Organic

Organic Geochemistry 35 (2004) 1025–1037

www.elsevier.com/locate/orggeochem

Geochemistry

Characterization of humic acids fractionated by ultrafiltration

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

a State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Wushan,

Guangzhou 510640, PR Chinab 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), ultravi-

olet–visible (UV–vis), and solid state 13C-nuclear magnetic resonance (13C-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.

� 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: +1-732-

932-8644.

E-mail address: whuang@envsci.rutgers.edu (W. Huang).

0146-6380/$ - see front matter � 2004 Elsevier Ltd. All rights reserv

doi:10.1016/j.orggeochem.2004.05.002

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 na-

ture, 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 sig-

nificantly affects the quality of the final portable water

ed.

1026 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 hydrody-

namic 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 ac-

cepted macromolecular model for HA, a recent model

proposed by Piccolo and his coworkers (Conte and

Piccolo, 1999; Piccolo et al., 1996, 1999, 2002) is predi-

cated on a hypothesis that HA is a supramolecular ag-

gregate of very small molecules with heterogeneous

functionality held together by weaker hydrophobic in-

teractions and hydrogen bonding. The model was pro-

posed primarily based on observations that the apparent

molecular weight of HA measured using high perfor-

mance 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. 13C

NMR and FTIR spectra indicated that the contents of

oxygen-containing and aromatic functional groups de-

crease and that the contents of aliphatic groups in-

creases, whereas the measured average apparent

molecular size (Mw) increases from 7.7 to 22.1 kDa, re-

spectively. 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 differ-

ent source materials under similar biogeochemical

conditions.

In this study, we employed an ultrafiltration tech-

nique 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 mo-

lecular 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 uti-

lized 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 (13C 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 Pigna-

tello, 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 ex-

traction lasting for 24 h. After each extraction, the

aqueous solution was separated from the solid by cen-

trifugation at 9400g. The supernatants of nine extrac-

tions 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 1027

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 so-

lution for 24 h, dialyzed against distilled water, freeze-

dried, and stored in a glass bottle.

2.2. Fractionation of HA by ultrafiltration

The BHA sample was fractionated into eight frac-

tions using a cross-flow ultrafiltration technique (Min-

itanTM, Millipore). The hydrophilic cellulose membranes

(Millipore) used had nominal molecular weight cutoffs

of 1, 3, 5, 10, 30, 100, 300 kDa. An aqueous BHA so-

lution at 600 mg/l was prepared by dissolving an ap-

propriate 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 mem-

brane 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 so-

lution. 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) pro-

duced at the beginning of the separation process, and

stored at 4 �C until completion of an entire ultrafiltra-

tion procedure. This separation process continued pro-

gressively 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 so-

lution 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-

dures for the four batches of BHA sample, the HA

fractions having the same molecular cutoffs were com-

bined 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 con-

ducted 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 stan-

dards (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 fil-

tered through a 0.22 lmmembrane before each run. The

system was operated at 1.0 ml/min, 25 �C and the in-

jected volume of sample was set at 20 ll. The wavelengthof the UV detector was set at 224 nm for standards and

at 254 nm for HA. A calibration equation,

logMw 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 polydis-

persity of HA molecules and the difference in chemical,

structural and molecular properties between the stan-

dards and HA samples.

2.3.2. Elemental composition, UV–visible, FT-IR, and 13C

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 com-

bustion procedure (Nelson and Sommers, 1982).

Spectrophotometric absorbance of the humic acid

solutions was measured on a Helios a dual-beam scan-

ning UV–vis spectrophotometer (Thermo Spectronic).

1028 L. Li et al. / Organic Geochemistry 35 (2004) 1025–1037

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

cm�1 for pellets prepared by mixing ground HA powder

(1 mg) with 60 mg KBr (FT-IR grade).

The 13C NMR spectra were recorded on a Bruker

DRX-400 NMR spectrometer operated at a 13C fre-

quency 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).

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 �Cat 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 li-

brary/literature data.

