Characterization of humic acids fractionated by ultrafiltration

Organic Geochemistry Organic Geochemistry 35 (2004) 1025–1037 www.elsevier.com/locate/orggeochem

Characterization of humic acids fractionated by ultrafiltration Li Li a

a,b

, Zhenye Zhao a, Weilin Huang c,*, Ping’an Peng a, Guoying Sheng a, Jiamo Fu a

State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Wushan, Guangzhou 510640, PR China b School of Ocean and Earth Science, Tongji University, Shanghai 200092, PR China c Department of Environmental Sciences, Cook College, Rutgers University, New Brunswick, NJ 08901-8551, USA Received 30 June 2003; accepted 5 May 2004 (returned to author for revision 17 November 2003) Available online 20 July 2004

Abstract Humic acid (HA) is a mixture of natural organic macromolecules having a range of physicochemical properties and exhibiting different reactivities in environmental systems. The objective of this study was to characterize chemical and molecular heterogeneity of HA by fractionating a bulk HA (BHA) into a series of subsamples, each having relatively homogeneous properties. The BHA was base extracted from Pahokee peat and was fractionated into eight fractions using an ultrafiltration apparatus with membranes having seven molecular cut-offs. The eight HA fractions obtained have apparent molecular sizes of <1, 1–3, 3–5, 5–10, 10–30, 30–100, 100–300 and >300 kDa, respectively, and their molecular size distributions were further calibrated using high performance size exclusion chromatography (HPSEC). The chemical and structural properties of the eight HA fractions were characterized systematically using elemental analysis, pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS), Fourier transform infrared (FTIR), ultraviolet–visible (UV–vis), and solid state 13 C-nuclear magnetic resonance (13 C-NMR) spectroscopy. The results show that each HA fraction has a relatively narrow distribution of molecular sizes on HPLC chromatographs, suggesting that ultrafiltration technique is effective for fractionating broadly heterogeneous humic macromolecules into relatively homogeneous fractions. UV–vis spectroscopy and Py-GC-MS analyses indicate that the fractions with lower molecular weights have more heterogeneous functional groups, greater O/C atomic ratios, and higher contents of oxygen and lignin-derived aromatic structural units. Conversely, the HA fractions with higher molecular weights have lower contents of oxygen and aromatic structural units that correspond to greater H/C and lower O/C atomic ratios. This study suggests that HAs formed under the same biogeochemical conditions may consist of macromolecules with a range of chemical, structural and molecular properties. Ó 2004 Elsevier Ltd. All rights reserved.

1. Introduction Humic acid (HA) is a mixture of natural organic macromolecules operationally defined as the fraction

*

Corresponding author. Tel.: +1-732-932-7928; fax: +1-732932-8644. E-mail address: [email protected] (W. Huang).

that is soluble in basic solutions (pH > 12) but insoluble in acidic solutions (pH < 2) (Stevenson, 1994). Due to its ubiquity in the environment and its polyelectrolytic nature, HA is an important colloid and sorbent phase controlling the speciation and fate of many organic and inorganic pollutants in surface aquatic and groundwater systems (Weber, 1988; Stevenson, 1994). Dissolved HA often interferes with water treatment processes and significantly affects the quality of the final portable water

0146-6380/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.orggeochem.2004.05.002

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L. Li et al. / Organic Geochemistry 35 (2004) 1025–1037

product (Reckhow et al., 1990). For instance, relatively high concentrations of HA in source water can cause formation of trihalogenated methanes that are possible carcinogens. Quantitative prediction and mechanistic description of its reactivities in both natural and engineered environmental systems require detailed delineation of chemical, structural, and molecular characteristics for HA. When dissolved in aqueous solutions, HA exhibits a range of properties similar to those of well-structured organic polymeric macromolecules and its hydrodynamic characteristics are highly dependent on solution properties (Ghosh and Schnitzer, 1980). At low pH and high concentrations of HA and background electrolytes, HA is believed to have a random coil structure, whereas at neutral pH and low concentrations of HA and background electrolytes HA are likely flexible and linear colloids. In addition to this well-known and widely accepted macromolecular model for HA, a recent model proposed by Piccolo and his coworkers (Conte and Piccolo, 1999; Piccolo et al., 1996, 1999, 2002) is predicated on a hypothesis that HA is a supramolecular aggregate of very small molecules with heterogeneous functionality held together by weaker hydrophobic interactions and hydrogen bonding. The model was proposed primarily based on observations that the apparent molecular weight of HA measured using high performance size exclusion chromatography (HPSEC) can be drastically reduced when the eluting solution contains a fraction of acetic acid. The organic acid was believed capable of breaking hydrogen bonding, disaggregating HA supramolecules. This study aims to elucidate the heterogeneous structural and compositional characteristics of HA with different apparent molecular sizes. It is known that the size or apparent molecular weight of HA is an important property correlated well with the reactivities of HA in both natural and engineered environmental systems. Molecular sizes of HA were shown to affect formation of harmful disinfection by-products in water chlorination process (Reckhow et al., 1990), color and background DOC removal efficiency in processes based on activated carbon adsorption (Kilduff et al., 1996) and membrane separation (Fan et al., 2001), organic pollutant-HA binding (Chin et al., 1997), and complexation with heavy metals in aquatic systems (Lakshman et al., 1996; Christl et al., 2001). In a prior study (Li et al., 2003), we found that eight HA fractions obtained by repeated base-extraction of Pahokee peat exhibit large variations in chemical, functional, and molecular properties. From the first HA fraction (Fr1) to the eighth HA fraction (Fr8) extracted repetitively from the same batch of the peat sample, the O/C atomic ratio decreases from 0.52 to 0.36 whereas the H/C atomic ratio increased from 1.1 to 1.5. 13 C NMR and FTIR spectra indicated that the contents of

