with those of Fig. 8 shows that more uncomplexed carboxyl groups are present in the Lake Bradford samples than in those of Volo Bog. The sample with the least amount of free carboxyls be-
A
. 4ooo
0
_ s6oo
~ 2o'oo
16oo
WAVENUMBERS
Fig. 9. The FTIR spectra of purified humics and fulvics obtained by acid-base extraction procedures for Lake Bradford samples. Spectra are for (A) humics and (B) fulvics.
CHARACTERIZATION OF HUM1C AND FULVIC MATERIALS IN AQUEOUS SAMPLES
175
ing the large (0.45-0.1/~m) particulates, due to the large amount of inorganic matrix present. The Volo Bog samples also have a prominent band centered at 1200-1000 cm-~, which is the C - O stretch of the polysaccharide units. The N - H band, due to the peptides, appears as a weak shoulder at 1520 cm-~; although it is present in the spectra, it cannot be reliably used to determine the amount of amino acid groups. The symmetric carboxylate stretch can occur between 1610 and 1550 cm -~. This band can be complicated by overlap from unsaturated CC functional groups, which lead to bands at 1640-1675 cm -~. The effect of these contributions is seen in the shift of the peak maximum toward higher frequencies. Figure 9 shows FTIR spectra of humic and fulvic acids extracted from Lake Bradford by acid-base precipitation methods, with subsequent purification with HC1 and HF to remove metals and silica [24]. Humic acids, after purification, show a much higher aliphatic content (2925-2825 cm-~). This is most likely due to decarboxylation during purification. Comparison of the carboxyl bands at 1720 cm -~ and 1400 cm -~ reveals that all measurable groups are uncomplexed and in the protonated form. The silica band at 1384 cm-~ is also absent. The band at 1560 cm-~ is shifted in frequency to 1620 cm -~, indicating that a larger percentage of unsaturated groups in the purified humics. Comparison of Fig. 9B with Fig. 8D clearly demonstrates that the purified fulvic acids contain mostly aliphatic character and that the broad band from hydrogen-bonded OH is greatly decreased. These observations are consistent with the humics undergoing substantial chemical change during the acid-base precipitation, as compared to the much milder ultrafiltration separations. CONCLUSIONS
In this work we have demonstrated the potential for ultrafiltration to separate colloidal and macromolecular sized humic and fulvics in natural waters. We have also shown that with hollow-fiber ultrafiltration, sufficient materials can be obtained for chemical characterization of both the natural organic materials and the associated inorganic substituents in the various size fractions. These methods offer a much milder approach to the collection and characterization of natural humic and fulvic acids than previous procedures and allow a much more detailed picture of the importance of the various sizes of materials in the transport of pollutants in surface and subsurface waters. ACKNOWLEDGEMENTS
The authors wish to acknowledge the help of Dr. Edmund A. Huff of
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Argonne National Laboratory and Mr. Lin Feng Rao of Florida State University in obtaining the inorganic and N M R data, respectively. We also wish to thank Ms. J. Jastrow and Dr. R.M. Miller of Argonne National Laboratory for reviewing the manuscript. This work was performed at Argonne National Laboratory and at Florida State University and was supported by the United States Department of Energy, Assistant Secretary for Energy Research, Office of Health and Environmental Research, under contract W-31-109-ENG-38. The authors also wish to acknowledge the continuing support and encouragement of Dr. Frank Wobber of the U.S. Department of Energy's Subsurface Science Program. REFERENCES 1 F.H. Frimmel and R.F. Christman, Humic Substances and their Role in the Environment, Wiley-lnterscience, New York, 1988. 2 E.M. Thurman and R.L. Malcolm, Preparative isolation of aquatic humic substances. Environ. Sci. Technol., 15 (1981) 463-466. 3 J.C. Lobartini, K.H. Tan, L.E. Asmussen, R.A. Leonard, D. Himmelsbach and A.R. Gingle, Humic matter isolated from soils and water by the XAD-8 resin and conventional NaOH methods. Commun. Soil Sci. Plant Anal., 20 (1989) 1453-1477. 