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pubs.acs.org/est

Quantification of Phenolic Antioxidant Moieties in Dissolved Organic

Matter by Flow-Injection Analysis with Electrochemical Detection
Nicolas Walpen, Martin H. Schroth, and Michael Sander*
Institute of Biogeochemistry and Pollutant Dynamics (IBP), Department of Environmental Systems Science, ETH Zurich, Zurich,
Switzerland 8092
*
S Supporting Information

ABSTRACT: Phenolic moieties in dissolved organic matter

(DOM) play important roles as antioxidants in oxidation
processes in natural and engineered systems. This work
presents an automated and highly sensitive flow injection
analysis (FIA) system coupled to both spectrophotometric and
electrochemical detection to quantify electron-donating
phenolic moieties in DOM by determining the number of
electrons that these moieties transfer to an added chemical
oxidant, the radical cation of 2,2′-azino-bis(3-ethylbenzothiazo-
line-6-sulfonic acid) (ABTS •+ ). The FIA system was
successfully validated using Trolox as a redox standard. Highest
method sensitivity was attained when combining the FIA with
chronoamperometric detection, resulting in limits of quantifi-
cation of picomolar amounts of Trolox and nanogram amounts
of DOM (corresponding to solutions with <1 mg carbon per liter). The analysis of DOM isolates showed a strong linear
correlation between the number of electrons donated and their titrated phenol contents, supporting oxidation of phenols by
ABTS•+. The broad application spectrum of the FIA system to dilute natural DOM samples was illustrated by analyzing water
samples collected from northern peatlands and by monitoring the oxidation of phenols in one peat sample upon incubation with
a phenol oxidase. The superior analytical capability of the FIA system allows quantifying phenols and monitoring phenol
dynamics in dilute DOM samples.

■ INTRODUCTION
Dissolved organic matter (DOM) is a complex mixture of low
molecular weight and macromolecular organic compounds that
originate from the incomplete degradation of plant and
microbial precursors.1,2 Among the various functional groups
in DOM are phenolic moieties (i.e., mono- or polyhydroxylated
benzene units).3−8 While their contributions to the acid−base
and the metal complexation chemistries of DOM have been
extensively studied, comparatively little is known about their
electron-donating (= antioxidant) properties in oxidation
reactions. Yet, recent studies showed that the antioxidant
properties of phenolic moieties play key roles in both pollutant
and biogeochemical redox reactions in engineered as well as
natural systems.9−12 For instance, in water-treatment systems,
phenols in DOM consume chemical oxidants that are added to
the water to remove micropollutants and pathogens.13−16 In
sunlit natural aquatic systems, phenolic moieties in DOM can
decelerate indirect photolysis of pollutants and biomolecules by
donating electrons to radical cations of these molecules formed
via DOM-sensitized photochemical oxidation, thereby reverting
the radical intermediates back to the parent compounds.17−21
In natural systems, oxidative processing of DOM may result in
a depletion of phenolic moieties.1,9,11 The oxidation of phenolic
moieties in DOM from northern peatlands has received
particular research interest: carbon sequestration in these
systems is considered to be linked to low activities of phenol
oxidases, leading to an accumulation of dissolved phenols that
inhibit the activity of hydrolases and microorganisms.22−26
Understanding the role of phenols in all of these processes
requires analytical methods to quantify these moieties and to
monitor changes in their concentrations during reactions.
Phenolic moieties in DOM can be directly quantified by
potentiometric acid−base titrations of DOM solutions.27−30
However, this approach requires large amounts of DOM (gram
quantities). Furthermore, because the approach is relatively
labor intensive and time-consuming, it is ill suited for a fast and
automated analysis. An alternative approach are reduction-
based assays (reviewed in refs 31−34), which are widely used in
physiology, pharmacology, food chemistry, and biogeochemis-
try.32−36 These assays quantify the reduction of an added
chemical oxidant by phenols in the analyzed samples. The most
commonly used assay uses the radical cation of 2,2′-azino-
bis(3-ethylbenzothiazoline-6-sulfonic acid) (i.e., ABTS•+) as an
Received: March 4, 2016
Revised: May 17, 2016
Accepted: May 26, 2016
Published: May 26, 2016