Table 1

Yields, averaged molecular-weight- (Mw), elemental composition (%),

Mass

(wt%)

Mw (kDa) Elemental comp

C N

UF1 (<1 kDa) 1.9 1.07 48.7 4.0

UF2 (1–3 kDa) 1.6 1.18 48.5 3.6

UF3 (3–5 kDa) 1.7 1.49 48.4 3.4

UF4 (5–10 kDa) 2.0 1.77 49.9 3.9

UF5 (10–30 kDa) 15.5 3.24 53.6 4.4

UF6 (30–100 kDa) 22.2 5.25 54.0 4.6

UF7 (100–300 kDa) 9.4 6.29 54.7 4.6

UF8 (>300 kDa) 45.8 18.56 57.0 5.3

BHA 10.68 56.1 3.9

BHAcalculated 10.86 55.1 4.8

3. Results and discussion

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 cut-

offs 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-molecular-

weight 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 po-

larity than soil HAs (Perminova et al., 1998).

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-

atomic ratio and E4=E6 of the UF fractions and BHA

osition (wt%) Atomic ratio E4=E6

H O H/C O/C N/C

3.3 44.0 0.81 0.68 0.070 13.6

3.5 44.3 0.87 0.69 0.064 13.2

3.8 44.4 0.94 0.69 0.060 12.6

3.9 42.3 0.94 0.63 0.067 11.7

4.1 37.9 0.92 0.53 0.070 7.0

4.3 37.1 0.96 0.52 0.073 6.1

4.4 36.2 0.96 0.50 0.072 5.7

5.3 32.4 1.12 0.43 0.080 4.5

5.0 35.0 1.06 0.47 0.060 5.8

4.7 35.5 1.02 0.48 0.075 6.0

UF1

UF2

BHA

UF8

UF7

UF6

UF5

UF4

UF3

4 6 8 10 12 14 min2 16

0.5k100k 10k 1k

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

L. Li et al. / Organic Geochemistry 35 (2004) 1025–1037 1029

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 condi-

tions between the two techniques. Ultrafiltration is op-

erated 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

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 stan-

dard, 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 aggre-

gate configurations appear in conditions of higher ionic

strength or low pH. While the solution chemistry re-

mains constant for HPSEC, the HA concentration in the

reservoir of an ultrafiltration apparatus changes over

time during the concentration mode, causing the ap-

parent 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 (Ev-

erett et al., 1999), a very important criterion for deter-

mining 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 re-

ported here. In addition, the calibration curve we es-

tablished 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

Table 2

Relative contents of different carbons in UF fractions and BHA sample calculated from 13C 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

UF1 1.7 24.5 4.6 33.9 11.2 5.3 18.7 38.5

UF2 2.0 24.1 5.5 34.3 11.4 5.1 17.6 39.8

UF3 2.1 20.6 5.3 33.5 12.3 7.6 18.7 38.8

UF4 2.1 17.6 5.2 34.0 10.7 8.3 22.1 39.2

UF5 1.6 17.4 6.8 30.3 12.8 9.3 21.7 37.1

UF6 1.8 17.0 6.6 30.1 14.5 8.9 21.1 36.7

UF7 1.9 15.8 5.8 29.3 14.9 9.3 23.0 35.1

UF8 1.6 13.2 5.0 22.0 13.5 9.8 34.9 27.0

BHA 1.6 13.9 4.7 24.2 12.6 9.7 33.3 28.9

BHAcalculated 1.7 15.6 5.7 26.7 13.6 9.3 27.6 32.4

1030 L. Li et al. / Organic Geochemistry 35 (2004) 1025–1037

wt%, respectively, whereas the HA fractions have varied

elemental compositions. As the molecular cutoff in-

creases from UF1 to UF8, the hydrogen content dis-

plays 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 ran-

ges 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 un-

ique 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, sug-

gesting 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 gradu-

ally 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 as-

signment of different peaks on a typical HA IR spectrum

includes H-bonded OH stretching (mO–H) at 3500–2500

cm�1, asymmetric and symmetric stretching vibration of

aliphatic C–H (masC–H, msC–H) from 3000 to 2800 cm�1,

carboxylic and ketonic carbonyl stretching (mC@O) at

1710 cm�1, stretching vibration of conjugated C@C

(mC@C) or H-bonded carbonyl C@O (mC@O) at 1630 cm�1,

O–H bending vibration of alcolols or carboxylic group

(dO–H) and C–O stretching of phenolic group (mC–O) at1400 cm�1, and C–O stretching vibration (mC–O) and O–

