FeII oxidation by molecular O2 during HCl extraction

Abstract

Environmental context
In the environment, iron exists mainly as FeII and FeIII and plays an important role in biogeochemical processes. The FeII and FeIII content is often quantified by hydrochloric acid extraction and the acid is thought to prevent FeII oxidation by oxygen. However, we found that with increasing HCl concentration and temperature, oxidation of FeII by oxygen is accelerated. Therefore, in order to obtain reliable results extractions should be performed with dilute HCl or in the absence of oxygen.

Abstract
HCl is commonly used to stabilise FeII under oxic conditions and is often included in Fe extractions. Although FeII oxidation by molecular O2 in HCl is described in the field of hydrometallurgy, this phenomenon has not been systematically studied in environmentally relevant systems. The extent of FeII oxidation by O2 during extraction of soils and magnetite by HCl and in HCl/FeCl2 solutions was therefore quantified. FeII was stable in 1 M HCl at room temperature for several days, whereas in 6 M HCl at 70°C, 90% of the FeII was oxidised within 24 h. In the absence of O2, no FeII oxidation occurred. Experiments at low pH with increasing H⁺ or Cl– concentration alone and geochemical modelling suggested that the formation of complexes of FeII and HCl may be responsible for the observed FeII oxidation. The use of strictly anoxic conditions for Fe extraction by HCl to obtain reliable Fe redox speciation data is therefore recommended.

Full text

Fe
II
oxidation by molecular O
2
during HCl extraction
Katharina Porsch
A
,
B
and Andreas Kappler
A
,
C
A
Geomicrobiology, Center for Applied Geosciences, University of Tuebingen,
Sigwartstrasse 10, D-72076 Tuebingen, Germany.
B
Present address: Helmholtz Centre for Environmental Research – UFZ,
Department of Bioenergy, Permoserstrasse 15, D-04318 Leipzig, Germany.
C
Corresponding author. Email: andreas.kappler@uni-tuebingen.de
Environmental context. In the environment, iron exists mainly as Fe
II
and Fe
III
and plays an important role
in biogeochemical processes. The Fe
II
and Fe
III
content is often quantified by hydrochloric acid extraction
and the acid is thought to prevent Fe
II
oxidation by oxygen. However, we found that with increasing HCl
concentration and temperature, oxidation of Fe
II
by oxygen is accelerated. Therefore, in order to obtain reliable
results extractions should be performed with dilute HCl or in the absence of oxygen.
Abstract. HCl is commonly used to stabilise Fe
II
under oxic conditions and is often included in Fe extractions. Although
Fe
II
oxidation by molecular O
2
in HCl is described in the field of hydrometallurgy, this phenomenon has not been
systematically studied in environmentally relevant systems. The extent of Fe
II
oxidation by O
2
during extraction of soils
and magnetite by HCl and in HCl/FeCl
2
solutions was therefore quantified. Fe
II
was stable in 1 M HCl at room temperature
for several days, whereas in 6 M HCl at 708C, 90% of the Fe
II
was oxidised within 24 h. In the absence of O
2
,noFe
II
oxidation occurred. Experiments at low pH with increasing H
þ
or Cl

concentration alone and geochemical modelling
suggested that the formation of complexes of Fe
II
and HCl may be responsible for the observed Fe
II
oxidation. The use of
strictly anoxic conditions for Fe extraction by HCl to obtain reliable Fe redox speciation data is therefore recommended.
Additional keywords: abiotic oxidation, biogeochemistry, geomicrobiology, iron minerals, soil extraction.
Introduction
In the environment, Fe mainly exists in the two redox states Fe
II
and Fe
III
and undergoes abiotic and microbial redox processes.
At neutral pH, Fe
II
can be oxidised abiotically by MnO
2
,NO
2

,
molecular O
2
and H
2
O
2
.
[1–4]
Fe
III
can be reduced chemically
under anoxic conditions e.g. by hydrogen sulfide, the superoxide
radical HO
2

