Environ. Sci. Technol.
2009, 43, 7068–7073
Removal of Acetaminophen Using
Enzyme-Mediated Oxidative Coupling
Processes: II. Cross-Coupling with
Natural Organic Matter
JUNHE LU AND QINGGUO HUANG*
Department of Crop and Soil Sciences, University of Georgia,
Griffin, Georgia 30223, USA
Received January 14, 2009. Revised manuscript received
July 31, 2009. Accepted July 31, 2009.
The influence of natural organic matter (NOM) on the
transformation of acetaminophen in laccase-mediated oxidative
coupling systems was investigated in this study. It was
found that the removal of acetaminophen was enhanced
while the self-coupling of acetaminophen was suppressed in
thepresenceofdissolvedNOM,likelyresultingfromcross-coupling
betweendissolvedNOMandacetaminophen.Inadditiontocross-
coupling with acetaminophen, NOM moieties could couple
to each other upon reaction with laccase. This was evidenced
by the development of a characteristic absorbance band
centered at 472 nm. According to the rate of the absorbance
change at 472 nm, the NOM coupling reactions in four different
NOM solutions were evaluated. Apparently, the tendency of
NOM coupling reactions correlates with the tendency of
acetaminophen cross coupling with NOM in these solutions.
Possible reaction pathways of cross-coupling were explored
using guaiacol as a model NOM proxy, and the products were
extracted and analyzed with mass spectrometry (MS). The
results suggested that acetaminophen and guaiacol molecules
were cross-coupled via the formation of CsOsC bonds.
Introduction
Trace levels of human-derived pharmaceuticals and endo-
crine disrupting chemicals (EDCs) in water represent an
emerging concern to the public and research communities.
Most of these micropollutants are relatively hydrophilic which
enables them to escape the conventional water treatment
processes (1-5). As a consequence, they are released to the
environment in a continuous mode and pose a potential risk
to the aquatic ecosystem and eventually to humans (3, 6).
The water/wastewater treatment industry faces a potential
challenge to develop effective and practicable methods to
address these contaminants in order to control and reduce
their environmental exposure. Chemical oxidation and UV-
enhanced oxidation have been found effective to remove
these micropollutants (1, 4, 7-9). However, the efficiency of
these methods is often limited by the extremely low con-
centration of the contaminants (ppt or lower). In addition,
large-scale application of these strategies can be costly due
to the requirement of high energy and reagent input.
As an alternative to oxidative degradation, enzyme-
mediated oxidative coupling processes have been proposed
to be a potentially cost-effective strategy to remove certain
* Corresponding author phone: (770) 229-3302; fax: (770) 412-
4734; e-mail: qhuang@uga.edu.
7068 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 18, 2009
micropollutants. Auriol et al. tested the feasibility of using
this process to remove estrogen hormones, and demonstrated
that both the hormone compounds and the estrogen activity
at environmentally relevant levels could be effectively
removed using horseradish peroxidases (HRP) or laccase to
catalyze oxidative coupling reactions under mild conditions
(10-12). Performance of the enzyme was not significantly
affected by municipal wastewater matrices in the laccase-
catalyzed systems (11). In our previous work (13), acetami-
nophen was found to be able to actively participate in laccase-
catalyzed oxidative coupling reactions and couple to each
other to form products with varying degrees of polymerization
via a radical reaction mechanism. Our result suggests that
laccase mediates a single-electron oxidation of the substrates
yielding free radicals, and the resultant radicals can then
couple with each other by CsC bonds formation resulting
in polymeric products. The products that are soluble can
continue to serve as substrates for further oxidative coupling
and form larger polymers that can be more readily removed
from water.
Laccase-catalyzed oxidative coupling processes require
little energy and minimal reagent input, and can be easily
integrated into conventional water treatment systems without
significant capital and operational costs. In real conditions,
however, a number of factors may affect the performance of
the enzyme and the efficiency of the transformation, which
have to be fully understood prior to a rational design. How
the presence of NOM could impact such coupling reactions
of micropollutants represents an important factor to be
understood in light of the facts that (i) NOM is ubiquitous
in water and often a critical factor influencing various
physicochemical treatment processes (14); and (ii) mi-
cropollutants are usually present in waters at concentrations
orders of magnitude lower than NOM. This study was
conducted with the attempt of addressing how the presence
of NOM may impact on laccase-mediated oxidative coupling
reactions of acetaminophen.