H bending deformation (dO–H) due mainly to carboxyl

groups at 1250 cm�1 (Aiken et al., 1985; Stevenson,

1994).

Further inspection of the IR spectra indicates ap-

preciable differences in resolution and strength of as-

signed peaks among the eight HA fractions. These

differences suggest that the fractions with greater mo-

lecular 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 cm�1 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 cm�1 on the same IR spectrum.

This shoulder peak is commonly assigned to C–H de-

formation vibration of aliphatic structure (dC–H). The

UF1

UF2

BHA

UF8

UF7

UF6

UF5

UF4

UF3

4000 2000 1000 cm-1

1450

2930 1710

1630

1400

1250

1180 990

1045

1100

970

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

ppm

5615230

72

130173

050100150200

UF3

UF4

UF5

UF6

UF7

UF8

WHA

UF2

UF1

Fig. 3. CP/MAS 13C NMR spectra of the eight UF fractions

and BHA.

L. Li et al. / Organic Geochemistry 35 (2004) 1025–1037 1031

peaks at 1400 and 1250 cm�1 are much stronger in the

lower molecular weight fractions than the higher mo-

lecular weight fractions, indicating higher contents of

carboxylic and other oxygen-containing groups associ-

ated with these HA fractions. Sharp peaks between 1200

and 900 cm�1 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 cm�1; (2) C–O

stretching vibration (mC–O) of carbohydrates and alco-

hols (1100 cm�1), or C–C stretching motions of aliphatic

groups (mC–C), or in-plane C–H bending of aromatic

rings (dC–H) at 1100 cm�1 (Miikki et al., 1997; Francioso

et al., 2002); (3) O–H stretching (m�OH) at 1050 cm�1;

and (4) an out-of-plane C–H deformation vibration

linked to alkenes (d@C–H) at 990 and 970 cm�1 (Aiken

et al., 1985; Pavia et al., 2001).

It should be noted that the band centered around

1050–1150 cm�1 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

cm�1 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 cm�1 may suggest that the two

peaks of 990 and 970 cm�1 should not be attributed to

phosphate salts (Pavia et al., 2001).

3.5. Solid state 13C NMR spectroscopy

The solid state 13C 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.