4 J.J. Alberts and Z. Filip, Sources and characteristics of fulvic and humic acids from a salt marsh estuary. Sci. Total Environ., 81/82 (1989) 353-361. 5 M.A. Arshad and M. Schnitzer, Chemical characteristics of humic acids from five soils in Kenya, Z. Pflanzenernahr. Bodenk., 152 (1989) 11-16. 6 K. Yonebayashi and T. Hattori, Chemical and biological studies on environmental humic acids. II. IH-NMR and IR spectra of humic acids. Soil Sci. Plant Nutr., 35 (1989) 383-392. 7 R. Candler, W. Zech and H.G. Alt, Characterization of water-soluble organic substances from a typic dystrochrept under spruce using GPC, IR, tH NMR, and 13NMR spectroscopy. Soil Science, 146 (1988) 445-452. 8 A.U. Baes and P.R. Bloom, Diffuse reflectance and transmission fourier transform infrared (DRIFT) spectroscopy of humic and fulvic acids. Soil Sci. Soc. Am. J., 53 (1989) 695-700. 9 A. Piccolo, Characteristics of soil humic extracts obtained by some organic and Inorganic solvents and purified by HC1-HF treatment. Soil Science, 146 (1988) 418-426. 10 P.G. Hatcher, M. Schnitzer, L.W. Dennis and G.E. Maciel, Aromaticity of humic substances in soils, Soil Sci. Soc. Am. J., 45 (1981) 1089-1094. 11 I. Kogel-Knabner and P.G. Hatcher, Characterization of alkyl 13C NMR spectroscopy and dipolar dephasing. Sci. Total Environ., 81/82 (1989) 169-177. 12 R. Frund and H.-D. Ludeman, The quantitative analysis of solution and CPMAS-C-13 NMR spectra of humic material. Sci. Total Environ., 81/82 (1989) 157-168. 13 G.K. Stearman, R.J. Lewis, L.J. Tortorelli and D.D. Tyler, Characterization of humic acid from no-tilled and tilled soils using carbon-13 nuclear magnetic resonance. Soil Sci. Soc. Am. J., 53 (1989) 744-749. 14 M.A. Wilson, R.P. Philp, A.H. Gillam, T.D. Gilbert and K.R. Tate, Comparison of the structures of humic substances from aquatic and terrestrial sources by pyrolysis gas chromatography mass spectrometry. Geochim. Cosmochim. Acta, 47 (1983) 497-502.
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15 A.C. Sigleo, T.C. Hoering and G.R. Helz, Composition of estuarine colloidal material: organic components. Geochim. Cosmochim. Acta, 46 (1982) 1619-1626. 16 C. Saiz-Jimenez and J.W. De Leeuw, Pyrolysis-gas chromatography-mass spectrometry of soil polysaccharides, soil fulvic acids and polymaleic acid. Org. Geochem., 6 (1984) 287-293. 17 J.M. Bracewell, G.W. Robertson and D.I. Welch, Polycarboxylic acids as the origin of some pyrolysis products characteristic of soil organic matter. J. Anal. Appl. Pyrolysis, 2 (1980) 239-248. 18 J. Alcaniz, J. Romera, L. Comellas, R. Munne and A. Puigbo, Effects of some mineral matrices on flash pyrolysis-GC of soil humic substances. Sci. Total Environ., 81/82 (1989) 81-90. 19 W. Michaelis, H.H. Richnow and A. Jenisch, Structural studies of marine and riverine humic matter by chemical degradation. Sci. Total Environ., 81/82 (1989) 41-50. 20 S. Morinaga and R. Ishiwatari, Gas chromatographic determination of CI-C 5 lowmolecular-weight organic acids in alkaline permanganate oxidation products of humic substances. J. Chromatogr., 403 (1987) 225-231. 21 F.J. Stevenson, Colloidal properties of humic substances, in Humus Chemistry, Wiley, New York, 1982, Ch. 12. 22 E.M. Thurman, R.L. Wershaw, R.L. Malcolm and D.J. Pinkney, Molecular size of aquatic humic substances. Org. Geochem., 4 (1982) 27-35. 23 J.I. Kim, G. Buckau, E. Bryant and R. Klenze, Complexation of Americium (III) with humic acid. Radiochim. Acta, 48 (1989) 135-143. 24 K.L. Nash and G.R. Choppin, Interaction of humic and fulvic acids with Th(IV), J. Inorg. Nucl. Chem., 42 (1980) 1045-1050. 25 R.G. Messerschmidt, Complete elimination of specular reflectance in infrared diffuse reflectance measurements. Appl. Spectrosc., 39 (1985) 737-739. 26 G.R. Choppin and B. Allard, Complexes of actinides with naturally occurring organic compounds, in A.J. Freeman, C. Keller, (Ed), Handbook on the Physics and Chemistry of the Actinides, North Holland, New York, 1985, pp. 407-429. 27 N.A. Marley, P. Bennett, D.R. Janecky and J.S. Gaffney, Spectroscopic evidence for organic diacid complexation with dissolved silica in aqueous systems I. Oxalic Acid, Org. Geochem., 14 (1989) 525-528.