oxygen-containing and aromatic functional groups decrease and that the contents of aliphatic groups increases, whereas the measured average apparent molecular size (Mw ) increases from 7.7 to 22.1 kDa, respectively. The results suggested that two subunits of humic acids may exist: an aliphatic subunit having larger apparent Mw and an aromatic subunit having smaller apparent Mw . Each subunit may be formed from different source materials under similar biogeochemical conditions. In this study, we employed an ultrafiltration technique to fractionate the bulk peat HA (BHA) sample on a basis of apparent molecular size. It is intuitive that the fractionated HA subsamples should have different molecular sizes and are relatively homogeneous compared to BHA, and that the differences in chemical and structural properties among the HA fractions should provide insight into the heterogeneity of BHA. We utilized HPSEC to determine the distribution of apparent molecular sizes for each fraction. Infrared, ultraviolet– visible (UV–vis), and solid-state carbon-13 nuclear magnetic resonance (13 C NMR) spectrometry were used for determining the functionalities of the HA fractions. Elemental analysis and pyrolysis-gas-chromatography (Py-GC-MS) were used to characterize the chemical compositions of the HA samples. Our study indicates that different source materials can cause large chemical, structural and molecular heterogeneity for HAs that were formed under the same biogeochemical conditions.

2. Materials and methods 2.1. Extraction of HA The HA sample used in this study was extracted from Pahokee peat (45.7% TOC). The peat was obtained from the International Humic Substances Society (IHSS). The peat sample was selected for this study because its HA has been extensively characterized previously (Mao et al., 2000; Li et al., 2003) and utilized in the study of organic pollutant sorption processes (Xing and Pignatello, 1997; Chiou et al., 2000; Xia and Pignatello, 2001; Mao et al., 2002). The BHA was extracted from the peat following a standard procedure recommended by IHSS (Swift, 1996). In brief, the peat was first treated with 0.1 M HCl (1:10 w/w), and was sequentially extracted nine times with 0.1 M NaOH under an N2 atmosphere, each extraction lasting for 24 h. After each extraction, the aqueous solution was separated from the solid by centrifugation at 9400g. The supernatants of nine extractions were combined and acidified with 6 M HCl to pH 1–2 for precipitating HA. After centrifugation, the HA precipitate was re-dissolved with a minimal volume of 0.1 M KOH under an N2 atmosphere and KCl was

L. Li et al. / Organic Geochemistry 35 (2004) 1025–1037

added to obtain a Kþ concentration of 0.3 M. After removal of fine insoluble particles by centrifugation, the HA supernatant was acidified, and the HA precipitate obtained was treated with 0.1 M HCl + 0.3 M HF solution for 24 h, dialyzed against distilled water, freezedried, and stored in a glass bottle. 2.2. Fractionation of HA by ultrafiltration The BHA sample was fractionated into eight fractions using a cross-flow ultrafiltration technique (MinitanTM , Millipore). The hydrophilic cellulose membranes (Millipore) used had nominal molecular weight cutoffs of 1, 3, 5, 10, 30, 100, 300 kDa. An aqueous BHA solution at 600 mg/l was prepared by dissolving an appropriate amount of the BHA into a buffer solution with 1 mM phosphate (pH 6.8), 0.01 M NaCl, and 100 mg/l NaN3 . The ultrafiltration process was operated at a flow rate of 300 ml/min with a combined diafiltration and concentration method (Kilduff and Weber, 1992). In brief, the ultrafiltration separation was achieved in four different batches; each batch followed an identical procedure and maintained the same solution chemistry. For each batch, the separation began with the membrane of 1 kDa molecular cutoff by concentrating the BHA solution from 8 to 2 l, and the 6 l filtered solution was retained, stored at 4 °C, and combined with the solutions of the same molecular cutoff obtained later. The concentrated 2 l solution with HA >1 kDa was then diafiltered with 2 l buffer solution. The retained solution with the HA fraction of >1 kDa was then diluted with 2 l of buffer solution. This diluted solution was then concentrated to 2 l with a larger molecular cutoff membrane (3 kDa), and diafiltered with 2 l buffer solution. The resulting 4 l solution with molecular cutoffs greater than 1 kDa but smaller than 3 kDa was then concentrated with the smaller cutoff membrane (1 kDa) to 1 l. The filtered solution (3 l) with HA <1 kDa was combined with the HA (<1 kDa) solutions (6 l) produced at the beginning of the separation process, and stored at 4 °C until completion of an entire ultrafiltration procedure. This separation process continued progressively for obtaining six HA fractions having greater molecular cutoffs. In the last step of the procedure, the solution (>100 kDa) was diluted with 2 l of buffer solution and was then concentrated to 1 l solution (>300 kDa fraction). Each of the resulting eight solutions was roto-evaporated at 38 °C to a volume of about 40 ml, which was dialyzed against distilled water (500 Da, Spectra/Por CE Dialysis Membranes), freeze-dried, and weighed for calculating mass distribution among the eight HA fractions. The eight HA fractions obtained from this procedure are UF1 (<1 kDa), UF2 (1–3 kDa), UF3 (3–5 kDa), UF4 (5–10 kDa), UF5 (10–30 kDa), UF6 (30–100 kDa), UF7 (100–300 kDa), and UF8 (>300 kDa). After completion of the separation proce-