1032 L. Li et al. / Organic Geochemistry 35 (2004) 1025–1037

The 13C-NMR spectra were also quantitatively pre-

sented 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 re-

mains unchanged. As a result, the sum of the total ar-

omatic carbons and hence the aromaticity decreases

from about 38.5% for UF1 to 27.0% for UF8. The

Table 3

Typical pyrolytical products of Py-GC-MS

No. Compounds Origin No

1 Pyridine P 44

2 Pyrrole P 45

3 Toluene P, L 46

4 2-Furaldehyde C 47

5 Methylpyrrole P 48

6 Methylpyrrole P 49

7 Dimethylbenzene P, L 50

8 Dimethylbenzene P, L 51

9 Styrene P, L 52

10 Dimethylbenzene P, L 53

11 1-Nonene Lp 54

12 Nonane Lp 55

13 Methyl-2-furaldehyde C 56

14 Trimethylbenzene P, L 57

15 Phenol C, P, L 58

16 Trimethylbenzene P, L 59

17 Decane Lp 60

18 o-Cresol L 61

19 Guaiacol L 62

20 m/p-Cresol L 63

21 1-Undecene Lp 64

22 Undecane Lp 65

23 Dimethylphenol L 66

24 Ethylphenol L 67

25 4-Methylguaiacol L 68

26 1-Dodecene Lp 69

27 Dodecane Lp 70

28 Catechol L 71

29 Vinylphenol L 72

30 Branched Tridecane Lp 73

31 2-Methoxy-benzenediol L 74

32 Ethylguaiacol L 75

33 Vinylguaiacol L 76

34 1-Tridecene Lp 77

35 Tridecane Lp 78

36 Syringol L 79

37 1-Tetradecene Lp 80

38 Tetradecane L 81

39 Methylsyringol L 82

40 Eugenol L 83

41 Acetoguaiacone L 84

42 1-Pentadecene Lp 85

43 Pentadecane Lp

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

aromaticity of BHA is about 28.9% which is slightly

greater than UF8 but lower than the rest of UF frac-

tions. 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 de-

creases from 24.5% for UF1 to 13.2% for UF8, indi-

cating higher contents of carboxylic functional groups

for lower molecular weight HA fractions. (see Table 3).

While the data listed in Table 2 provide detailed in-

formation on the differences of functionalities among the

. Compound Origin

Propioguaiacone L

1-Hexadecene Lp

Hexadecane Lp

Diketodipyrrole P

Propenylsyringol L

Acetosyringone L

1-Heptadecene Lp

Heptadecane Lp

Prist-1-ene Lp

Prist-2-ene Lp

Tetradecanoic acid Lp

1-Octadecene Lp

Octadecane Lp

Pentadecanoic acid Lp

Pentadecanoic acid Lp

1-Nonadecene Lp

Nonadecane Lp

Branched Hexadecanoic acid Lp

Hexadecanoic acid Lp

1-Eicosene Lp

Eicosane Lp

Branched Heptadecanoic acid Lp

Heptadecanoic acid Lp

1-Heneicosene Lp

Heneicosane Lp

Octadecaoic acid Lp

1-Docosene Lp

Docosane Lp

1-Tricosene Lp

Tricosane Lp

1-Tetracosene Lp

Tetracosane Lp

1-Pentacosene Lp

Pentacosane Lp

1-Hexacosene Lp

Hexacosane Lp

1-Heptacosene Lp

Heptacosane Lp

1-Octacosene Lp

Octacosane Lp

1-Nonacosene Lp

Nonacosane Lp

L. Li et al. / Organic Geochemistry 35 (2004) 1025–1037 1033

eight HA fractions, they might be interpreted qualita-

tively because the 13C NMR data were collected at a

fixed recycle time of 1.2 s for all the HA samples. In

general, quantitative interpretations of 13C NMR spec-

tra require that the contact time should be less than the

relaxation times (T1) of all 13C atoms on a sample. Since

the relaxation times (T1) may vary among a set of sam-

3

403020 10

18

12

14 14

13

6

4

7

13

15

1+2

5

29

20

20 21

2323

28

31

3738

36

3332

19 100

rela

tive

abun

danc

e, %

50

0

C9

C10

C11

30

C12

C13

C1

403020 10

100

rela

tive

abun

danc

e, %

50

0

Fig. 4. Total ion chromatogram (TIC) and specific ion chromatogr

numbers refer to the compounds listed in Table 4. Cn refers to the ca

ples, 13C NMR data collected using a fixed contact time

may be biased.

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

min 80 706050

62

48

39

41

4044

453

54

52

TIC

min

SIC m/z57

4

C21

C15

C16 C17

C19

C20

C23

C24 C18

C22

C26

C29

C27

C28

C25

52

62

80 706050

am (SIC, m/z 57) of the pyrolysates obtained for BHA. Peak

rbon chain number of the n-alkanes.

∗ UF1

min 80 7060504030 20 10

100re

lativ

e ab

unda

nce,

%

50

0

∗100

C11 C12

C13

C14 C21C15

C16

C17 C19

C10 C20

C23

C24

C29

C18

C25 C22

C26

C27

UF8

rela

tive

abun

danc

e, %

50

0min 40 50 70 80 10 20 6030

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.