Water-soluble NOM is primarily comprised of organic
acids that are often referred to as humic substances, which
can be operationally categorized into a relatively smaller and
more soluble fulvic acid fraction, and a larger and less soluble
humic acid fraction (14, 15). NOM, depending on origin,
contains varying amounts of aromatic moieties with various
functional substituents, including carboxyl, hydroxyl, meth-
oxyl, carbonyl, and amino groups (16). It has been suggested
that NOM is present in water as supramolecular assemblies,
collections of relatively small molecules that are self-
associated (17-21). Because NOM almost invariably contains
phenolic functionalities, it can potentially serve as active
substrates to laccase and other oxidative coupling catalysts.
Experimental evidence indicates that NOM molecules can
under appropriate conditions be effectively reconfigured at
both molecular and supramolecular levels by oxidative
coupling, yielding products with larger molecular sizes
(21-26). Molecular characterization suggested that oxidative
coupling resulted in a decrease of aromatic hydroxyl (phe-
nolic) groups and an increase of aromatic ether groups,
indicating the cross-linking of NOM moieties via CsOsC
bonds (22, 24). Cross-linking leads to a significant reduction
of the potential of the NOM to form disinfection byproducts
(DBPs) upon chlorination, since phenolic moieties in NOM
molecules are major precursors that cause DBP formation
(24). Because both NOM and acetaminophen can serve as
active substrates of laccase, we hypothesize that cross-
coupling can occur in laccase-mediated reaction systems.
This possibility was systematically investigated in the present
10.1021/es9001295 CCC: $40.75  2009 American Chemical Society
Published on Web 08/13/2009
study, with a particular aim of examining how such cross-
coupling may be dependent on NOM characteristics and
how it may impact acetaminophen removal.
Experimental Section
Reagents. All reagents were ACS grade or better. Acetamin-
ophen (98%) and guaiacol (99%) was obtained from Sigma-
Aldrich Co (St. Louis, MO). A stock solution of each at the
concentration of 1 mM was prepared and stored at 4 °C.
Laccase was also purchased from Sigma-Aldrich, and its
activity was determined immediately prior to each experi-
ment, using a photometric method as described in the
companion paper (13).
NOM. Fulvic and humic materials were kindly provided by
the Department of Chemical Engineering at the University of
Michigan which were extracted from Canadian peat and Chelsea
soil using the standard International Humic Substances Society
(IHSS) protocol up to the steps before desalting (27). Both fulvate
and humate fractions extracted from each soil were used in the
experiments as model NOM materials, which were respectively
designated as Canadian peat fulvates (CPFV), Canadian peat
humates (CPHM), Chelsea soil fulvates (CSFV), and Chelsea
soil humates (CSHM). Each model NOM was reconstituted in
0.01 M phosphate buffer (pH 7.0) and filtered through 0.45 µM
membrane to obtain NOM solutions that were used throughout
the study. The total dissolved organic carbon (DOC) content
of each NOM solution was determined using a Shimadzu 5050A
TOC analyzer (Shimadzu, Columbia, MD) and the UV absor-
bance measured with a Beckman DU640B spectrophotometer
(Beckman, Fullerton, CA). The data are given in Supporting
Information (SI) Table S1 for each NOM material. It should be
noted that the model NOM materials are of terrestrial origin
and may differ from those typically present in water or
wastewater in terms of certain property and reactivity.
Coupling Reaction. Acetaminophen coupling reactions
mediated by laccase in NOM and NOM-free control solutions
were conducted in 50 mL flasks at room temperature. Each
reactor contained 20 mL of a model NOM solution (DOC
concentrations given in SI Table S1) containing acetamin-
ophen (initial concentration 20 µM). The initial dosage of
enzyme was 1.0 unit/mL. After 1 h of reaction, 0.5 mL solution
was withdrawn from the reactor and immediately mixed with
0.5 mL methanol which was found to be sufficient to
completely denature the enzyme and stop the reaction. Blank
control samples were also prepared that contain acetamin-
ophen in NOM solutions in the absence of laccase. No
acetaminophen loss was observed in these control samples
after 1 h of incubation.