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dures for the four batches of BHA sample, the HA fractions having the same molecular cutoffs were combined for the characterization studies described below. 2.3. Characterization of the fractionated HA samples The eight HA fractions and BHA were characterized for their chemical, structural, and molecular properties. The procedures for characterizing the HA materials are briefly described below. 2.3.1. Apparent average molecular weight Apparent average molecular weight was quantified for BHA and the eight UF fractions with HPSEC conducted on a Biosep-Sec-S2000 column (300
7.8 mm, Phenomenex) with a guard column of the same packing materials (30
7.8 mm, Phenomenex). The mobile phase consisted of 2 mM phosphate at pH 6.8, with an ionic strength of 0.1 M adjusted with NaCl. The system was calibrated against polystyrene sulfonates sodium standards (PSS, Scientific Polymer Inc.) with molecular weights of 5, 8, 15, 35, and 60 kDa. In addition, blue dextran (2000 kDa, Sigma) and acetone (58 Da, HPLC grade, Fisher) were also used as probes for the void volume (V0 ¼ 5:72 ml) and total permeation volume (Vt ¼ 12:35 ml), respectively. The standards and HA samples were dissolved in a buffer solution identical to the HPSEC mobile phase at a sample concentration of 100 mg/l. The eluent and all sample solutions were filtered through a 0.22 lm membrane before each run. The system was operated at 1.0 ml/min, 25 °C and the injected volume of sample was set at 20 ll. The wavelength of the UV detector was set at 224 nm for standards and at 254 nm for HA. A calibration equation, log Mw ¼ 0:4439Rt þ 7:4083ðR2 ¼ 0:99Þ, was obtained based on the chromatograms of the five PSS standards and acetone. Here, Rt is the retention time. The apparent Mw value was determined for each HA solution using the equations given by Yau et al. (1979). The term ‘‘apparent’’ was used here because the Mw measured for HA macromolecules using this technique may differ variously from their actual Mw due to both the effect of solution chemistry on the configuration and polydispersity of HA molecules and the difference in chemical, structural and molecular properties between the standards and HA samples. 2.3.2. Elemental composition, UV–visible, FT-IR, and 13 C NMR spectra The elemental composition (C, H, N and O) was determined with a CHN-O-RAPID Elemental Analyzer (Heraeus) following a standard high-temperature combustion procedure (Nelson and Sommers, 1982). Spectrophotometric absorbance of the humic acid solutions was measured on a Helios a dual-beam scanning UV–vis spectrophotometer (Thermo Spectronic).

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L. Li et al. / Organic Geochemistry 35 (2004) 1025–1037

3. Results and discussion

Aqueous solutions of each HA fraction were prepared in a way similar to that for the above HPSEC study. The spectra were obtained by scanning from 200 to 700 nm, and the absorbance at 465 and 665 nm was recorded. The infrared spectra were recorded on a Perkin– Elmer 1725 X FT-IR spectrometer at a resolution of 4 cm1 for pellets prepared by mixing ground HA powder (1 mg) with 60 mg KBr (FT-IR grade). The 13 C NMR spectra were recorded on a Bruker DRX-400 NMR spectrometer operated at a 13 C frequency of 100.63 MHz and at a magic-angle-spinning (MAS) rate of 6.0 kHz. The solid HA samples were filled in a 4-mm diameter ZrO2 rotor with a Kel-F cap. A 1.2-s recycle time and a 1.5-ms contact time were used. Each spectrum consisted of 2400 data points and the chemical shifts were referenced externally to glycine (176.03 ppm).

3.1. Mass distribution of the fractionated HA The yields of all UF fractions and their elemental compositions are listed in Table 1. This table shows that the four fractions (UF5–8) with greater molecular cutoffs constitute 93 wt% of the total HA recovered from the ultrafiltration process, and that UF8 constitutes 45.8% of the BHA. Our result that the majority of HA has sizes within a narrow range of molecular sizes is consistent with several studies of different soil HAs using ultrafiltration techniques (Shin et al., 1999; Tomb
acz, 1999; Christl et al., 2000; Francioso et al., 2002). For example, Christl et al. (2000) reported that the soil HA fractions with >300 kDa and 30–100 kDa contained 52% and 34% of the total carbon, respectively, and that two other HA fractions with 100–300 and 10–30 kDa contributed less than 10% of the total carbon of the humic acid. Our result is different from a report showing that 75% of marine organic carbon was low-molecularweight DOM and 24% was high-molecular-weight DOM (Benner et al., 1997). This difference is likely because DOM generally has much smaller Mw and higher polarity than soil HAs (Perminova et al., 1998).