or different organic compounds such as hydro-
quinones in humic substances.
[4–6]
Both, Fe
II
and Fe
III
can be
microbially oxidised and reduced in anoxic and microoxic
habitats such as aquifers, sediments and soils.
[7,8]
The miner-
alogy of the Fe precipitates formed by abiotic and microbial
redox processes strongly depends on geochemical conditions
such as pH, the presence of other ions and of humic substances,
as well as on Fe oxidation and reduction rate.
[9–13]
The mineralogy and crystallinity of the Fe precipitates
formed during these reactions affect the fate of organic and
inorganic nutrients and pollutants in the environment. Arsenic,
for example, co-precipitates during Fe
III
mineral formation and
is released when these minerals are dissolved.
[14,15]
Furthermore
arsenic can undergo redox reactions with Fe phases, such as Fe
II
sorbed to goethite.
[16]
The reactivity and therefore the identity of
the Fe minerals involved in these redox processes ultimately
control the fate of the arsenic. In order to identify and quantify
microbial and abiotic Fe mineral (trans)formation and Fe redox
processes, the concentrations of the different Fe redox species
and Fe minerals present have to be quantified in a reliable and
reproducible manner.
Different methods are used to quantify Fe redox species and
to characterise Fe mineralogy, e.g. X-ray diffraction (XRD),
Mo¨ssbauer spectroscopy or X-ray absorption spectroscopy.
[17]
As these methods require sophisticated analytical equipment,
wet chemical Fe extraction methods are also commonly used.
Extraction protocols vary in type of extraction agent, incubation
time, temperature, presence or absence of light, and shaking
of the sample.
[18–20]
Different fractions of Fe minerals are
dissolved by different extraction agents, e.g. ‘ion-exchangeable
Fe’ by 1 M MgCl
2
(pH 7), ‘adsorbed Fe’ and Fe carbonates
by 1 M Na-acetate (pH 5), ‘low crystalline Fe minerals’ by
0.5–1 M HCl or hydroxylamine-HCl, and ‘highly crystalline Fe
minerals’ by 5–12 M HCl or dithionite.
[18–22]
If extraction
agents with circumneutral pH are applied, the presence of O
2
must be excluded to avoid Fe
II
oxidation by O
2
. It is generally
believed that extraction with HCl circumvents this problem, as
Fe
II
oxidation is reported to be very slow at pH ,3.
[4]
However,
extraction of Fe-containing soils with 6 M HCl performed in our
laboratory (Fig. 1a,b) revealed that significant Fe
II
oxidation
occurred in the presence of O
2
even at acidic pH. Fe
II
oxidation
by molecular O
2
in HCl of high concentration has already been
described in the field of hydrometallurgy. Several studies
determined the kinetics of this process and influencing factors
such as O
2
concentration, temperature, and effect of catalysts
under defined conditions.
[23–25]
Most of these studies were
performed with pure FeCl
2
solutions, pure O
2
and under con-
stant stirring, conditions which are usually not used for Fe
extraction procedures of environmental samples or samples
from biogeochemical Fe mineral (trans)formation experiments.
Hence, the aim of our study was (i) to determine the extent of
Fe
II
oxidation during widely used HCl extraction procedures of
CSIRO PUBLISHING
K. Porsch and A. Kappler, Environ. Chem. 2011,8, 190–197. doi:10.1071/EN10125 www.publish.csiro.au/journals/env
ÓCSIRO 2011 1448-2517/11/020190190
Research Paper

environmentally relevant samples, (ii) to identify the key factors
controlling Fe
II
oxidation under these conditions, and (iii) to
define conditions under which Fe
II
oxidation is minimised
during HCl extraction.
Experimental methods
Fe minerals and chemicals
Magnetite was purchased from Lanxess GmbH, Germany. It
was pure according to XRD (Bruker D8 Discover X-ray dif-
fraction instrument, Bruker AXS GmbH, Karlsruhe, Germany)
and had a Fe
II
/total Fe (Fe
tot
) ratio of 0.27 determined by
Mo¨ssbauer spectroscopy (WissEl – Wissenschaftliche Elek-
tronik GmbH, Starnberg, Germany; for details of Mo¨ssbauer
spectroscopy see Hohmann et al.
[14]
). FeCl
2
stock solutions of
90 mM (.93% Fe
II
) were prepared by dissolving FeCl
2
4H
2
Oin
1 M HCl and were stored at 48C in the dark. 1–6 M HCl solutions
were obtained by dilution of a 37% HCl solution with de-ionised
water. A 95–97% H
2
SO
4
solution was diluted with de-ionised
water to 3 M.
Soil sampling and characterisation
Top soil (,20 cm) was sampled from Waldenbuch (Wabu)
and the Schoenbuch forest (Sbu) (both located in south-west
Germany). The soil was stored in plastic bags at 48C in the dark
until further use. For experiments the soil fraction ,2 mm was
used. A detailed characterisation of the soils is given in Table A1
of the Accessory publication.
Fe extraction from soils under oxic conditions
Field moist soil (0.5 g) was extracted with 25mL of 1–6 M HCl
in closed 60-mL serum bottles at 708C in a water bath in the
dark for 24 h. After short mixing, 1.8 mL of the suspension was
sampled and centrifuged (Centrifuge 5417C, Eppendorf AG,
Hamburg, Germany) for 15 min at 20 817gand room tempera-
ture (258C) and Fe
II
and Fe
tot
concentrations were quantified in
the supernatant. In the experiment with 6 M HCl, Fe
II
and Fe
tot
concentrations were followed over time. Before sampling, the
bottles were taken from the water bath, mixed and left for 5 min
to allow soil particles to sediment. From the liquid, 0.5-mL
samples were taken, centrifuged as described above and Fe
II
and
Fe
tot
were quantified in the supernatant.
Fe
II
oxidation experiments under oxic conditions
In order to determine the influence of HCl concentration,
temperature and proton (H
þ
) and chloride (Cl