1034 L. Li et al. / Organic Geochemistry 35 (2004) 1025–1037

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 in-

dicators of lignin material. Several homologous pyroly-

sates of these compounds are also present. The aliphatic

Table 4

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

Protein Benzenes A-Phenols Guaiacols Syringols AliphaticsP

Ar

UF1 9.93 15.59 28.78 25.91 9.77 0.31 80.05

UF2 10.08 16.68 28.03 24.86 7.95 0.39 77.52

UF3 18.46 16.44 27.40 22.04 6.42 0.27 72.31

UF4 15.90 19.67 28.21 19.03 5.40 0.23 72.32

UF5 16.72 19.01 26.87 18.97 5.76 1.74 70.60

UF6 13.99 18.47 28.08 18.69 5.50 2.16 70.73

UF7 15.40 18.88 27.91 17.53 5.24 1.85 69.56

UF8 20.84 14.52 15.66 20.51 6.53 7.06 57.22

BHA 18.56 16.98 16.94 20.74 5.43 6.85 60.10

BHAcalculated 17.67 16.71 22.22 19.77 6.13 4.18 64.83

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

L. Li et al. / Organic Geochemistry 35 (2004) 1025–1037 1035

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€ogel-Knabner, 2000; Chefetz et al., 2002).

In addition to the lignin-derived compounds, pyrol-

ysates 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 microorgan-

isms and plant cutins or suberins. Other significant

pyrolysates identified in this study include protein-de-

rived 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 nitro-

gen-containing compounds and aliphatic compounds

increase from 9.9% and 0.3% to 20.8% and 7.1%, re-

spectively, whereas contents of phenols decrease from

64.5% to 42.7%. The content of benzenes has no ap-

parent 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 13C 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 13C NMR tech-

nique. 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 aroma-

ticity with the 13C 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 (Saiz-

Jimenez, 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 com-

positions. They can be fractionated by ultrafiltration

into subsamples, each having relatively less heteroge-

neous properties. As the molecular cutoff of the ultra-

filtration fractionated HA fraction increases, the

apparent Mw increases accordingly. Chemical, spectro-

scopic 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 ap-

parent Mw have lower O/C but higher H/C atomic ra-

tios and lower contents of oxygen-containing

functional groups. Careful assignments of peaks on Py-

GC-MS pyrograms show that the HAs with smaller

1036 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 condi-

tions remain the same, different source materials can

lead to formation of chemically, structurally, and mo-

lecularly very different HA.

Acknowledgements

We are grateful to Prof. Weijun Wang of the South

China Agricultural University for his technical assis-

tance and his kindness for providing equipment neces-

sary 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

References

Aiken, G.R., McKnight, D.M., Wershaw, R.L., MacCarthy, P.,

1985. Humic Substances in Soils, Sediments and Water.

John Wiley & Sons, New York.

Benner, R., Biddanda, B., Black, B., McCarthy, M., 1997.

Abundance, distribution, and stable carbon and nitrogen

isotopic compositions of marine organic matter isolated by

tangential-flow ultrafiltration. Marine Chemistry 57, 243–

263.

Chefetz, B., Salloum, M.J., Deshmukh, A.P., Hatcher, P.G.,

2002. Structural components of humic acids as determined

by chemical modifications and carbon-13 NMR, pyrolysis-

and thermochemolysis-gas chromatography/mass spectrom-

etry. Soil Science Society of America Journal 66, 1159–1171.

Chen, Y., Senesi, N., Schnitzer, M., 1977. Information provided

on humic substances by E4=E6 ratios. Soil Science Society of

America Journal 41, 352–358.

Chin, Y.-P., Gschwend, P.M., 1991. The abundance, distribu-

tion, and configuration of porewater organic colloids in

recent sediments. Geochimica et Cosmochimica. Acta 55,

1309–1317.