2.3.3. Pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) Py-GC-MS was performed on a Finnigan GC-8000 TOP-Voyager gas chromatography-mass spectrometry coupled with a CDS-1500 pyrolyzer. During analysis, about 1 mg of HA sample was placed in a quartz tube that was introduced into a CDS-2000 pyroprobe. The probe was inserted into the injector whose temperature maintained at 250 °C. The probe was then heated to 610 °C at 5 °C/ms and kept for 10 s. Helium was used as a carrier gas to flush the pyrolytic compounds into a fused silica column coated with DB-5MS (30 m
0.32 mm
0.25 lm, J&W). The column temperature was kept initially at 35 °C for 5 min, then programmed to 200 °C at 2.5 °C /min, later to 300 °C at 5 °C /min and held at 300 °C for 5 min. The ion source was operated at 70 eV with a mass detection range of m/z 40–500. The products were identified by comparison of mass spectra with library/literature data.

3.2. Molecular weight distribution of the bulk HA and the fractionated HAs The HPSEC chromatograms for the eight UF HA fractions and BHA are shown in Fig. 1, along with characteristic molecular sizes of 0.5, 1, 10, and 100 kDa. The apparent Mw values calculated from the HPSEC chromatograms are listed in Table 1. The data show that BHA has an average apparent Mw of 10.68 kDa and the UF HA fractions have apparent Mw ranging from 1.07 to 18.56 kDa. All UF HA fractions have HPSEC peaks sharper and narrower than that of BHA due to frac-

Table 1 Yields, averaged molecular-weight- (Mw ), elemental composition (%), atomic ratio and E4 =E6 of the UF fractions and BHA

UF1 (<1 kDa) UF2 (1–3 kDa) UF3 (3–5 kDa) UF4 (5–10 kDa) UF5 (10–30 kDa) UF6 (30–100 kDa) UF7 (100–300 kDa) UF8 (>300 kDa) BHA BHAcalculated

Mass (wt%)

Mw (kDa)

1.9 1.6 1.7 2.0 15.5 22.2 9.4 45.8

1.07 1.18 1.49 1.77 3.24 5.25 6.29 18.56 10.68 10.86

E4 =E6

Elemental composition (wt%)

Atomic ratio

C

N

H

O

H/C

O/C

N/C

48.7 48.5 48.4 49.9 53.6 54.0 54.7 57.0 56.1 55.1

4.0 3.6 3.4 3.9 4.4 4.6 4.6 5.3 3.9 4.8

3.3 3.5 3.8 3.9 4.1 4.3 4.4 5.3 5.0 4.7

44.0 44.3 44.4 42.3 37.9 37.1 36.2 32.4 35.0 35.5

0.81 0.87 0.94 0.94 0.92 0.96 0.96 1.12 1.06 1.02

0.68 0.69 0.69 0.63 0.53 0.52 0.50 0.43 0.47 0.48

0.070 0.064 0.060 0.067 0.070 0.073 0.072 0.080 0.060 0.075

13.6 13.2 12.6 11.7 7.0 6.1 5.7 4.5 5.8 6.0

L. Li et al. / Organic Geochemistry 35 (2004) 1025–1037

100k

10k

1k

0.5k

UF1

UF2

UF3

UF4

UF5

UF6

UF7

UF8

BHA 2

4

6

8

10

12

14

16 min

Fig. 1. Size distribution of the eight UF fractions and BHA.

tionation with the molecular size-based ultrafiltration technique. One major feature of this study is that the apparent Mw values measured by HPSEC are dramatically lower than those indicated by the nominal molecular cutoff of the ultrafiltration membranes. This is consistent with several prior studies on DOM (Chin and Gschwend, 1991; Chin et al., 1994; Everett et al., 1999) and soil HA (Christl et al., 2000; Francioso et al., 2002). It is possible that the observed difference may result directly from the difference in separation principles and operating conditions between the two techniques. Ultrafiltration is operated under a semi-batch condition. Each UF membrane has a characteristic nominal molecular size cut-off level, which is operationally defined as the mass of a molecule whose retention is 90% on this membrane. Globular proteins are often used as standards for testing

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macromolecule retention by a membrane. Because they are chemically and structurally different from protein, dissolved humic acids are expected to have molecular configurations different from protein when dissolved in aqueous solution. It is likely that their polyelectrolytic nature may result in relatively larger apparent molecular sizes than their actual sizes during ultrafiltration. In the HPSEC technique, the measured apparent molecular sizes are calculated against an external standard, and the specific operating conditions are different from ultrafiltration. In this study, HPSEC was operated under the conditions of higher ionic strength and lower HA concentration in order to minimize the interactions between HA and the stationary phase of the column (Chin et al., 1994). It is known that the apparent sizes of HA strongly depend on the solution chemistry such as HA concentration, pH, and the background electrolyte concentration (Ghosh and Schnitzer, 1980). Flexible and random-coil configurations correspond to lower ionic strength or high pH condition whereas compact aggregate configurations appear in conditions of higher ionic strength or low pH. While the solution chemistry remains constant for HPSEC, the HA concentration in the reservoir of an ultrafiltration apparatus changes over time during the concentration mode, causing the apparent HA sizes to change over time. This suggests that the apparent molecular sizes measured with HPSEC are relatively more reliable and accurate than the sizes given by the ultrafiltration technique. It should also be pointed out that, although the HPSEC is likely more reliable and well documented, the absolute values of the weight-averaged molecular sizes determined and presented in Table 1 may have various uncertainties. In this study, we selected PSS as the standard because prior studies showed that PSS has a negative charge density (5.4 mmol/g) (Perminova et al., 1998) similar to typical dissolved humic materials (Everett et al., 1999), a very important criterion for determining the Mw for humic substances. However, soil HA has charge densities ranging between 2 and 5 mmol/g and its carboxylic functional groups may have much smaller dissociation constants than sulfonate groups on PSS. These differences between PSS and the soil HA samples may cause inaccuracy of the calculated Mw reported here. In addition, the calibration curve we established for the given HPSEC conditions was based on the PSS standards having sizes of 5–60 kDa. The first five UF fractions (UF1–UF5) actually have estimated Mw < 5 kDa; this may also cause potential uncertainty in the calculated molecular sizes reported here. 3.3. Chemical compositions The elemental compositions of BHA and the eight HA fractions shown in Table 1 indicate that BHA has contents of C, N, H, and O of 56.1, 3.9, 5.0, and 35.0