) concentrations
on Fe
II
oxidation, an FeCl
2
solution (8–9 mM) or magnetite
(9–10 mM Fe
tot
) were incubated in closed 23-mL test tubes with
a headspace of air at 708C in a water bath for 24 h in the dark
in duplicates or triplicates (Table A2 of the Accessory publi-
cation). In order to determine the influence of HCl concentra-
tion, 9–10 mL of 1–6 M HCl was added to FeCl
2
and magnetite
respectively. The initial phase of Fe
II
oxidation was investigated
in short-time experiments at 708C with maximum incubation
times of 15 min (FeCl
2
in 1–6 M HCl), 30 min (magnetite in
5–6 M HCl), and 60 min (magnetite in 4M HCl). For incubation
times #15 min, the samples were mixed every minute. For
incubations times .15 min, the samples were mixed once dur-
ing incubation. After incubation, 2 mL of each sample were
taken and aliquots of 100 mL were immediately diluted 1 : 10
with 1 M HCl. In order to test the effect of storage at room
temperature, the remaining 1.9 mL of selected samples (samples
taken after 2.5 min for FeCl
2
in 1–6 M HCl, after 15 min for
magnetite in 4 M HCl and after 5 min for magnetite in 5–6 M
HCl) were incubated undiluted at room temperature in the dark
and Fe
II
and Fe
tot
were followed over time (Fig. A1 of the
Accessory publication). In order to differentiate between
the effects of H
þ
and Cl

, FeCl
2
and magnetite were incubated
at 708C for 24 h either with 10 mL of 3 M H
2
SO
4
or with 1.17 g
of NaCl (end concentration ,2 M) in 10 mL of 1 M HCl.
Samples from all experiments were immediately diluted 1 : 10
with 1 M HCl after incubation and analysed for Fe
II
and Fe
tot
.
Fe oxidation experiments under anoxic conditions
In order to determine if Fe
II
in HCl is oxidised by molecular O
2
,
soil Sbu, FeCl
2
and magnetite were incubated in 6 M HCl for
24 h at 708C in an anoxic glovebox (M. Braun Inertgas-Systeme
GmbH, Garching, Germany, 100% N
2
). All solutions were made
anoxic by purging with N
2
. As the experiments were performed
in a glovebox (100% N
2
) with anoxic solutions, O
2
was present
neither in the solutions nor in the headspaces.
0
0
0
100
200
300
Fetot FeII Oxic Anoxic
400
10
20
30
Sbu
Wabu
Oxic cond.
Oxic cond.
0 4 8 12 16 20 24
0
13
HCI concentration (M)
4566
4812
Extraction time (h)
16 20 24
100
Fetot (µmol g⫺1 wet soil)Fe (µmol g⫺1 wet soil) FeII (µmol g⫺1 wet soil)
200
300
400
(a)
(b)
(c)
Fig. 1. (a) Fe
tot
and (b) Fe
II
extracted from Schoenbuch forest (Sbu) (&)
and Waldenbuch (Wabu) (J) soils with oxic 6 M HCl at 708C under oxic
conditions over time. At each time point, a sub-sample was taken from the
extraction bottles. (c) Fe
tot
and Fe
II
extracted from soil Sbu with oxic and
anoxic 1–6 M HCl for 24 h at 708C under oxic and anoxic conditions. Data
for oxic 6 M HCl are the same as in (a) and (b) at 24 h. (a–c) Bars indicate the
range of duplicates (oxic conditions) or the standard deviation of triplicates
(anoxic conditions).
Fe
II
oxidation by O
2
in HCl
191

Analytical methods
Fe
II
and Fe
tot
concentrations in the extracts and of the FeCl
2
stock solutions were determined by the ferrozine assay (see
Hegler et al.
[26,27]
). The initial Fe
II
concentration of defined
Fe-HCl mixtures before Fe
II
oxidation was calculated from the
measured Fe
tot
concentration based on the determined initial
Fe
II
/Fe
tot
ratio of the FeCl
2
solutions and magnetite. The Fe
II
recovery after Fe
II
oxidation was defined as the ratio between
the Fe
II
measured in the experiments and the calculated initial
Fe
II
concentration.
Geochemical equilibrium calculation
The chemical speciation in anoxic HCl-FeCl
2
solutions at 25
and 708C in the absence and presence of NaCl was calculated
using the REACT module of ‘The Geochemist’s Workbench 6.0’
package (RockWare, Inc., Golden, CO, USA) and the ‘thermo’
database (for details see the ‘Geochemical equilibrium calcu-
lation’ section of the Accessory publication).
Results
Oxidation of Fe
II
in soils during Fe extraction with HCl
Two different soils (Table A1 of the Accessory publication)
were extracted with oxic 6 M HCl at 708C for 24 h under oxic
conditions (Fig. 1a,b). Fe
tot
quantification over time showed that
after 1 h, more than 90% of the Fe
tot
was already extracted rel-
ative to the maximum extractable Fe
tot
concentration that was
obtained after 21.5 h (275.0 10.5 (Sbu) and 347.1 23.0
(Wabu) mmol Fe per g wet soil). In contrast, the Fe
II
concen-
tration was highest after 1 h (28.1 2.9 (Sbu) and 14.8 0.1
(Wabu) mmol Fe
II
per g wet soil) and decreased significantly
over time. After 24 h, only 24.5 1.9% (Sbu) and 22.4 6.5%
(Wabu) of the Fe
II
content measured after 1 h were detectable.
As the Fe
tot
concentration did not decrease over time, the
observed Fe
II
decrease suggests that Fe
II
oxidation occurred in
6 M HCl.
In order to determine the influence of the HCl concentration
on Fe
II
oxidation, soil Sbu was extracted under oxic conditions
for 24 h at 708C with oxic 1–6 M HCl (Fig. 1c). The use of
3–6 M HCl extracted a similar Fe
tot
concentration, whereas with
1 M HCl the Fe
tot
concentration was slightly lower. In contrast,
the highest Fe
II
concentration was found in extracts with
1 M HCl (161.3 4.3 mmol Fe per g wet soil). The Fe
II
concen-
tration decreased with increasing HCl concentration and the
measured concentration of Fe
II
in 6 M HCl was only 4% of that
measured in 1 M HCl. Building on these results with soils, two
defined Fe
II
-containing compounds, an FeCl
2
solution and the
mixed Fe
II
–Fe
III
–mineral magnetite, were used to quantify
abiotic Fe
II
oxidation depending on (i) the presence of molecular
O
2
, (ii) HCl concentration, (iii) Cl