© 2016 American Chemical Society 6423 DOI: 10.1021/acs.est.6b01120

Environ. Sci. Technol. 2016, 50, 6423−6432
Environmental Science & Technology
oxidant, because it has a high water solubility and a standard
reduction potential (i.e., Eh0 (ABTS•+/ABTS) = 0.70 V)
sufficiently high to oxidize phenols. In these assays, ABTS•+
is obtained through either chemical, enzymatic, or electro-
chemical one-electron oxidation of ABTS.37−39 The reduction
of ABTS•+ to ABTS by an analyte can be quantified either
electrochemically37,38 or spectrophotometrically39 as ABTS•+
has a strong absorbance in the red (λmax = 728 nm), whereas
ABTS is colorless.40
We recently used the ABTS•+/ABTS redox couple in
mediated electrochemical oxidation (MEO), a constant-
potential amperometric technique, to quantify phenolic
moieties in DOM.8 In these measurements, the addition of
DOM samples to electrochemical cells resulted in the reduction
of preoxidized ABTS•+ to ABTS, which subsequently was
reoxidized at the working electrode (WE) of the cell to
ABTS•+. Integration of the resulting oxidative current peaks
directly provided the electron-donating capacity (EDC) of the
DOM sample (i.e., the amount of electrons transferred per
amount of DOM analyzed). The EDC of several humic
substances correlated strongly with their titrated phenol
contents, supporting the hypothesis that mainly phenolic
groups donated electrons to ABTS•+.
While the EDC values of as little as a few micrograms of
DOM could be quantified by MEO,6 the sensitivity of the
method was insufficient to analyze dilute DOM samples (i.e.,
below approximately 10 mgC L−1). Furthermore, MEO relies
on manual sample addition to the electrochemical cells with
little opportunity for automation. Finally, the MEO setup has
limited portability and hence cannot easily be used for
measurements outside the laboratory. These limitations of
MEO may, in principle, be overcome by implementing the
reduction-based approach on a flow-injection analysis (FIA)
platform. Such platforms have been successfully used to
quantify antioxidants in other disciplines, including food
chemistry.37−39,41−43 Compact, robust, and field-deployable
FIA systems can be designed that allow for automated sample
analysis.
The objective of this work was to develop and validate a FIA
system for the accurate and precise quantification of antioxidant
phenolic moieties in dilute DOM samples. The system used
ABTS•+ as chemical oxidant and spectrophotometric and
electrochemical flow cells to detect the extents of ABTS•+
reduction. The system response was validated using Trolox, a
redox standard with a known EDC, and using a series of model
DOM isolates with published phenol contents and EDC values
that were previously quantified by MEO. Finally, the FIA
system was used to quantify the EDC values of dilute DOM
samples collected from three ombrotrophic bogs and, for one of
the peat samples, to monitor changes in the phenol contents of
the peat DOM during incubation with a phenol oxidase.
■
MATERIALS AND METHODS
Chemicals. ABTS (≥98%), (±)-6-hydroxy-2,5,7,8-tetrame-
thylchromane-2-carboxylic acid (Trolox, 97%), potassium
chloride (≥99%), acetic acid (≥99.8%), and potassium
phosphate dibasic (≥99%) were obtained from Sigma-Aldrich
(Switzerland). Potassium dihydrogen phosphate was obtained
from Merck (Switzerland). Laccase from Trametes versicolor was
from Fluka (activity, 22.4 U mg−1) and used as received. The
laccase stock solution (0.745 mg mL−1) was prepared in pH
4.75 acetate buffer (1 mM).
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Organic Matter Samples. Humic substances (HS) and
natural organic matter (NOM) samples were purchased from
the International Humic Substances Society (IHSS, St. Paul,
MN) and included Suwanee River II Standard humic acid (HA)
(SRHA), Elliot Soil Standard HA (ESHA), Pahokee Peat
Standard HA (PPHAS), Leonardite Standard HA (LHA),
Suwannee River I Standard fulvic acid (FA) (SRFA I),
Suwannee River II Standard FA (SRFA II), Pahokee Peat
Reference HA (PPHAR), Nordic Lake Reference HA (NLHA),
Nordic Lake Reference FA (NLFA), Pony Lake Reference FA
(PLFA), Suwannee River I Aquatic NOM (SRNOM I),
Suwannee River II Aquatic NOM (SRNOM II), and Upper
Mississippi River Aquatic NOM (UMNOM).
Stock solutions of HS and NOM (100 mg of HS or NOM
L−1) were prepared by dissolving the respective material in 0.1
M phosphate buffer (pH 7), followed by filtration through
syringe filters (polypropylene, 0.45 μm, Pall, Switzerland). The
HS/NOM samples were diluted 20- to 50-fold to 2−5 mg HS
or NOM L−1 prior to analysis on the FIA system.
Natural dissolved organic matter (DOM) samples were
collected in July 2014 from three peat bogs in Värmland,
Sweden (i.e., Lungsmossen (LM), Norra Romyren (NR), and
Likstamossen (LK); see Figure S1 and Table S1 in the
Supporting Information for bog locations and exact sampling
coordinates). DOM samples were collected from the peat pore
water (three locations per peat, 125 cm below the peat surface)
and from open water pools in the peats (three to five pools per
peat, 40 cm (NR) or 120 cm (LM and LK) below the water
surface). All samples were immediately syringe filtered
(polypropylene, 0.45 μm), frozen, and stored at −25 °C until
analysis.
Solutions. All solutions were prepared in ultrapure water
(resistivity >18.2 MΩ cm) from a Barnstead NANOpure
Diamond system. All solutions used in electrochemical
measurements contained 0.1 M KCl. The FIA reagent and
carrier solutions (see details below) were pH-buffered with
0.001 M acetate (pH 5) and 0.1 M phosphate (pH 7),
respectively.
Characterization of Organic Matter Samples. UV−
visible light-absorbance spectra of all samples were collected
from 200 to 800 nm on a Varian Cary 100 Bio. The
nonpurgable organic carbon (NPOC) was measured on a
Shimadzu total organic carbon (TOC-L) analyzer calibrated
with a TOC standard (Sigma-Aldrich, Switzerland). Titrated
phenol contents for the IHSS HS/NOM samples were
obtained from the literature.29,30,44
MEO. Mediated electrochemical oxidation of SRHA was
conducted in a glassy carbon cylinder (volume, 9 mL; Sigradur
G, HTW, Germany) that served as the WE.45 The cylinder was
filled with pH 7 buffer (0.1 M phosphate and 0.1 M KCl) and
was polarized to Eh = +0.7 V. A platinum wire was used as a
counter electrode and separated from the WE compartment by
a porous glass frit. An Ag/AgCl reference electrode was used
(ALS, Japan). All potentials are referenced versus the standard
hydrogen electrode (SHE). The EDC values were quantified by
integrating the oxidative current peaks according to
EDC =
1
mSRHA
∫ FI dt (1)
where mSRHA is the mass of analyzed SRHA, I (A) is the
baseline-corrected current, and F (= 96 485 C mol−1) is the
Faraday constant.
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Figure 1. Scheme of the flow-injection analysis (FIA) system for quantification of the electron-donating capacity (EDC) of dissolved organic matter
(DOM) samples. The system contains two separate lines for the reagent (i.e., the oxidant ABTS•+) solution (green syringe) and the carrier solution
(blue syringe). The DOM samples are injected into the carrier-solution stream using an injector valve with a 100 μL sample loop. A third syringe
(red syringe) is used to load the sample loop. Up to 10 samples can be automatically injected using a 11-port/10-position selector valve. Following
mixing of the reagent and carrier streams in a mixing tee, the solution is passed through a 10-m long knitted open-tubular reaction coil. Reduction of
the oxidant ABTS•+ to ABTS by electron-donating moieties in DOM (bottom left panel) are detected either via current responses in an
electrochemical flow cell (bottom, middle panel) operated in chronoamperometry mode or via absorbance loss at a wavelength of λ = 728 nm in an
optical flow cell (bottom, right panel). The absorbance loss reflects the reductive decolorization of ABTS•+ (absorbance in the red) to ABTS
(colorless).