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L. Li et al. / Organic Geochemistry 35 (2004) 1025–1037

Table 2 Relative contents of different carbons in UF fractions and BHA sample calculated from

UF1 UF2 UF3 UF4 UF5 UF6 UF7 UF8 BHA BHAcalculated

13

C NMR spectra (%)

Carbonyl C 220–190

Carboxyl C 190–160

O,N-aryl C 160–145

H,C-aryl C 145–110

O-alkyl C 110–65

OCH3 65–45

Alkyl C 45–0

Aromaticity

1.7 2.0 2.1 2.1 1.6 1.8 1.9 1.6 1.6 1.7

24.5 24.1 20.6 17.6 17.4 17.0 15.8 13.2 13.9 15.6

4.6 5.5 5.3 5.2 6.8 6.6 5.8 5.0 4.7 5.7

33.9 34.3 33.5 34.0 30.3 30.1 29.3 22.0 24.2 26.7

11.2 11.4 12.3 10.7 12.8 14.5 14.9 13.5 12.6 13.6

5.3 5.1 7.6 8.3 9.3 8.9 9.3 9.8 9.7 9.3

18.7 17.6 18.7 22.1 21.7 21.1 23.0 34.9 33.3 27.6

38.5 39.8 38.8 39.2 37.1 36.7 35.1 27.0 28.9 32.4

wt%, respectively, whereas the HA fractions have varied elemental compositions. As the molecular cutoff increases from UF1 to UF8, the hydrogen content displays a gradual increase from 3.3 wt% for UF1 to 5.3 wt% for UF8. The carbon and oxygen contents for the first three fractions (UF1–UF3) are within narrow ranges of 48.7–48.4 and 44.0–44.4 wt%, respectively. From UF4 to UF8, the carbon content increases from 49.9 to 57.0 wt% whereas the oxygen content decreases from 42.3 to 32.4 wt%. The nitrogen content manifests a unique trend: decreasing initially from 4.0 to 3.4 wt% from UF1 to UF3, then increasing from 3.9 to 5.3 wt% from UF4 to UF8. The change in elemental compositions is also well reflected by an increase of H/C atomic ratio from 0.81 to 1.12 and decrease of O/C atomic ratio from 0.68 to 0.43 as the molecular cutoff increases from UF1 to UF8. Such changes indicate that the HA fractions of smaller molecular cutoffs have greater polarity and aromaticity than those of larger molecular cutoffs. The calculated average properties of BHA based on mass fraction and characteristics of each HA fraction are listed in Tables 1 and 2 as BHAcalculated . The results are approximately the same as the actual BHA, suggesting that the fractionation may have not altered the properties of HA. 3.4. UV–visible and infrared spectroscopy The UV–vis absorbance of HA typically decreases as the wavelength increases and its absorption spectrum is commonly broad and featureless. An absorbance ratio measured at wavelengths of 465 and 665 nm, commonly designated as the E4 =E6 ratio, is generally <8.5. This ratio is independent of HA concentration. According to prior studies, this ratio is inversely proportional to the degree of condensation or the molecular weight (Chen et al., 1977; Stevenson, 1994; Tomb
acz, 1999). Table 1 lists the E4 =E6 ratios measured for BHA and the eight HA fractions. The E4 =E6 ratio of BHA is 5.8 whereas the

ratios of the eight HA fractions exhibit large variations. According to Table 1, the E4 =E6 ratio decreases gradually from 13.6 for UF1 to 11.7 for UF4, then drops to 7.0 for UF5 and gradually decreases to 4.5 for UF8. The first four HA fractions have much greater E4 =E6 ratios than BHA and those reported in the literature for HA and fulvic acid, suggesting that more chromophores (i.e., carboxylic and ketonic C@O, aromatic C@C) may be concentrated in those lower molecular weight fractions. The FTIR spectra shown in Fig. 2 for the eight UF fractions and BHA indicate that all HA samples exhibit generally similar IR spectra, suggesting that they have very similar structures and functional groups. The assignment of different peaks on a typical HA IR spectrum includes H-bonded OH stretching (mO–H ) at 3500–2500 cm1 , asymmetric and symmetric stretching vibration of aliphatic C–H (masC–H , msC–H ) from 3000 to 2800 cm1 , carboxylic and ketonic carbonyl stretching (mC@O ) at 1710 cm1 , stretching vibration of conjugated C@C (mC@C ) or H-bonded carbonyl C@O (mC@O ) at 1630 cm1 , O–H bending vibration of alcolols or carboxylic group (dO–H ) and C–O stretching of phenolic group (mC–O ) at 1400 cm1 , and C–O stretching vibration (mC–O ) and O– H bending deformation (dO–H ) due mainly to carboxyl groups at 1250 cm1 (Aiken et al., 1985; Stevenson, 1994). Further inspection of the IR spectra indicates appreciable differences in resolution and strength of assigned peaks among the eight HA fractions. These differences suggest that the fractions with greater molecular weights have higher contents of aliphatic carbon structures and that the fractions with lower molecular weights have higher contents of O-containing structures. The peak at 2930 cm1 on the IR spectrum of UF8 is very strong compared to other UF fractions, indicating higher content of aliphatic structures. The high aliphatic content in UF8 is also indicated by an identifiable shoulder peak near 1450 cm1 on the same IR spectrum. This shoulder peak is commonly assigned to C–H deformation vibration of aliphatic structure (dC–H ). The