All samples were analyzed using a Shimadzu LC 20AT
HPLC equipped with a Shimadzu 20A photo diode array (PDA)
detector (Shimadzu, Columbia, MD) as described in the
companion paper (13). Acetaminophen was quantified by
the chromatogram peak areas measured by absorbance at
260 nm with an external calibration. The relative yields of
three major self-coupling products, including a dimer, a
trimer, and a tetramer were also determined based on their
respective peak areas at 260 nm normalized to the peak area
of acetaminophen before reaction. These selected products
are those shown in Figure 6 of the companion paper (13).
The Impact of Oxidative Coupling Reactions on NOM.
The oxidative coupling reactions of NOM were induced by
dosing 1.0 unit/mL laccase to 20 mL of each model NOM
solution without acetaminophen added.The reaction mixture
was then transferred to a 1 cm quartz cuvette. At each 10 min
interval over a one-hour period, the UV absorbance spectrum
of the solution in the cuvette was scanned using a Beckman
DU640B spectrophotometer.
Characterization of Cross-Coupling Products. Oxidative
coupling reactions were induced by adding 1.0 unit/mL
FIGURE 1. Yields of coupling products in laccase catalyzed
systems when acetaminophen was incubated with NOM as
cosubstrates or without NOM. Concentrations of NOM as DOC
were given in SI Table S1. Acetaminophen was initially at 50
µM, laccase was dosed at 1.0 unit/mL, pH 7.0, and reaction
time 1 h. (a) Relative concentrations of residual acetaminophen
remaining after the reaction and the relative yields of the three
major coupling products; (b) Yields of the coupling products in
relative to the total removal of acetaminophen. Error bars
represent the standard deviation of three replicates.
laccase to a 50 mL phosphate buffer solution (pH 7.0)
containing 50 µM acetaminophen and 10 µM guaiacol as a
model NOM compound. After 60 min, a few drops of
concentrated H2SO4 were added to adjust the pH to below
2. The solution was then loaded onto a C18 solid-phase
extraction (SPE) cartridge (Restek, 6 mL, 500 mg) that had
been preconditioned with 5 mL methanol at a flow rate of
10 mL/min. After loading, the SPE cartridge was eluted with
5 mL methanol and the elution was collected, dried by a
gentle nitrogen gas flow, and then reconstituted to 3 mL
with methanol. A reaction system that contained only
guaiacol but not acetaminophen was also tested following
the same procedure described above.
A Waters Micromass Quattra LC tandem mass spectrom-
eter (MS/MS) (Waters, Milford, MA) was used to characterize
the coupling products extracted by SPE. The MS was operated
in positive electronspray ionization mode (ESI+), with the
capillary potential set at 3.5 kV. Nitrogen (Airgas, >99.999%
purity) was used as nebulizer and drying gas and maintained
at flow rates of 45 and 300 L/h, respectively. Desolvation
temperature was at 350 °C and source block temperature at
100 °C. Cone voltage was set to 58 V and the extractor 4 V.
The mass analyzer was first run at scanning mode, and
secondary MS spectra were then generated for selected ions
using collision induced dissociation. The collision gas was
Ar (Airgas, >99.999% purity). Fragmentor voltages and
collision energy (CE) were experimentally optimized.
Results and Discussion
Oxidative Coupling Reactions of Acetaminophen in the
Presence of NOM. Figure 1a displays the fraction of acet-
aminophen remaining (A) after one hour reaction mediated
by laccase as well as the relative yields (Pi) of three major
self-coupling products in reaction systems that contained
different NOM and a reaction system that did not contain
NOM. It is evident that the removal of acetaminophen was
VOL. 43, NO. 18, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 7069
enhanced in the reaction systems with NOM as a cosubstrate.