v. H
þ
concentration, and
(iv) temperature (an overview of all experiments is given in
Table A2 of the Accessory publication).
Influence of molecular O
2
on Fe
II
oxidation in HCl
In order to determine if Fe
II
in HCl is oxidised by molecular O
2
present in air, an FeCl
2
solution, magnetite (Table 1) and soil
Sbu (Fig. 1c) were incubated in oxic 6 M HCl under oxic con-
ditions and in anoxic 6 M HCl under anoxic conditions, each at
708C. Fe
II
present in 8–9 mM FeCl
2
and 9–10 mM magnetite
was completely recovered under anoxic conditions whereas
under oxic conditions 90% of the Fe
II
was oxidised within 24 h.
The Fe
II
/Fe
tot
ratio determined after anoxic extraction with
6 M HCl from soil Sbu (67.4 0.3%) was similar to the Fe
II
/
Fe
tot
ratio obtained with oxic 1 M HCl (69.7 1.6%). In com-
bination with the fact that the total amount of extractable
Fe (Fe
tot
) was only slightly higher for anoxic 6 M HCl compared
with oxic 1 M HCl (Fig. 1c), this indicates that Fe
II
was not
oxidised in soil Sbu under oxic conditions in 1 M HCl.
As no Fe
II
oxidation occurred in 6 M HCl under O
2
-free
conditions for any of the three Fe
II
-containing samples, we
concluded that (i) Fe
II
is stable in HCl under anoxic conditions
and (ii) Fe
II
in HCl of high concentration is oxidised by
molecular O
2
. In order to rule out that during flushing of the
HCl with N
2
for deoxygenation, HCl outgassed and a lower HCl
concentration was responsible for the absence of Fe
II
oxidation
under anoxic conditions, magnetite was extracted for 24 h at
708C with deoxygenated 6 M HCl under oxic conditions. For
this purpose deoxygenated HCl was added to magnetite in test
tubes outside the glovebox, so that O
2
was present in the
headspace and diffused back into solution. After the incubation,
only 2.8 2.2% of the Fe
II
was recovered demonstrating that the
HCl concentration was high enough to allow significant Fe
II
oxidation.
Effect of HCl, H
1
and Cl
2
concentration on abiotic
Fe
II
oxidation
The effect of HCl concentration on Fe
II
oxidation was deter-
mined by incubation of dissolved FeCl
2
and dissolution of
magnetite in oxic 1–6 M HCl for 24 h at 708C under oxic
Table 1. Recovery of Fe
tot
and Fe
II
from 8]9 mM dissolved FeCl
2
and 9]10 mM magnetite in different HCl and H
2
SO
4
solutions after 24 h at 708C
The Fe
II
recovery was calculated based on the measured Fe
tot
concentration and the stoichiometry of the Fe phases, mean s.d. are given, n¼3, dash (–) means
that these set-ups were not tested. Oxic means that O
2
was present in solution and headspace (air), anoxic means that no O
2
was present in the solution and
headspace
Set-up O
2
FeCl
2
Magnetite
Fe
tot
(%) Fe
II
(%) Fe
tot
(%) Fe
II
(%)
1 M HCl Oxic 101.7 0.1 92.1 1.8 93.3 12.7 101.3 2.0
2 M HCl Oxic – – 103.4 6.4 85.8 0.4
3 M HCl Oxic 99.3 1.3 59.2 2.4 103.2 6.5 60.5 1.2
4 M HCl Oxic – – 94.4 12.3 34.0 7.0
5 M HCl Oxic – – 95.4 9.7 19.6 8.7
6 M HCl Oxic 99.9 3.0 9.9 2.3 96.0 2.4 10.7 8.7
6 M HCl Anoxic 104.4 1.8 100.3 2.0 97.7 36.5 97.5 2.9
3MH
2
SO
4
Oxic 102.5 4.1 90.9 3.2 101.5 1.6 99.4 0.9
1 M HCl þ2 M NaCl Oxic 99.5 2.3 80.4 0.4 79.9 4.6 88.9 1.6
K. Porsch and A. Kappler
192