L. Li et al. / Organic Geochemistry 35 (2004) 1025–1037 1630 2930

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peaks of 990 and 970 cm1 should not be attributed to phosphate salts (Pavia et al., 2001).

1045 1250 1100 1180 990 1400 970

3.5. Solid state

UF1 UF2 UF3 UF4 UF5 UF6 1450

UF7

UF8 BHA 4000

2000

1000

1031

13

C NMR spectroscopy

The solid state 13 C NMR spectra are shown in Fig. 3 for all the HA samples. The peaks are generally assigned to aliphatic carbon (0–45 ppm), oxygenated aliphatic carbon (45–110 ppm), aromatic carbon (110–160 ppm), carboxylic carbon (160–190 ppm), and carbonyl carbon (190–220 ppm). The assigned peaks and the estimated peak areas are listed in Table 2. According to Fig. 3, the aliphatic carbon region (0– 45 ppm) exhibits a broad peak likely resulting from a mixture of various paraffinic carbons. The integrated area of this peak region is 33.3% for BHA and is about 18.7–34.9% for the eight UF fractions. The oxygenated aliphatic carbon region has two peaks corresponding to methoxyl groups (56 ppm) and O-alkyl groups (72 ppm) (i.e., –CH(OH)– or –CH2 –O–C). The intensity of the methoxylic carbon peak appears to increase from UF1 to UF8, but no change of peak intensity is observed for the O-alkyl carbons. Two peaks at 130 and 152 ppm can be found in the aromatic carbon region, corresponding to hydrogen/carbon substituted and oxygen-substituted aryl carbons, respectively.

cm -1

Fig. 2. FTIR spectra of the eight UF fractions and BHA. 173

peaks at 1400 and 1250 cm1 are much stronger in the lower molecular weight fractions than the higher molecular weight fractions, indicating higher contents of carboxylic and other oxygen-containing groups associated with these HA fractions. Sharp peaks between 1200 and 900 cm1 on the IR spectra of UF1–UF4 are often assigned as following: (1) C–O stretching vibration (mC–O ) of OCH3 , alcohols, or ether at 1180 cm1 ; (2) C–O stretching vibration (mC–O ) of carbohydrates and alcohols (1100 cm1 ), or C–C stretching motions of aliphatic groups (mC–C ), or in-plane C–H bending of aromatic rings (dC–H ) at 1100 cm1 (Miikki et al., 1997; Francioso et al., 2002); (3) O–H stretching (mOH ) at 1050 cm1 ; and (4) an out-of-plane C–H deformation vibration linked to alkenes (d@C–H ) at 990 and 970 cm1 (Aiken et al., 1985; Pavia et al., 2001). It should be noted that the band centered around 1050–1150 cm1 may be due to Si–O–Si vibration if silicate is present in the HA samples (Senesi and Sipos, 1985; Shin et al., 1999). The two peaks at 990 and 970 cm1 may also be assigned to stretching vibration of P–O (mP–O ) if phosphate were present in the HA samples. However, the lack of a strong stretching peak of P@O (mP@O ) and a moderately strong stretching peak of P–O– H (mP–O–H ) at 1300–1240 cm1 may suggest that the two

130

152

72

56

30

UF1 UF2 UF3 UF4 UF5 UF6 UF7 UF8

WHA 200 Fig. 3. CP/MAS and BHA.

13

150

100

50

0

ppm

C NMR spectra of the eight UF fractions

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L. Li et al. / Organic Geochemistry 35 (2004) 1025–1037

The 13 C-NMR spectra were also quantitatively presented in Table 2. It shows that the H- and C-substituted aryl carbons may account for more than 80% and that the O-substituted aryl carbon is <20% of the total aryl carbons. The intensity of the H- and C-substituted aryl carbon peak appears decreasing from UF1 to UF8 whereas the intensity of the oxygen-substituted aryl remains unchanged. As a result, the sum of the total aromatic carbons and hence the aromaticity decreases from about 38.5% for UF1 to 27.0% for UF8. The

aromaticity of BHA is about 28.9% which is slightly greater than UF8 but lower than the rest of UF fractions. The peaks in 160–190 ppm are mainly due to carboxylic carbon, with some contribution from amine and ester carbons. The peak area shown in Table 2 decreases from 24.5% for UF1 to 13.2% for UF8, indicating higher contents of carboxylic functional groups for lower molecular weight HA fractions. (see Table 3). While the data listed in Table 2 provide detailed information on the differences of functionalities among the

Table 3 Typical pyrolytical products of Py-GC-MS No.