Interestingly, the relative yields of the three major self-
coupling products were all suppressed in the presence of
NOM, although the total acetaminophen conversion was
increased. Based on the results, we hypothesize that NOM
can participate in the reactions. As potential substrates toward
laccase and other oxidative coupling catalysts, NOM mol-
ecules bearing phenolic functionalities can be oxidized to
form free radicals. These radicals can further react with
acetaminophen through hydrogen exchange mechanism to
convert them to free radicals, resulting in enhanced removal
of acetaminophen. In addition, NOM radicals can cross-
couple with acetaminophen radicals. Hence relatively less
self-coupling products of acetaminophen were generated
despite more acetaminophen was transformed in systems
containing NOM. To quantitatively compare this effect in
systems with different NOM, the yield of each product in FIGURE 2. Time based differential UV absorbance of CSFV
each reaction system was further normalized against the solution treated by laccase. Laccase dosage was 1.0 unit/mL,
removal ratio of acetaminophen using eq 1. pH 7.0.

Pi
Yi ) (1)
1-A

It is evident in Figure 1b that the yields of self-coupling

products relative to total transformed acetaminophen (Yi)
were significantly reduced in the presence of NOM. Among
the different reaction systems, such an effect of reduction in
self-coupling products formation seems to be the least strong
in CSHM solution while those in the other three are similar
to each other. The difference in this effect in different NOM
solutions may reflect the tendency of NOM to participate in
cross-coupling reactions dependent on the NOM concentra-
tion and reactivity. In this regard, CSHM solution appears
to be least reactive among the four tested.
Differential UV Absorbance Analysis. In addition to cross-
coupling with other contaminants, NOM molecules are
presumed to be able to couple with each other during
oxidative coupling processes (21, 22). As described in the
Experimental Section, we conducted experiments in which
each NOM solution in the absence of acetaminophen was
incubated with laccase and the UV absorbance spectrum
was scanned every ten min to probe the reaction. Differential
UV absorbance spectra, a measure that has often been used
in NOM studies, were calculated using eq 2 to quantify the
NOM spectral changes.
∆Aλ ) Aλ - Ainit
λ (2)
In eq 2, Ainit
λ and Aλ are the absorbance of light at wavelength
λ prior to and after reaction, respectively, and ∆Aλ is the
differential UV absorbance at that wavelength (28-32).
The differential UV spectra for each of the NOM solutions
show a similar pattern when incubated with laccase, as
illustrated in Figure 2 and SI Figure S1. Two prominent
features stand out in these Figures. First, no time-dependent
changes of absorbance occurred at wavelength below 320
nm, where structural features related to aromaticity is located.
The ∆Aλ at this range is primarily the contribution of enzyme
which is not included in Ainit
λ . Second, a chromophore at 472
nm was generated regardless of the origin and types of NOM,
and ∆A472 nm increased linearly with the incubation time
(Figure 3). This wavelength is within the visible range and
corresponds to long-range conjugation of electrons. The
pattern of the observed spectral changes appears to be
indicative of the coupling between NOM molecules. As noted
earlier, oxidative coupling facilitates polymerization reactions
by forming CsOsC, CsNsC, and/or CsC bonds (22, 33-36).
This process bridges aromatic rings directly or indirectly,
thus making the conjugation of electrons between two or
more aromatic rings possible. Because this process did not
FIGURE 3. Formation of ∆A472 nm against reaction time due to
the coupling of NOM molecules during laccase-catalyzed
oxidative coupling process. DOC of each of the NOM solution
is given in SI Table S1, and laccase dosage was 1.0 unit/mL,
pH 7.0. The inset plot indicates the rate of change of ∆A472 nm
for each NOM solution that is calculated by linear regression.
Error bars represents 95% confidence intervals.
break down aromatic moieties per se, it did not cause time-
dependent changes in absorbance below 320 nm. The
differential spectra exhibit greater noise around 370 nm,
which may arise primarily from the fact that the light sources
switch (UV-visible) within this range. Nonetheless, noise in
this range does not obscure the two spectral features
discussed above, which occur respectively below 320 nm
and above 420 nm.
Based on this analysis, the increase in absorbance at 472
nm likely resulted from NOM coupling. As such, the rate of
such absorbance change ∆A472 nm/∆t as shown in Figure 3
may be indicative of the relative tendency of coupling
reactions between NOM molecules in each model NOM
solution. The ∆A472 nm/∆t values related to each of the NOM
solutions, obtained by linear regression analysis of the
∆A472 nm data against time, are also presented in Figure 3.
Because the reactivity of NOM toward laccase is determined
primarily by the abundance of phenolic functionalities, which
does not necessarily correlate directly to organic carbon
content, we did not further normalize ∆A472 nm/∆t to DOC.