conditions (Table 1). At all HCl concentrations, Fe
tot
from
magnetite and FeCl
2
was recovered to $93%. The magnetite
used dissolved completely in 1 M HCl and 101.3 2.0% of the
expected Fe
II
was recovered, indicating that no Fe
II
oxidation
occurred. However, in 2 M HCl, a significant fraction of Fe
II
was oxidised as the Fe
II
recovery was only 85.8 0.4%. The
Fe
II
concentration decreased further with increasing HCl con-
centration and with 6 M HCl, only 10.7 8.7% of the Fe
II
was
recovered. For FeCl
2
in 1, 3 and 6 M HCl, similar results were
obtained. But in contrast to magnetite, some Fe
II
was oxidised in
1 M HCl after 24 h with a Fe
II
recovery of 92.1 1.8%.
In order to determine whether high concentrations of H
þ
or
Cl

cause Fe
II
oxidation, FeCl
2
and magnetite were respectively
incubated and dissolved under oxic conditions either with oxic
3MH
2
SO
4
or with oxic 1 M HCl containing 2 M NaCl for 24 h
at 708C (Table 1). A 3-M solution of H
2
SO
4
has a free H
þ
concentration of ,3 M, and also dissolved the magnetite. After
24 h, 90.9 3.2% and 99.4 0.9% of the total Fe
II
concentra-
tion of FeCl
2
and magnetite were recovered respectively, similar
to the results obtained for 1 M HCl. This indicates that no or
only minor Fe
II
oxidation took place. When 1 M HCl with
2 M NaCl was used for extraction, the Fe
II
recovery for FeCl
2
and magnetite lay between the recoveries obtained for 1 and
3 M HCl. These results indicate that the increasing Fe
II
oxidation
observed with increasing HCl concentration is neither triggered
by the increasing H
þ
concentration nor the increasing Cl

concentration alone.
Kinetics of O
2
-dependent abiotic Fe
II
oxidation in HCl
In order to determine the kinetics of Fe
II
oxidation at 708C, the
recovery of Fe
II
from FeCl
2
and magnetite was quantified over
time in short-time experiments with oxic HCl and air as head-
space (Fig. 2a,b). Set-ups with FeCl
2
were incubated for up to
15 min, and set-ups with magnetite for a maximum of 60 min.
For both FeCl
2
and magnetite, the Fe
II
recovery decreased over
time with a faster decrease at higher HCl concentrations. For
FeCl
2
in 1 M HCl, Fe
II
was completely recovered at each time
point, indicating that no Fe
II
oxidation took place within the first
15 min (Fig. 2a). For FeCl
2
in 3 and 4 M HCl a slight decrease
in the Fe
II
concentration was observed, whereas the decrease in
5 and 6 M HCl was already significant within 15 min. In the case
of magnetite, only 4–6 M HCl was used, as magnetite dissolved
only slowly in 1–3 M HCl and we intended to avoid a mixture of
dissolved Fe
2þ
, remaining magnetite and magnetite-sorbed Fe
II
.
In 5 and 6 M HCl, magnetite was dissolved completely within
5 min, and in 4 M HCl within 15 min. As observed for FeCl
2
,
Fe
II
oxidation was faster with increasing HCl concentration
(Fig. 2b).
Influence of sample storage at room temperature
on Fe
II
oxidation in HCl
Fe concentrations in samples from extraction procedures are
often not quantified directly after extraction, but are collected
and analysed together. To quantify Fe
II
oxidation during sample
storage, selected samples from the short-time experiments at
708C (samples taken after 2.5 min for FeCl
2
in 1–6 M HCl, after
15 min for magnetite in 4 M HCl and after 5 min for magnetite in
5–6 M HCl) were stored undiluted in the dark at room temper-
ature under oxic conditions (Fig. A1 of the Accessory publica-
tion). The Fe
II
recovery during sample storage decreased over
time, similar to what had been observed at 708C, with faster Fe
II
oxidation at higher HCl concentrations (Fig. 2c,d). However,
0
(a) (b)
(c) (d)
0 5 10 0 102030
Incubation time at 70°C (min)
40 50 60
Incubation time at 70°C (min)
Dissolved FeCI2Dissolved magnetite
15
20
40
60
80
100
0
20
40
60
80
100
6 M HCI
4 M HCI
5 M HCI
3 M HCI
1 M HCI
FeII recovery (%)
0
024681012141618
Incubation time at room temp. (days)
20
40
60
80
100
0
024681012141618
Incubation time at room temp. (days)
20
40
60
80
100
FeII recovery (%)
Fig. 2. Fe
II
recovery after incubation of (a) 8–9 mM FeCl
2
and (b) 9–10 mM magnetite in oxic HCl of
different concentrations at 708C under oxic conditions. HCl with magnetite was sampled for the first time
after complete dissolution of the mineral phase. For each time point a separate set of two parallel samples was
incubated. (c) For FeCl
2
in 1–6 M HCl the samples incubated for 2.5 min at 708C were kept at room
temperature under oxic conditions and the Fe
II
recovery was followed over time. (d) Magnetite samples that
were dissolved for 15 min in 4 M HCl at 708C and magnetite samples that were dissolved for 5 min in
5–6 M HCl at 708C were kept at room temperature under oxic conditions. The Fe
II
recovery was followed
over time. (a–d) Results are means of duplicates. Bars indicate the range of duplicates.
Fe
II
oxidation by O
2
in HCl
193