Enzymatic Oxidation Experiments. One DOM sample
from LM bog was thawed and split into six subsamples of 8 mL
each. The pH was adjusted to 4.75 by addition of concentrated
acetate buffer (0.1 M acetate; final acetate concentration, 1
mM). This pH falls within the pH range of highest laccase
activity46 and was close to the pH of the original sample (pH
4.0). Two subsamples were kept as negative controls (no
addition of laccase). Laccase was added to four of the aliquots
(final activity of 0.5 U mL−1; initial ratio of enzyme activity to
NPOC (excluding the acetate buffer) of 13.4 U mgC−1 (with
NPOC = 37.28 mgC L−1)). Two of these aliquots also received
ABTS (final concentration, 7.5 μM). All samples were
incubated on a horizontal shaker (300 rpm, 25 ± 1 °C) in
amber vials for 3 days. Aliquots of 150 μL were repeatedly
withdrawn from the subsamples and mixed with phosphate
buffer (final concentration 0.1 M phosphate, pH 7, 0.1 M KCl)
to inactivate the laccase.46 The quenched aliquots were stored
at 4 °C until analysis on the FIA system (Eh = 0.71 V; pH 7).
reduction potential of Eh = +0.82 V. The counter and reference
electrodes were the same as used in MEO of SRHA (see
above). The bulk electrolysis was terminated when 60% of the
added ABTS was oxidized (i.e., CABTS•+/(CABTS + CABTS•+) =
0.6).
The total volumetric flow rate (qV = qreagent + qcarrier) was set
to 90 μL min−1 unless indicated differently. A volumetric
mixing ratio of qcarrier/qreagent = 5 was used in all analyses to
minimize sample broadening that results from mixing the
carrier and the reagent solutions. All samples were adjusted to
have the same pH and electrical conductivity as the carrier
solution prior to injection.
Sample Selection and Injection. The carrier solution was
delivered through an injector valve connected to a 10-position
selector valve (both Cetoni, Germany) used for automated
sample injection. The injection loop (volume, 100 μL) was
filled with sample solutions using a third syringe pump (Cetoni,
Germany). The samples were introduced to the carrier stream