The ∆A472 nm/∆t data in Figure 3 in fact reflect the reactivity
of each NOM solution resulting from the collective effect of
NOM intrinsic reactivity and its quantity in the solution. The
data reveal that CSHM solution is less reactive than the other
three NOM solutions, which have similar extent of reactions.
Apparently, less NOM radicals were available in the reaction
system containing CSHM and the extent of cross-coupling

7070 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 18, 2009
FIGURE 4. MS spectra of the products resulting from laccase-catalyzed reaction systems, laccase dosage 1.0 unit/mL, pH 7.0. (a)
Guaiacol (10 µM) along as a control; (b). Acetaminophen (50 µM) incubated with guaiacol (10 µM) as NOM model compound; (c)
MS/MS spectrum of the cross-coupling products between acetaminophen and guaiacol.

with other compound would be less. This is consistent with
the data presented in Figure 1 in which the removal of
acetaminophen was less (Figure 1a) but the yields of acet-
aminophen self-coupling products relative to the total
acetaminophen removal (Figure 1b) were greater in CSHM
solution than those in the other three NOM solutions.
Reaction Pathways. It is generally believed that phenolic/
anilinic moieties are the active electron donors enabling a
substrate active toward laccase (21, 24). It may thus be
presumed that the relative reactivity of NOM with respect to
laccase catalysis is correlated to such functionalities present
in NOM molecules. Because of the complexity and uncer-
tainty of NOM molecular structures, model compounds
having well-defined structures representing the reactive
moieties in NOM molecules have been widely employed in
NOM studies (37-42). We used guaiacol in this study to mimic
the phenolic moieties in NOM molecules to elucidate the
possible cross-coupling reaction pathways. Guaiacol was
incubated in reaction systems mediated by laccase in the
presence and absence of acetaminophen. The product
solutions after one hour of reactions were extracted and
analyzed using positive ESI-MS, and the MS spectra are
presented in Figure 4. As shown in Figure 4a, a series of
products (m/z ) 489, 611, 733, 855) with a periodical
increment of mass (122) were prominent when guaiacol alone
was incubated with laccase. Such a pattern of mass distribu-
tion likely resulted from the self-coupling of guaiacol (M )
124) with 4-8 guaiacol monomer units. However, the relative
yields of self-coupling products with a lower degree of
polymerization were not significant in comparison to the
higher degree coupling products. When acetaminophen was
present in the system (Figure 4b), the self-coupling of guaiacol
was largely suppressed with the relative intensities of the
mass peaks corresponding to self-coupling products having
more than three guaiacol units significantly reduced in
comparison to a peak corresponding to dimer (m/z 247).
This suggests that the self-coupling led to primarily dimer
products with a lesser extent to form higher degree polymers,
likely resulting from cross-coupling interferences. A peak
(m/z ) 274) is pronounced in Figure 4b for the system with
acetaminophen, but is absent in Figure 4a for the system
without acetaminophen. This peak likely corresponds to a
product that resulted from cross-coupling between guaiacol
and acetaminophen. Unlike other products, the compound

VOL. 43, NO. 18, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 7071

FIGURE 5. Fragmentation pathways of the cross-coupling
products between acetaminophen and guaiacol during MS/MS
analysis.
corresponding to this peak has an odd number of molecular
weight (M ) 273), indicating that it contains an odd number
of nitrogen atoms based on nitrogen rule. The nitrogen was
apparently from an acetaminophen molecule (M ) 151)
which, if coupled with a guaiacol molecule (M ) 124) via
radical-radical mechanism, would form a product with a
molecule weight of 273.
Upon oxidation by the laccase, a substrate loses an
electron and subsequently a proton from the hydroxyl oxygen
or acetamide nitrogen to form a radical. The unpaired electron
in the nitrogen/oxygen atom tends to relocate to the
conjugated sites on the benzene ring through resonance. In
theory, a total of four resonance structures of acetaminophen
radicals and three resonance structures of guaiacol radicals
can be formed in this way as shown in SI Figure S2. Therefore,
a total of 12 isomeric products could possibly be formed via
cross-coupling between acetaminophen and guaiacol by
CsC, CsOsC, CsNsC, NsO, or OsO bonds. However,
given the energy status of different resonance structures as
well as the steric hindrance and electrostatic interactions
when two radicals approaching to each other, only some of
them are favored energetically and/or kinetically.
