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: [email protected] (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
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