■ FLOW-INJECTION ANALYSIS SYSTEM

General Setup. The FIA system setup is shown schemati-
cally in Figure 1 and contains four units in sequential
alignment: solution delivery, sample selection and injection,
mixing and reaction, and detection.
Solution Delivery. Reagent and carrier solutions were
continuously delivered through the system by computer-
controlled syringe pumps (Cetoni, Germany). The carrier
solution was buffered to pH 7 (0.1 M phosphate and 0.1 M
KCl). The reagent solution contained the oxidant ABTS•+
(0.75 mM) and was buffered to pH 4 (1 mM acetate; 0.1 M
KCl), because the stability of the ABTS•+ in solution was found
to decrease with increasing pH (Supporting Information,
Figure S2). The reagent solution was obtained by bulk
electrolysis of the respective ABTS solution in a 25 mL glassy
carbon WE cylinder (Sigradur G, HTW, Germany) at a
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by redirecting the carrier stream through the injection loop.
Mixing and Reaction. The carrier and reagent solutions
were combined in a mixing tee (Vici, Switzerland). The mixed
solution had a pH of 7 due to the higher buffering capacity of
the carrier than the reagent solution. Following the mixing tee,
the solution passed through a PTFE knitted open tubular
reaction coil (length, 10 m; Biotech, Sweden) that served to
increase the reaction time in the system prior to detection.
Detection. Two complementary detection methods were
used to quantify the extents of ABTS•+ reduction: absorbance
measurements in a spectroscopic flow cell and chronoamper-
ometry in an electrochemical flow cell. Spectrophotometric
detection was conducted in a Z-flow cell with a 1 cm path
length (FIAlab Instruments, Seattle, WA), an HL-2000 light
source, and an STS-Vis detector (both Ocean Optics, Dunedin,
FL). We detected the reductive decolorization of ABTS•+ to
the colorless ABTS. The absorbance was monitored at 728 nm,
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Figure 2. (a) Change in the solution absorbance at 728 nm in the spectrophotometric detector resulting from triplicate injections of Trolox
standards with amounts ranging from 6 to 1 nmol. Inset: Linear increase in the amounts of ABTS•+ reduced, Δn ABTS•+, with increasing amounts of
injected Trolox, nTrolox. (b) Linear increase in the heights, h, of the negative absorbance peaks (shown in panel a) with increasing amounts of injected
Trolox, nTrolox, between 6 and 1 nmol. (c) Oxidative current responses in chronoamperometric detection resulting from triplicate injections of Trolox
standards with amounts ranging from 4.6 to 0.02 nmol. (d) Linear increase in the heights h of the oxidative current peaks (shown in panel c) with
increasing amounts of injected Trolox, nTrolox, between 4.6 and 0.02 nmol. LOQ and σ correspond to the limits of quantification and the
measurement noises, respectively.

the absorbance maximum of ABTS•+ (Supporting Information, analyzer (CH Instruments, Austin, TX). All reduction
Figure S3a). The EDC value of Trolox, a redox standard, was potentials (Eh) applied to the WE are reported against the
obtained by integrating the negative absorbance peak using standard hydrogen electrode. The chronoamperometric
OriginPro (version 9.1, OriginLab, Northhampton, MA) responses were analyzed using OriginPro. The EDCj value
according to (mmole‑ gC−1) of an organic matter sample j was obtained from
qV the heights of its oxidative current peak(s), following
1 A(728 nm)
EDCTrolox =
n Trolox l
∫ ε(728 nm)
dt
(2)
calibration of the detector with Trolox standards:

where nTrolox (molTrolox) is the injected amount of Trolox, qV
(mL min−1) is the total volumetric flow rate, ε(728 nm) (=
14 000 M−1 cm−1) is the molar absorption coefficient of
ABTS•+ (Supporting Information, Figure S3b), l (cm) is the
path length in the Z-flow cell, and A(728 nm) is the measured
absorbance.
The electrochemical cross-flow cell consisted of a glassy
carbon WE, a steel tube counter electrode, and a Ag/AgCl
reference electrode (all ALS, Japan) and was run in
chronoamperometry mode using a 630D electrochemical
6426
hj 1
EDCj = EDCTrolox
mj a Trolox (3)
where hj (nA) is the height of the oxidative current peak for
sample j, mj (gC,j) is the injected amount of carbon in j
determined by NPOC measurements (see above), aTrolox (nA
mmolTrolox−1) is the slope of the linear calibration curve of
oxidative current peak heights of Trolox standards versus the
respective injected Trolox amounts, and EDCTrolox (= 2 mmole‑
mmolTrolox−1) is the electron donating capacity of Trolox.
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■ RESULTS AND DISCUSSION
Spectrophotometric Detection and Validation of the
FIA System. The baseline absorbance A in the spectrophoto-
metric cell at 728 nm was relatively constant over several hours
at values of A = 1.07 (Figure 2a). Yet, the baseline absorbance
showed systematic oscillations that resulted in a relatively large
measurement noise of σ = 0.011 (see baseline in Figure 2a
labeled “0 nmol” and Figure S4a in the Supporting
Information). These oscillations possibly reflected variations
in the volumetric mixing ratios of the reagent and carrier
solutions in the mixing tee, which resulted in periodic changes
in the absolute ABTS•+ concentration entering the spectro-
photometric cell. No attempts were made to eliminate these
oscillations because they were absent in the more sensitive
electrochemical detection and, therefore, did not interfere with
the electrochemical quantification of EDC values (see below).
We validated the FIA system based on absorbance loss
resulting from injections of six different Trolox standard
solutions. Trolox was chosen because it is rapidly and
completely oxidized to its quinone structure by ABTS•+ in a
two-electron transfer reaction.8,47,48 The spectrophotometric
detection was used for validation, because, in contrast to the
electrochemical detection, it allows quantifying the total
amount of ABTS•+ molecules that were converted to ABTS
(eq 2). Triplicate injections of decreasing amounts of Trolox
from 6 to 1 nmol resulted in decreasing heights of the negative
absorbance peaks (Figure 2a). Integration of the peaks yielded
an EDC value of 2.06 ± 0.03 mole‑ molTrolox−1 (inset in Figure
2a), which was in very good agreement with the expected EDC
value of 2 mole‑ molTrolox−1.8 These values correspond to a
Trolox recovery of 103 ± 2%, thereby validating the FIA
system.
Chronoamperometric Detection. Electron transfer from
electron-donating molecules in the injected samples to ABTS•+
lowered the ratio of ABTS•+/ABTS and, as a consequence, the
Eh of the solution volume that contained the injected sample.
The decrease of the solution Eh relative to the constant Eh
applied to the WE in the electrochemical detector resulted in
current peak responses relative to the baseline current
measured in the absence of injected samples. To maximize
sensitivity, the WE was polarized to the open-circuit potential
(OCP) that was measured for the ABTS•+/ABTS ratio in the
mixed reagent and carrier solutions delivered to the electro-
chemical cell at the onset of each FIA analysis. We note that the
OCP values were between +0.71 V and +0.72 V for all
experiments, demonstrating high reproducibility in the electro-
chemical oxidation of ABTS used to generate the ABTS•+
reagent. As expected, the baseline currents increased with
increasing difference between the Eh applied to the WE and the
OCP (Supporting Information, Figure S4b,c). Polarizing the
WE to the OCP values resulted in very low baseline currents of
Ibaseline < 10 nA in the subsequent chronoamperometric
detection. While the current baselines typically showed small
drifts (<50 nA) toward more oxidative values over the course of
several hours of continuous analysis, these drifts were always
baseline corrected and hence did not affect EDC quantification.
The drifts likely resulted from a slow loss of ABTS•+ in the
reagent solution (despite its low pH of 4) over time.
In contrast to the spectrophotometric detection, no baseline
oscillations were observed in the electrochemical detection,
suggesting that mixing of the reagent and carrier solutions
resulted in constant ratios of ABTS•+ to ABTS (note that the
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current only changes if the ABTS•+/ABTS ratio changes). The
noise in the baseline current was σ = 0.39 nA (Supporting
Information, Figure S4d).
Injections of standard solutions with decreasing amounts of
Trolox from 4.6 to 0.02 nmol resulted in decreasing heights of
the oxidative current responses (Figure 2c). The highest Trolox
amount of 4.6 nmol was chosen such that the ABTS•+/ABTS
ratio after reaction with Trolox did not decrease below 0.33
molABTS•+ molABTS−1 and, therefore, that the solution Eh
decreased by at most ΔEh = 0.04 V from its initial value (i.e.,
between 0.71 and 0.72 V; see the Supporting Information,
Figure S5 for details). The heights of the oxidative current
peaks linearly increased with increasing injected amounts of
Trolox, nTrolox (Figure 2d). The linear response range covered
more than 2 orders of magnitude in nTrolox.
We note that the measured current responses resulted from
the re-equilibration of the ABTS•+-ABTS redox couple only in
the solution volume that was in direct contact with the WE and
not the entire solution volume in the detector. We
independently determined that for the electrochemical flow
cell used in this work, approximately 15% of the ABTS formed
during reaction of Trolox with ABTS•+ was reoxidized to
ABTS•+ while passing through the cell (Supporting Informa-
tion, Figure S6). For this reason, a series of Trolox standards
was analyzed in all subsequent DOM analyses to calibrate the
electrochemical detection (eq 3).
Determination of the Limits of Quantification. The
limits of quantification (LOQ) of the FIA method for both the
spectrophotometric and electrochemical detections were
determined based on the heights of negative absorbance and
oxidative current peaks of Trolox standards, using a statistical
approach:49−51
LOQ = kσ /RF (4)
where k (= 10) is a constant, σ is the noise in the baseline
readings (i.e., σ = 0.011 and 0.39 nA for the spectrophotometric
and electrochemical detection, respectively), and RF is the
response factor (i.e., RF = 0.114 molTrolox−1 and 178.65 A
molTrolox−1 for the spectrophotometric and electrochemical
detection, respectively).
The LOQ of the FIA system with spectrophotometric
detection was approximately 980 pmol Trolox (= 1.96 nmol of
transferred electrons). The LOQ for the electrochemical
detection was 22 pmol Trolox (= 44 pmol of transferred
electrons) and thus approximately 50 times lower than for the
spectroscopic detection. The much lower LOQ obtained by
chronoamperometry reflected the significantly smaller noise in
the electrochemical detection. Because of the superior
sensitivity of the electrochemical detection, it was used for all
subsequent analyses.
Analysis of SRHA by FIA and MEO. We further validated
the FIA method by quantifying the EDC of Suwannee River
humic acid (SRHA) as a model DOM. In a first step, we
systematically assessed the effect of volumetric flow rate (varied
between 90 and 270 μL min−1) on the current responses.
These flow rates corresponded to reaction times in the FIA
system from 25 min down to 8.5 min, respectively. Volumetric
flow rates above 270 μL min−1 were not tested because they
resulted in very short reaction times and an unacceptably high
backpressure (>1 bar) in the system. Volumetric flow rates
below 90 μL min−1 were omitted because they resulted in
broader current peaks and increased the intervals between
injections, thereby minimizing sample throughput. For each
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Figure 3. (a) Oxidative current responses to injections of different amounts of Suwannee River Humic Acid (SRHA) into the flow injection analysis
(FIA) system at total volumetric flow rates of 270 μL min−1 (left; reaction time tReaction= 8.5 min)) and 90 μL min−1 (right; tReaction= 25 min). (b)
Electron donating capacity (EDC) values for SRHA obtained by FIA analysis at different flow rates (from 90 to 270 μL min−1) and by mediated
electrochemical oxidation (MEO). The EDC values are given in absolute values (left ordinate) and normalized to the EDC values obtained by MEO
for integration over 60 min. Inset: Overlaid baseline-corrected oxidative current responses in MEO to triplicate additions of 10 μg of SRHA. (c)
Oxidative current responses in the FIA system to injections of Trolox calibration standards (first five peaks) and SRHA (triplicate injections each) at
amounts between 600 and 5 ng of SRHA. (d) Linear correlation between the heights of the oxidative current peaks (determined from data in panel
c) and the amount of injected SRHA, mSRHA. LOQ and σ correspond to the limits of quantification and the measurement noise, respectively.