Fe
II
oxidation at room temperature was much slower compared
with Fe
II
oxidation at 708C. The Fe
tot
concentration remained
constant over time (data not shown).
Geochemical modelling of Fe
II
speciation
and undissociated HCl
In order to identify the reactive Fe
II
species that might be
responsible for the observed Fe
II
oxidation in HCl, the Fe
II
species present under anoxic conditions and their relative con-
centrations were estimated by geochemical modelling at differ-
ent Cl

concentrations at 25 and 708C with FeCl
2
as a source of
Fe
II
. In all scenarios analysed, the predominant Fe
II
species were
Fe
2þ
, FeCl
þ
and FeCl
2
(Fig. 3a,b). At 258C, the Fe
2þ
concen-
tration decreased with increasing HCl concentration, while
the FeCl
2
concentration increased simultaneously (Fig. 3a). The
FeCl
þ
concentration varied between 31 and 41% with a maxi-
mum concentration of 3 M total Cl

.At708C, the Fe
2þ
con-
centration at low Cl

concentrations was lower than at 258C but
also decreased with increasing HCl concentration accompanied
by an increase of FeCl
2
(Fig. 3b). In contrast, the FeCl
þ
con-
centration was higher at low Cl

concentrations compared with
258C and decreased with increasing HCl concentration. When
the Cl

concentration was increased by adding NaCl to FeCl
2
in
1 M HCl at 708C, the change in concentration of the three Fe
II
species was similar to the changes observed with increasing HCl
concentration at 708C (Fig. 3b). The change in undissociated
HCl concentration was also calculated for the three different
experimental systems and increased in all scenarios with
increasing total Cl

concentration (Fig. 3c). At 258C with
increasing HCl concentration, the concentration of undissociated
HCl did not exceed 50 mM. In contrast, at 708C the increase
of undissociated HCl was up to two orders of magnitude
higher and reached the millimolar range. With increasing NaCl
concentration in 1 M HCl at 708C, less undissociated HCl was
formed than with increasing HCl concentration at 708C.
Discussion
Thermodynamics of Fe
II
oxidation by O
2
During the oxidation of Fe
II
by O
2
four electrons are transferred:
4Fe2þþO2þ4Hþ!4Fe3þþ2H2O
ðDG0¼178 kJ mol1Þð1Þ
Considering the E
h
–pH diagram for Fe
II
/Fe
III
and O
2
/H
2
O
at 258C and a concentration of 1 M of all involved compounds
(Fig. A2 of the Accessory publication), from a thermodynamic
standpoint Fe
II
oxidation is expected to occur at all pH values.
However, Weiss suggested that the electrons are transferred in
four one-electron steps.
[28]
The free energies of each step were
calculated for standard conditions based on the DG
0
values
given by Stumm and Morgan
[4]
:
Fe2þþO2ðaqÞ!Fe3þþO
2ðaqÞ
ðDG0¼þ90 kJ mol1Þð2Þ
Fe2þþO
2ðaqÞþ2Hþ!Fe3þþH2O2ðaqÞ
ðDG0¼92 kJ mol1Þð3Þ
Fe2þþH2O2ðaqÞþHþ!Fe3þþOHðaqÞþH2O
ðDG0¼21 kJ mol1Þð4Þ
Fe2þþOHðaqÞþHþ!Fe3þþH2O
ðDG0¼171 kJ mol1Þð5Þ
0
0
1000
2000
3000
Undissociated HCI (µmol L⫺1)
log(initial FeII oxidation rate)
(mM day⫺1)
4000
0246810
Total CI⫺ (M)
0246810
Total CI⫺ (M)
20
40
60
Fe species (%)
80
0
⫺2.5
⫺1.5
⫺0.5
0.5
⫺0.5 0.0 0.5 1.0 1.5
log(undissociated HCI) (µmol L⫺1)
2.0 2.5 3.0 3.5
1.5
2.5
0246810
Total CI⫺ (M)
20
40
60
Fe species (%)
80
FeCI2
FeCI⫹
70°C, HCI 25°C, HCI
70°C, HCI
y ⫽ 1.37x ⫺ 1.52
R2 ⫽ 0.96
70°C, NaCI
in 1 M HCI
25°C, HCI
Fe2⫹
FeCI2
FeCI⫹
Fe2⫹
(a)
(c) (d)
(b)
Fig. 3. Concentrations of Fe
II
species for 10 mM FeCl
2
under anoxic conditions calculated using the REACT module of ‘The
Geochemist’s Workbench 6.0’ package (a) at 258C with increasing HCl concentration and (b) at 708C with increasing HCl
concentration (solid lines) and increasing NaCl concentration in 1 M HCl (dashed lines). (c) Calculated concentration of
undissociated HCl for the same conditions as in (a) and (b). (d) Initial Fe
II
oxidation rate of FeCl
2
in 1–6 M HCl at 258C(&) and
708C(r) versus the concentration of formed undissociated HCl. Initial Fe
II
oxidation rates were calculated from the Fe
II
concentrations measured at day 0 and 2 at 258C (Fig. 2c), and 0 and 2.5 min at 708C (Fig. 2a) respectively.
K. Porsch and A. Kappler
194