In order to further elucidate the structures of the cross-
coupling products, the species corresponding to the peak of
m/z 274 was analyzed by secondary MS, and the spectrum
is given in Figure 4c. It is noted that the primary MS is not
capable of differentiating isomers. The peak of m/z 274 in
Figure 4b is in fact contributed by a mixture of isomers.
Consequently, the MS/MS spectrum generated from m/z
274 (Figure 4c) represents a mixture of MS/MS spectra
originating from different isomers. Because each isomer may
have its characteristic secondary fragmentation pattern. MS/
MS spectrum in Figure 4c does provide hints as to how
acetaminophen and guaiacol are cross-coupled. One of the
principal fragments exhibits an m/z value of 200, which
corresponds to the loss of a neutral methyl acetyl molecule
(M ) 74) from the parent ion (m/z ) 274). This was most
likely to occur by eliminating the methoxyl group from the
guaiacol moiety and the acetate group from the acetami-
nophen moiety via formation of an intramolecular bond that
bridges the two aromatic rings as shown in Figure 5a. This
requires the two moieties in facile orientation that allows
them to be able to approach to each other. Based on the
above analysis, the parent ion of this fragment (m/z ) 200)
was most likely originated from a cross-coupling product
that was formed by covalently bonding between the hydroxyl
oxygen in guaiacol and one of the ortho carbons of the
acetamino group in the acetaminophen molecule. As shown
7072 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 18, 2009
in Figure 5a, this structure allows the formation of a 6-member
ring, which is a thermodynamically favorable configuration,
through the elimination of a methyl acetyl molecule during
MS fragmentation. None of the other possible configuration
seems to be able to favor such a fragmentation pathway.
Another fragment distinct in the MS/MS shown in Figure 4c
has an m/z value of 242. This fragmentation corresponds to
the loss of a neutral methanol molecule (M ) 32) from the
parent ion (m/z ) 274). As indicated in Figure 5b, such a
fragmentation pathway could happen only if the parent ion
was originated from a cross-coupling product formed via
bonding between the hydroxyl oxygen of guaiacol and one
of the ortho carbons of the hydroxyl group in the acetami-
nophen molecule. A methanol molecule can be eliminated
from such a configuration through the formation of a
6-member ring during the MS fragmentation (Figure 5b). In
summary, the cross-coupling between acetaminophen and
guaiacol is likely to proceed primarily via CsOsC bond
formation. No direct evidence supporting the presence of
other cross-coupling species is present in the MS/MS spectra.
Nonetheless, the cross-coupling through CsC, CsNsC, or
other bonds are also possible in real systems given the
diversity of the functionalities and structural properties of
NOM molecules (43-45).
Both NOM and model compound studies suggest the
possibility of formation of cross-coupling products between
NOM and acetaminophen during laccase-mediated oxidative
coupling processes. Such cross-coupling between NOM and
pharmaceuticals could very likely play a more dominant role
than self-coupling in micropollutant transformation because
typical NOM concentrations (∼mg/L as TOC in water/
wastewater) are often 10 orders of magnitude higher than
micropollutants (ppt or lower). Incorporation of the con-
taminants into NOM molecules via oxidative coupling will
lead to deactivation of the biological effects of these
contaminants, whereas the cross coupling between NOM
molecules has been proven to enhance the removal of NOM
from water (21, 22). This in fact provides a potential strategy
to remove NOM and micropollutants simultaneously in
water/wastewater treatment.
Acknowledgments
The study was supported in part by U.S. EPA STAR grant
G6M10518 and by HATCH funds. The content of the paper
does not necessarily represent the views of the funding
agencies. We thank Mr. Roger A. Pinto in the Department of
Chemical Engineering at the University of Michigan for
providing NOM samples.
Supporting Information Available
(i) Time-dependent differential UV spectra of the model NOM
upon oxidation by laccase; (ii) resonance structures of acet-
aminophen and guaiacol radicals; (iii) model NOM materials
used in the experiment. This material is available free of
charge via the Internet at http://pubs.acs.org.
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