tested flow rate, four Trolox calibration standards with amounts
between 2 and 0.1 nmol were injected prior to SRHA.
The heights of the oxidative current peaks obtained from
injections of SRHA solutions decreased linearly with decreasing
injected SRHA masses (from 400 to 300 and 200 ng) at all
tested flow rates (shown for the highest and lowest tested flow
rates of 270 and 90 μL min−1 in Figure 3a). The broadening of
the current peaks from 270 to 90 μL min−1 was due to
increased time for longitudinal dispersion of ABTS and ABTS•+
during advective transport through the FIA system. While
sharper current peaks are preferred because of their higher
signal-to-noise ratios, the increase in EDC values of SRHA with
decreasing flow rates (Figure 3b) demonstrated that electron
transfer from electron donating phenolic moieties in SRHA to
ABTS•+ was incomplete at the higher tested flow rates.
Conversely, the EDC value of SRHA obtained at the lowest
flow rate was in good quantitative agreement with the EDC
value of SRHA obtained by MEO (i.e., EDCFIA ≈ 0.96
EDCMEO), which has until now been the state-of-the art
approach to determine EDC values of DOM. For comparison,
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Figure 3b also shows the baseline-corrected oxidative current
responses in MEO to triplicate injections of 10 μg of SRHA
(inset) and the corresponding cumulative numbers of electrons
transferred obtained by integrating of the current peaks up to
60 min after SRHA injections. The good agreement of EDC
values obtained by FIA and MEO confirmed accurate
quantification of the EDC values of DOM by the FIA system.
Oxidation at a slightly higher Eh in the FIA than MEO systems
(i.e., + 0.71 to +0.72 V vs +0.70 V, respectively) likely
contributed to the slightly faster reaction in FIA system
(reaction time, 25 min at 90 μL min−1) than in the MEO cell.
The quantification of EDC values fundamentally differed in
the FIA and MEO systems in that ABTS•+ reduction is
detected after it has occurred in the reaction coil whereas it is
directly monitored over time in MEO. This difference results in
very different shapes of the current responses in FIA and MEO.
The current peaks in FIA were relatively narrow (peak width of
Δt < 10 min) and symmetrical (Figure 3a,c). These peak
shapes resulted from small longitudinal dispersion during
advective transport of the samples through the FIA system.
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Environmental Science & Technology Article