Although reaction steps 2–4 are thermodynamically favourable,
the first step is thermodynamically unfavourable and is assumed
to be the rate limiting step.
[4]
The free energy of reactions 1 and 2
calculated for our experimental conditions show that the overall
reaction (reaction 1) is thermodynamically favourable, whereas
the first electron transfer step (reaction 2) is thermodynamically
unfavourable both at 25 and 708C (see ‘Free energy calcula-
tions’ section of the Accessory publication, Table A3). Thus
thermodynamic considerations do not explain why Fe
II
oxida-
tion occurs at circumneutral pH and at high HCl concentrations,
but not at lower HCl concentrations.
Kinetics of Fe
II
oxidation by O
2
The Fe
II
oxidation rate is strongly influenced by pH. Under
circumneutral conditions (pH 6–8), the oxidation of Fe
II
is
described by the rate law
[3]
:
d½FeII=dt¼k½FeII pO2½OH2ð6Þ
where [Fe
II
] and [OH

] are the Fe
II
and OH

concentrations; t,
time; k, the reaction constant; and p
O
2
, the partial pressure of O
2
.
Accordingly, the oxidation rate increases 100-fold when the pH
increases by one unit. OH

ions enhance Fe
II
oxidation because
of the pH dependent formation of Fe
II
–hydroxo complexes. For
pH ,2 the rate is independent of the OH

concentration, and
therefore does not change with pH.
[29]
Besides OH

ions, the
kinetics of Fe
II
oxidation was also shown to depend on other
anions present. Cl

and SO
4
2
ions, for example, decrease the
Fe
II
oxidation rate.
[29,30]
As slight changes in pH at pH ,0
should have no effect on the Fe
II
oxidation rate and addition
of Cl

is expected to decrease it, one would expect that with
increasing HCl concentrations, the Fe
II
oxidation rate decreases.
However, we observed the opposite – an increasing Fe
II
oxi-
dation rate with increasing HCl concentration. Our results
therefore suggest that in the presence of high HCl concentrations
a reactive Fe
II
species is formed that is easily oxidised, and that
the concentration of this species increases with increasing HCl
concentration. This is in line with other publications that sug-
gested that the Fe
II
oxidation rate depends among other factors
on the present Fe
II
species, which are determined by the present
anions, but depends not on the formation of a more reactive
oxidant species (see e.g. Trapp and Millero
[31]
).
Role of Fe
II
–Cl species for Fe
II
oxidation
In order to identify this reactive Fe
II
species, the concentrations
of different Fe
II
species present in our experimental systems
were estimated by geochemical modelling showing that Fe
2þ
,
FeCl
þ
and FeCl
2
are the main dissolved Fe
II
species at all HCl
concentrations at 25 and 708C (Fig. 3a,b). Although the calcu-
lated concentrations of these three Fe
II
species varies between
this and three previous studies,
[32–34]
all studies exhibit the same
trend whereby the concentration of Fe
II
–Cl

complexes and
the number of Cl

ligands in these complexes increase with
increasing Cl

concentration. Hypothesising that the concen-
tration of an easily oxidisable Fe
II
species increases with
increasing HCl concentration, our modelling results suggest that
FeCl
2
might be this species (Fig. 3a,b). Although the formation
of Fe
II
–Cl

ion pairs slow down Fe
II
oxidation at circumneutral
pH,
[29,30]
the oxidation kinetics of these complexes may be
different at acidic pH. If the formation of FeCl
2
(or another Fe
II
–
Cl

complex) stimulates Fe
II
oxidation under acidic conditions,
one would hypothesise that (i) in the absence of Cl

at low pH,
no significant Fe
II
oxidation occurs as no Fe
II
–Cl

complex can
form and (ii) at low pH, i.e. at pH ,0, the formation of the Fe
II
–
Cl

species and thus the Fe
II
oxidation are influenced mainly
by the Cl

concentration and not by the H
þ
concentration.
Hypothesis (i) was tested by incubating Fe
II
in 3 M H
2
SO
4
(H
þ
concentration ,3 M) and indeed, only minor or no oxida-
tion of Fe
II
occurred compared with the 3 M HCl set-up
(Table 1), indicating that Cl

must be present to enhance Fe
II
oxidation under acidic conditions. This is supported by Awakura
et al.
[23]
who observed no increase in Fe
II
oxidation rates with
increasing H
2
SO
4
concentration (1–3 M) at 808C.
Hypothesis (ii) presumes that at low pH, the reactive Fe
II
species forms with increasing Cl

concentration. And indeed,
geochemical modelling yielded almost the same distribution
of Fe–Cl

species with increasing NaCl concentrations as for
increasing HCl concentrations (Fig. 3b). Furthermore, Fe
II
oxidation was enhanced in the presence of 2 M NaCl in
1 M HCl in comparison to set-ups with 1 M HCl alone but not
as much as by 3 M HCl (Table 1). These results indicate that the
Fe
II
oxidation rate is influenced by the Cl