Chin, Y-P., Aiken, G., O’Loughlin, E., 1994. Molecular weight,

polydispersity, and spectroscopic properties of aquatic

humic substances. Environmental Science and Technology

28, 1853–1858.

Chin, Y.-P., Aiken, G., Danielsen, K.M., 1997. Binding of

pyrene to aquatic and commercial humic substancesthe role

of molecular weight and humic structure. Environmental

Science and Technology 31, 1630–1635.

Chiou, C.T., Kile, D.E., Rutherford, D.W., Sheng, G., Boyd,

S.A., 2000. Sorption of selected organic compounds from

water to a peat soil and its humic-acid and humin fractions:

Potential sources of the sorption nonlinearity. Environmen-

tal Science and Technology 34, 1254–1258.

Christl, I., Knicker, H., K€ogel-Knabner, I., Kretzschmar, R.,

2000. Chemical heterogeneity of humic substances: charac-

terization of size fractions obtained by hollow-fiber ultra-

filtration. European Journal of Soil Science 51,

617–625.

Christl, I., Milne, C.J., Kinniburgh, D.G., Kretzschmar, R.,

2001. Relating ion binding by fulvic and humic acids to

chemical composition and molecular size. 2. Metal binding.

Environmental Science and Technology 35, 2512–2517.

Conte, P., Piccolo, A., 1999. Conformational arrangement of

dissolved humic substances. influence of solution composi-

tion on association of humic molecules. Environmental

Science and Technology 33, 1682–1690.

Everett, C.R., Chin, Y.-P., Aiken, G., 1999. High pressure size

exclusion chromatography analysis of dissolved organic

matter isolated by tangential-flow ultrafiltration. Limnology

and Oceanography 44, 1316–1322.

Fan, L., Harris, J.L., Roddick, E.A., Booker, N.A., 2001.

Influence of the characteristics of natural organic matter on

the fouling of microfiltration membranes. Water Research

35, 4455–4463.

Francioso, O., S�anchez-Cort�es, S., Casarini, D., Garcia-Ramos,

J.V., Ciavatta, C., Gessa, C., 2002. Spectroscopic study of

humic acids fractionated by means of tangential ultrafiltra-

tion. Journal of Molecular Structure 609, 137–147.

Ghosh, K., Schnitzer, M., 1980. Macromolecular structures of

humic substances. Soil Science 129, 266–276.

Kilduff, J.E., Weber Jr., W.J., 1992. Transport and separation

of organic macromolecules in ultrafiltration processes.

Environmental Science and Technology 26, 569–577.

Kilduff, J.E., Karanfil, T., Chin, Y.-P., Weber Jr., W.J., 1996.

Adsorption of natural organic polyelectrolytes by activated

carbon: A high-performance size exclusion chromatography

study. Environmental Science and Technology 30, 1336–

1343.

K€ogel-Knabner, I., 2000. Analytical approaches for character-

izing soil organic matter. Organic Geochemistry 31, 609–

625.

Lakshman, S., Mills, R., Feng, F., Patterson, H., Cronan, C.,

1996. Use of fluorescence polarization to probe the structure

and aluminum complexation of three molecular weight

fractions of a soil fulvic acid. Analytica Chimica Acta 321,

113–119.

Li, L., Huang, W., Peng, P., Sheng, G., Fu, J., 2003. Chemical

and molecular heterogeneity of humic acids repeatedly

extracted from Pahokee peat. Soil Science Society of

America Journal 67, 740–746.

Mao, J.-D., Hu, W.-G., Schmidt-Rohr, K., Davies, G.,

Ghabbour, E.A., Xing, B., 2000. Quantitative characteriza-

tion of humic substances by solid-state carbon-13 nuclear

magnetic resonance. Soil Science Society of America Jour-

nal 64, 873–884.

Mao, J.-D., Hundal, L.S., Thompson, M.L., Schmidt-Rohr, K.,

2002. Correlation of poly(methylene)-rich amorphous ali-

phatic domains in humic substances with sorption of a

nonpolar organic contaminant, phenanthrene. Environmen-

tal Science and Technology 36, 929–936.