Figure 4. (a) Electron donating capacities (EDC) of selected humic and fulvic acid (HA and FA) and natural organic matter (NOM) isolates used as
models for dissolved organic matter (DOM), as determined by flow injection analysis (EDCFIA; Eh= 0.71 V, pH 7) and mediated electrochemical
oxidation (EDCMEO; Eh= 0.73 V, pH 7). The EDCMEO data was taken from a previous publication.8 The brown and the blue symbols represent
DOM from terrestrial and aquatic systems, respectively. Error bars represent standard deviations of triplicate measurements. (b) EDC values of
model DOM isolates and DOM collected from three ombrotrophic bogs versus their specific UV absorbance values at 254 nm (SUVA254). The peat
DOM samples were collected from the peat pore water at 125 cm depth (dark green symbols) and from open water pools within the bog at 40 (NR)
or 120 cm (LK, LM) depth (light green symbols). Error bars represent standard deviations of triplicate measurements. (c) Comparison of EDC
values from tested DOM samples after normalization to SUVA254. The error bars for HS/NOM isolates (blue and brown bars) represent standard
deviations of triplicate measurements. For peat DOM sample data, the error bars represent the standard deviations among the EDC averages shown
in panel b (either n = 3 or 5 average EDC values, as specified on the data bars). (d) Changes in the EDC values of a selected peatland DOM (peat
pore water from LM) incubated at pH 4.75 without laccase (red symbols; control), with laccase (blue symbols) and with laccase and ABTS as
electron transfer mediator (green symbols). Shown also are selected oxidative current peak responses for samples from each of the incubations for
four selected incubation times. All EDC values were calibrated with five injections of Trolox standards. Error bars represent ranges of duplicate
incubations.