concentration but
that the formation of Fe
II
–Cl

complexes alone does not explain
the experimental data. This suggests that another reactive Fe
II
species besides Fe
II
–Cl

complexes is formed.
Role of undissociated HCl for Fe
II
oxidation
The formation of a complex of Fe
II
with HCl (Fe–HCl complex)
was previously suggested to explain an increase in Fe
II
oxidation
rate at room temperature with increasing HCl concentration.
[23–25]
Fast Fe
II
oxidation in 4 M HCl was also observed by Astanina
and Rudenko.
[35]
They proposed that Fe
II
oxidation occurs by
the formation of Fe
II
–(hydroxo)aquo complexes and suggested
that at high HCl concentrations, undissociated HCl rather than
anions enters these complexes, and thereby changes the oxida-
tion kinetics. In our modelled scenarios, the concentration of
undissociated HCl increased with increasing Cl

concentration
but the increase was temperature dependent (Fig. 3c). The
higher concentration of undissociated HCl at 708C than at 258C
may be the reason for the higher Fe
II
oxidation rates observed at
708C. This is supported by the linear relationship of the initial
Fe
II
oxidation rates of FeCl
2
in 1–6 M HCl and the concentration
of undissociated HCl formed under these conditions (Fig. 3d).
As the same relationship can be observed at 25 and 708C, the
formation of more undissociated HCl with increasing temper-
ature is probably the main reason for the increase in Fe
II
oxidation rates rather than temperature alone (which is expected
to increase the oxidation rates according to the Arrhenius
equation). The potentially important role of undissociated HCl
in Fe
II
oxidation is also supported by the fact that an increase in
Cl

by addition of NaCl to 1 M HCl at 708C resulted in much
lower concentrations of undissociated HCl compared with
the experiments with increasing HCl concentration (Fig. 3c).
Accordingly, we observed lower Fe
II
oxidation in 1 M HCl
amended with 2 M NaCl than in 3 M HCl (Table 1). In
conclusion, the formation of undissociated HCl and hence the
potential formation of a Fe–HCl complex depends on H
þ
and
Cl

concentration and on temperature. This formation explains
well the observed Fe
II
oxidation in HCl and its increase with
increasing HCl concentration and temperature.
Conclusion
The results of this study showed that extraction of Fe from
environmental samples with oxic and highly concentrated HCl
leads to incorrect quantities of Fe
II
and Fe
III
in the Fe redox
Fe
II
oxidation by O
2
in HCl
195

speciation analysis. However, dissolution of Fe minerals in
6 M HCl has been applied in numerous studies to follow
microbial changes in Fe mineralogy and Fe redox speciation
and no Fe
II
oxidation was mentioned in these studies.
[9,36,37]
This may be due to several reasons. First, for extractions with
6 M HCl performed at ambient temperatures we found initial
Fe
II
oxidation rates of ,2 mM day
1
(Fig. 3d). Thus, if
extraction procedures including Fe quantification take only a
few hours, Fe
II
oxidation is probably too low to be recognised
but can still be significant (,50–100 mMh
1
). Second, in
some experimental systems, the total concentration of Fe
II
is
unknown, therefore, partial Fe
II
oxidation will be unrecognised.
Third, if Fe is extracted with highly concentrated HCl under
anoxic conditions, no Fe
II
oxidation will occur.
However, there are a few studies in which Fe
II
oxidation in
HCl was indeed observed but not discussed in detail. Matthews
et al.
[38]
for example, quantified stable isotope fractionation
between Fe
II
–Cl

and Fe
III
–Cl

complexes and observed Fe
II
oxidation in 6 M HCl. Based on our results and previous studies,
we recommend that in order to prevent Fe
II
oxidation during Fe
extraction with highly concentrated HCl, the extraction should
be performed under anoxic conditions or at least high tempera-
tures should be avoided. If the samples must be stored under
oxic conditions, even if it is only for a few days, a dilution of
the samples to a HCl concentration of 3 M HCl or lower is
suggested.
Accessory publication
Accessory material includes soil properties and experimental
methods for their determination (Table A1), an overview of all
experiments (Table A2), the experimental set-up for quantifi-
cation of initial Fe
II
oxidation at 708C and Fe
II
oxidation over
time at room temperature (Fig. A1), geochemical modelling of
HCl–FeCl
2
solutions at 25 and 708C in the absence and presence
of NaCl (see ‘Geochemical equilibrium calculation’ section of
the Accessory publication), E
h
–pH diagram for Fe
III
/Fe
II
,O
2
,H
2
and O
2
/O
2

(Fig. A2), and free energy calculations for our
experimental conditions (see ‘Free energy calculations’ section
of the Accessory publication, Table A3). This material is
available free of charge online at http://www.publish.csiro.au/
?act¼view_file&file_id¼EN10125_AC.pdf.
Acknowledgements
This work was funded by the Deutsche Forschungsgemeinschaft (DFG) and
the Stifterverband fu
¨r die Deutsche Wissenschaft. The authors thank Martin
Obst, Thilo Behrends, Philip Larese-Casanova and Jutta Meier for their
helpful comments concerning this manuscript.
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Manuscript received 11 November 2010, accepted 18 February 2011
Fe
II
oxidation by O
2
in HCl
197