Compounds

Origin

No.

Compound

Origin

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

Pyridine Pyrrole Toluene 2-Furaldehyde Methylpyrrole Methylpyrrole Dimethylbenzene Dimethylbenzene Styrene Dimethylbenzene 1-Nonene Nonane Methyl-2-furaldehyde Trimethylbenzene Phenol Trimethylbenzene Decane o-Cresol Guaiacol m/p-Cresol 1-Undecene Undecane Dimethylphenol Ethylphenol 4-Methylguaiacol 1-Dodecene Dodecane Catechol Vinylphenol Branched Tridecane 2-Methoxy-benzenediol Ethylguaiacol Vinylguaiacol 1-Tridecene Tridecane Syringol 1-Tetradecene Tetradecane Methylsyringol Eugenol Acetoguaiacone 1-Pentadecene Pentadecane

P P P, L C P P P, L P, L P, L P, L Lp Lp C P, L C, P, L P, L Lp L L L Lp Lp L L L Lp Lp L L Lp L L L Lp Lp L Lp L L L L Lp Lp

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85

Propioguaiacone 1-Hexadecene Hexadecane Diketodipyrrole Propenylsyringol Acetosyringone 1-Heptadecene Heptadecane Prist-1-ene Prist-2-ene Tetradecanoic acid 1-Octadecene Octadecane Pentadecanoic acid Pentadecanoic acid 1-Nonadecene Nonadecane Branched Hexadecanoic acid Hexadecanoic acid 1-Eicosene Eicosane Branched Heptadecanoic acid Heptadecanoic acid 1-Heneicosene Heneicosane Octadecaoic acid 1-Docosene Docosane 1-Tricosene Tricosane 1-Tetracosene Tetracosane 1-Pentacosene Pentacosane 1-Hexacosene Hexacosane 1-Heptacosene Heptacosane 1-Octacosene Octacosane 1-Nonacosene Nonacosane

L Lp Lp P L L Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp Lp

C: Carbohydrate; P: Protein; L: Lignin; Lp: Lipid.

L. Li et al. / Organic Geochemistry 35 (2004) 1025–1037

ples, 13 C NMR data collected using a fixed contact time may be biased.

eight HA fractions, they might be interpreted qualitatively because the 13 C NMR data were collected at a fixed recycle time of 1.2 s for all the HA samples. In general, quantitative interpretations of 13 C NMR spectra require that the contact time should be less than the relaxation times (T1 ) of all 13 C atoms on a sample. Since the relaxation times (T1 ) may vary among a set of sam-

100

1033

3.6. Py-GC-MS Fig. 4 shows the pyrogram obtained for BHA using the Py-GC-MS technique, and Fig. 5 shows two specific

3

TIC

19

relative abundance, %

1+2

50 5

15

6 4

20 13

14

7 12

36

28

52

29 23 21 20 23

14 18

31

41 33

37

32

13

38

40 39

48 53

44 4

54 62

0 10

20

50

40

30

70

60

30

100

SIC m/z57

C12

relative abundance, %

C11

min

80

C13 C22 C14

C10

C15

C9

C21

62

50

C17

C25

C24

C18 C16

C23

C20 C26

C19

C27

52

C28 C29

0 10

20

30

40

50

60

70

80

min

Fig. 4. Total ion chromatogram (TIC) and specific ion chromatogram (SIC, m/z 57) of the pyrolysates obtained for BHA. Peak numbers refer to the compounds listed in Table 4. Cn refers to the carbon chain number of the n-alkanes.

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L. Li et al. / Organic Geochemistry 35 (2004) 1025–1037

∗

relative abundance, %

100

UF1

50

0 10

20

50

40

30

70

60

min

80 ∗

100

relative abundance, %

UF8 C11

C12 C13 C14 C21

C15 C16

50

C10

C17

C18

C23

C20 C22 C19

C25

C27

C24 C29 C26

0 10

20

30

40

50

60

70

80

min

Fig. 5. Specific ion chromatogram (SIC, m/z 57) of the ultrafiltration fractions UF1 and UF8. Cn refers to the carbon chain number of the n-alkanes. The sign * refers to the phthalate contaminant during the ultrafiltration process.

ion chromatograms (SIC) of m=z 57 for UF1 and UF8. The pyrograms consist of more than 100 pyrolysates at different abundances, among which 85 were identified and listed in Table 4. In order to compare the pyrograms among different HA fractions, relative abundances of the pyrolysates in each HA sample are calculated by normalizing each individual peak area to the total peak area of all the identifiable pyrolysates. These pyrolysates are divided into several groups according to their pos-

sible sources or their original structures. The relative contents of each group are summed and the results are listed in Table 4. The results of Py-GC-MS analysis show that BHA has significant contributions from higher plant material. Prominent peaks 15, 19, and 36 on the pyrogram (Fig. 4) represent phenol, guaiacol, and syringol, which are indicators of lignin material. Several homologous pyrolysates of these compounds are also present. The aliphatic

L. Li et al. / Organic Geochemistry 35 (2004) 1025–1037

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Table 4 Mass distribution of the major groups of pyrolytic products in the UF fractions and the BHA (%)