Conversely, the current responses in MEO were much wider
(width of Δt = 60 min) and asymmetric, a fast initial increase in
the current to maximum values was followed by prolonged,
approximately exponential decays in the currents over time
(Figure 3b). The prolonged current decay challenges accurate
integration of small peaks that result from the analysis of small
analyte amounts.
We estimated the LOQ of the FIA system for DOM by
triplicate injections of SRHA at amounts between 600 and 5
ngSRHA (Figure 3c). Each of the triplicate injections resulted in
nearly identical current responses, highlighting the high
reproducibility of the FIA analysis. Note that the first five
6429
current peaks resulted from Tolox injections and served to
calibrate the system (from 2 to 0.092 nmol of Trolox). The
LOQ of SRHA was approximately 19 ngSRHA (calculated with σ
= 0.39 nA, and RF = 0.206 A gSRHA−1 in eq 4 (Figure 3d)),
which corresponds to an approximate NPOC concentration of
0.4 mgC L−1 (using the measured carbon content of SRHA of
0.53 gC gSRHA−1 and an injection volume of 100 μL). These low
DOC values imply that the FIA system is sufficiently sensitive
to determine the EDC values of dilute DOM samples collected
from natural systems. Such dilute DOM solutions cannot be
accurately analyzed using MEO. In fact, the addition of 600 ng
DOI: 10.1021/acs.est.6b01120
Environ. Sci. Technol. 2016, 50, 6423−6432
Environmental Science & Technology
of SRHA into the MEO cell did not result in quantifiable
current responses (Supporting Information, Figure S7).
FIA Analysis of a Diverse Set of DOM Samples. Figure
4a shows good agreement in the EDC values of 13 HS and
NOM isolates quantified by FIA (y-axis) and MEO (x-axis).8
This result demonstrates that the FIA method can be used to
quantify EDC values of DOM from very different sources. The
EDC values of the HS/NOM determined by FIA also showed a
strong linear correlation (R2 = 0.94) with their titrated phenol
contents29,30,44 (Supporting Information, Figure S8a), strongly
supporting that phenolic moieties in DOM donated electrons
to ABTS•+.8
The EDC values of the model DOM quantified by FIA are
replotted in Figure 4b versus their specific UV absorbance
values at 254 nm (SUVA254). This parameter is linearly
correlated to the reported aromaticity values of these DOM (R2
= 0.97, Figure S8b). Combining this information with the linear
correlation of EDC values to the phenol contents, Figure 4b,c
suggests that electron-donating phenolic moieties in DOM per
unit aromaticity (i.e., SUVA254) decrease from aquatic to
terrestrial materials. This trend was previously reported and
explained by a depletion of phenolic moieties in the older, more
oxidatively processed terrestrial DOM than in the younger, less
processed aquatic DOM.8,52
DOM samples collected from the pore waters and open-
water pools of three ombrotrophic bogs had comparatively high
EDC values between 5.34 mmole‑ gC−1 and 7.14 mmole‑ gC−1
(green points, Figure 4b). When normalized to SUVA254 values
(from 3.45 to 4.02 L mgC−1 m−1; see Table S2 for DOC and
absorbance values used in the calculation of SUVA254 values),
the peat EDC values were higher than those of the aquatic and
terrestrial HS/NOM isolates (Figure 4c). The concentrations
of iron and sulfide in the peat samples were too small for these
species to have had major contributions to the measured EDC
values (Table S2, Supporting Information). The finding of
comparatively high EDC values for peat DOM is consistent
with the overall high contents of phenols in peatlands and their
low extents of oxidative transformation due to anoxic
conditions that prevail in the permanently water-saturated
pores of these systems.53−55 Interestingly, the lower SUVA254-
normalized EDC values of peat-pool than peat-pore water
possibly reflected more extensive oxidation of DOM in the oxic
peat pools as compared with the anoxic peat-pore water. The
analysis of peat DOM demonstrates that the FIA system allows
quantifying EDC values of DOM from natural sources.
Furthermore, the data suggests that EDC measurements by
FIA can be used to monitor the extents of oxidative processing
of DOM in natural systems.
Enzymatic Oxidation of Phenols in Peatland DOM. To
demonstrate that the FIA system can be used to monitor the
oxidation of phenolic moieties in DOM, we incubated one of
the peat pore-water samples from LM with laccase from
Trametes versicolor and monitored the resulting change in EDC
values over 3 days. As expected, the EDC values of control
DOM samples (i.e., no laccase added) stayed approximately
constant during the incubation (Figure 4d; red squares).
Conversely, the EDC values of the DOM incubated with
laccase decreased by nearly one-third over 2 days and thereafter
remained approximately constant (blue lines). Addition of
minute amounts of ABTS to samples containing laccase
facilitated the initial decrease in the EDC values of the DOM
(green lines), consistent with mediated oxidation of the
phenolic moieties by ABTS•+ formed via the oxidation of
6430
Article
ABTS by laccase (i.e., ABTS is a known substrate for laccase56
and is oxidized by the copper atoms in the active site of the
enzyme57,58). The final decrease in EDC values in the presence
of ABTS and laccase (i.e., 38%) was, however, only slightly
larger than in the systems containing laccase but no ABTS,
suggesting that most phenolic moieties in the DOM were
directly oxidizable in the active site of the enzyme.
The considerable EDC values that remained after laccase
treatment likely resulted from the significantly lower solution
pH during incubation (pH 4.75) than in the FIA system for
EDC quantification (pH 7.0). Because increasing pH favors
phenol oxidation both thermodynamically and kinetically,59 the
DOM likely contained a subpool of phenolic moieties that were
oxidizable by ABTS•+ at pH 7 but not oxidizable by laccase or
ABTS•+ at pH 4.75. We note that the FIA analysis could not be
conducted at pH 4.75 because laccase from the incubation
samples would have oxidized ABTS at this pH and hence
interfered with the EDC analysis. Conversely, laccase is inactive
at pH 7 and hence did not interfere with the EDC
quantification at this pH.
■ IMPLICATIONS
This work presents a highly sensitive FIA system with
chronoamperometric detection for the automated quantifica-
tion of electron donating phenolic moieties in DOM. The very
low LOQ of this method (i.e., around 20 ngDOM) allows
quantifying EDC values for natural samples with NPOC
concentrations below 1 mgC L−1. As such, this work largely
advances the analytical capabilities to characterize the redox
properties of DOM. The superior sensitivity of the FIA method
compared to any previously published method is in large part
due to using an electrochemical flow cell detector with low
background current noise.
The FIA system allows characterizing the redox properties of
DOM in dilute samples using only very small solution volumes.
As such, this technique opens new possibilities to monitor
changes in the phenol contents of DOM in oxidation processes
at high temporal and spatial resolution. We anticipate that
future work will employ the FIA system to monitor changes in
the EDC values of DOM during its enzymatic, photochemical,
or chemical oxidation. Direct analytical access to the dynamics
in DOM phenol pools will contribute to a more holistic
understanding of their role in oxidation processes in both
natural and engineered systems.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.est.6b01120.
Additional information and data on peat sampling
locations and peat DOM properties, the FIA system,
the FIA system, the redox properties of the ABTS•+/
ABTS couple, and the EDC values of model HS and
NOM isolates (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*Phone: +41 (0)44 632-8314; fax: +41 (0)44 633-1122; e-mail:
michael.sander@env.ethz.ch.
Notes
The authors declare no competing financial interest.
DOI: 10.1021/acs.est.6b01120
Environ. Sci. Technol. 2016, 50, 6423−6432
Environmental Science & Technology
■ ACKNOWLEDGMENTS
The project was funded by the Swiss National Science
Foundation (Project 200020_159692). We thank ETH Zurich
for funding the FIA system via the Scientific Equipment
Program. We further thank L. Klüpfel, K.H. Jacobson, P. Nauer
(all ETH Zurich), M. Lau (IGB Berlin), D. Cervenka, and R.
Sander for support during DOM sample collection, L. Klüpfel
for helpful discussions, and Ashley Brown (Eawag) for the
quantification of total iron in the peat DOM samples.
■
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6432 DOI: 10.1021/acs.est.6b01120

Environ. Sci. Technol. 2016, 50, 6423−6432