Miikki, V., Senesi, N., H€anninen, K., 1997. Characterization of

humic material formed by composting of domestic and

industrial biowastes. Part 2 Spectroscopic evaluation of

humic acid structures. Chemosphere 34, 1639–1651.

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

Nelson, D.W., Sommers, L.E., 1982. Total carbon, organic

carbon, and organic matter. in: Methods of Soil Analysis,

Part 2. Chemical and Microbiological Properties. SSSA

Agronomy Monograph no. 9. SSSA and ASA, Madison,

WI.

Pavia, D.L., Lampman, G.M., Kriz, G.S., 2001. Introduction

to Spectroscopy, third ed. Harcourt College Publishers,

Orlando.

Perminova, I.V., Frimmel, F.H., Kovalevskii, D.V., Abbt-

Braun, G., Kudryavtsev, A.V., Hesse, S., 1998. Develop-

ment of a predictive model for calculation of molecular

weight of humic substances. Water Research 32, 872–881.

Piccolo, A., Nardi, S., Concheri, G., 1996. Micelle-like confor-

mation of humic substances as revealed by size exclusion

chromatography. Chemosphere 33, 595–602.

Piccolo, A., Conte, P., Cozzolino, A., 1999. Effects of mineral

and monocarboxylic acids on the molecular association of

dissolved humic substances. European Journal of Soil

Science 50, 687–694.

Piccolo, A., Conte, P., Trivellone, E., van Lagen, B., Buurman,

P., 2002. Reduced heterogeneity of a lignite humic acid by

preparative HPSEC following interaction with an organic

acid. Characterization of size-separates by Pyr-GC-MS and1H-NMR spectroscopy. Environmental Science and Tech-

nology 36, 76–84.

Reckhow, D.A., Singer, P.C., Malcolm, R.L., 1990. Chlorina-

tion of humic materials: Byproduct formation and chemical

interpretations. Environmental Science and Technology 24,

1655–1664.

Saiz-Jimenez, C., 1994. Analytical pyrolysis of humic sub-

stances: pitfalls, limitations, and possible solutions. Envi-

ronmental Science and Technology 28, 1773–1780.

Senesi, N., Sipos, S., 1985. Molecular weight distribution,

analytical and spectroscopic characterization of humic

fractions sequentially isolated by organic solvents from a

brown coal humic acid. Organic Geochemistry 8,

157–162.

Shin, H.-S., Monsallier, J.M., Choppin, G.R., 1999. Spectro-

scopic and chemical characterizations of molecular size

fractionated humic acid. Talanta 50, 641–647.

Stevenson, F.J., 1994. Humus Chemistry: Genesis, Composi-

tion, Reactions, second ed. John Wiley & Sons,

New York.

Swift, R.S., 1996. Organic matter characterization. in: Sparks,

D.L., et al., (Ed.), Methods of Soil Analysis. Part 3:

Chemical methods. SSSA Book Series No. 5. SSSA and

ASA, Madison.

Tomb�acz, E., 1999. Colloidal properties of humic acids and

spontaneous change of their colloidal state under variable

solution conditions. Soil Science 164, 814–824.

Weber, J.H., 1988. Binding and transport of metals by humic

materials. In: Frimmel, F.H., Christman, R.F. (Eds.),

Humic Substances and Their Role in the Environment.

John Wiley & Sons, Chichester.

Xia, G., Pignatello, J.J., 2001. Detailed sorption isotherms of

polar and apolar compounds in a high organic soil.

Environmental Science and Technology 35, 84–94.

Xing, B., Pignatello, J.J., 1997. Dual-mode sorption of low-

polarity compounds in glassy poly(vinyl chloride) and soil

organic matter. Environmental Science and Technology 31,

792–799.

Yau, W.W., Kirkland, J.J., Bly, D.D., 1979. Modern Size

Exclusion Liquid Chromatography. John Wiley & Sons,

New York.