UF1 UF2 UF3 UF4 UF5 UF6 UF7 UF8 BHA BHAcalculated

Protein

Benzenes

A-Phenols

Guaiacols

Syringols

Aliphatics

P Ar

9.93 10.08 18.46 15.90 16.72 13.99 15.40 20.84 18.56 17.67

15.59 16.68 16.44 19.67 19.01 18.47 18.88 14.52 16.98 16.71

28.78 28.03 27.40 28.21 26.87 28.08 27.91 15.66 16.94 22.22

25.91 24.86 22.04 19.03 18.97 18.69 17.53 20.51 20.74 19.77

9.77 7.95 6.42 5.40 5.76 5.50 5.24 6.53 5.43 6.13

0.31 0.39 0.27 0.23 1.74 2.16 1.85 7.06 6.85 4.18

80.05 77.52 72.31 72.32 70.60 70.73 69.56 57.22 60.10 64.83

A-Phenols: aliphatic substituted phenols. P Ar: sum of benzenes, phenols, guaiacols, and syringols.

substituted phenols are peaks 18, 20, 23, 24, and 29, the guaiacol homolog are peaks 25, 28, 31, 32, 33, 40, 41, and 44, and the syringol homolog are peaks 39, 48, and 49. These lignin-derived compounds are also reported in prior studies for humic acids of different origins (K€ ogelKnabner, 2000; Chefetz et al., 2002). In addition to the lignin-derived compounds, pyrolysates of various 1-alkene/n-alkane doublets with carbon number ranging from 9 to 29 (Fig. 4, SIC of m=z 57) indicated their possible derivations from microorganisms and plant cutins or suberins. Other significant pyrolysates identified in this study include protein-derived nitrogen-containing compounds (Peaks 1, 2, 5, 6, and 47), carbohydrate-derived furaldehyde compounds (Peaks 4 and 13), and chlorophyll derived compounds (Peaks 52 and 53). Although the pyrolysates identified are quite similar among the eight UF HA fractions, the relative contents of the pyrolysates are very different. As the molecular weight increases from UF1 to UF8, contents of nitrogen-containing compounds and aliphatic compounds increase from 9.9% and 0.3% to 20.8% and 7.1%, respectively, whereas contents of phenols decrease from 64.5% to 42.7%. The content of benzenes has no apparent change among the eight UF fractions. Fig. 5 shows the chromatograms of m=z 57 for UF1 and UF8 for aliphatic pyrolysates. The chromatogram for UF1 has only two or three peaks, indicating very low contents of aliphatic pyrolysates. Conversely, the chromatogram for UF8 has more than 20 peaks with large peak areas, indicating very diverse and high contents of aliphatic pyrolysates. The Py-GC-MS data are consistent with elemental compositions and functionality presented above. It should be pointed out, however, that the contents of some pyrolysates measured by Py-GC-MS are lower than those calculated from the 13 C NMR spectrum for each HA sample. For example, the contents of 1-alkene/ n-alkane doublets quantified from the pyrograms are no

more than 10% for all the HA samples tested here, but the aliphatic carbon content is 33% for BHA and 18– 35% for the UF fractions based on the 13 C NMR technique. In particular, no aliphatic carbon compound was identified on the pyrogram obtained for UF1 (Fig. 5) and less than 1% of aliphatic carbon compounds could be found in UF2–UF4. Such differences between the two techniques may result from underestimation of aromaticity with the 13 C NMR technique due to the reason discussed above. In addition, incomplete pyrolysis of HA under the testing conditions might have increased such differences. It is known that humic acid can only be pyrolyzed partially due to rapid charring of the organic matter in the pyrotube. In this case, the pyrolysates only represent a fraction of the HA components and are unlikely representative of the bulk HA chemistry (SaizJimenez, 1994).

4. Conclusion This study showed that the humic acid extracted from Pahokee peat is composed of macromolecules with a range of molecular weights and chemical compositions. They can be fractionated by ultrafiltration into subsamples, each having relatively less heterogeneous properties. As the molecular cutoff of the ultrafiltration fractionated HA fraction increases, the apparent Mw increases accordingly. Chemical, spectroscopic and Py-GC-MS analyses show that, as apparent Mw increases, the HA fraction becomes less polar and less aromatic. The HA fractions with smaller Mw have much greater O/C and lower H/C atomic ratios, and higher contents of oxygen-containing functional groups. Conversely, the HA fractions with larger apparent Mw have lower O/C but higher H/C atomic ratios and lower contents of oxygen-containing functional groups. Careful assignments of peaks on PyGC-MS pyrograms show that the HAs with smaller

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L. Li et al. / Organic Geochemistry 35 (2004) 1025–1037

apparent Mw may derive from lignin materials and that the HAs with greater apparent Mw may be originated from lipid-rich biopolymers. This suggests that, even though the biogeochemical and environmental conditions remain the same, different source materials can lead to formation of chemically, structurally, and molecularly very different HA.

Acknowledgements We are grateful to Prof. Weijun Wang of the South China Agricultural University for his technical assistance and his kindness for providing equipment necessary for fractionation of humic acid. This work was financially supported by Chinese Academy of Sciences (SKL 001108), Chinese Natural Science Foundation (40133010 and 40128002), and USDA/CSREES (Grant 2001-35107-11129). Associate Editor – Jim Rice

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