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Geochimica et Cosmochimica Acta 72 (2008) 50015008

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Limitations of the ferrozine method for quantitative assay

of mineral systems for ferrous and total iron
Alexandre S. Anastacio a, Brittany Harris a, Hae-In Yoo a, Jose Domingos Fabris b,
Joseph W. Stucki a,*
a
Department of Natural Resources and Environmental Sciences, University of Illinois, W-321 Turner Hall, 1102 South Goodwin Avenue,
Urbana, IL 61801, USA
b
Department of Chemistry, Federal University of Minas Gerais, Campus Pampulha, Belo Horizonte, MG, Brazil

Received 22 April 2008; accepted in revised form 10 July 2008; available online 24 July 2008

Abstract

The quantitative assay of clay minerals, soils, and sediments for Fe(II) and total Fe is fundamental to understanding bio-
geochemical cycles occurring therein. The commonly used ferrozine method was originally designed to assay extracted forms
of Fe(II) from non-silicate aqueous systems. It is becoming, however, increasingly the method of choice to report the total
reduced state of Fe in soils and sediments. Because Fe in soils and sediments commonly exists in the structural framework
of silicates, extraction by HCl, as used in the ferrozine method, fails to dissolve all of the Fe. The phenanthroline (phen) meth-
od, on the other hand, was designed to assay silicate minerals for Fe(II) and total Fe and has been proven to be highly reliable.
In the present study potential sources of error in the ferrozine method were evaluated by comparing its results to those
obtained by the phen method. Both methods were used to analyze clay mineral and soil samples for Fe(II) and total Fe.
Results revealed that the conventional ferrozine method under reports total Fe in samples containing Fe in silicates and gives
erratic results for Fe(II). The sources of error in the ferrozine method are: (1) HCl fails to dissolve silicates and (2) if the ana-
lyte solution contains Fe3+, the analysis for Fe2+ will be photosensitive, and reported Fe(II) values will likely be greater than
the actual amount in solution. Another diculty with the ferrozine method is that it is tedious and much more labor intensive
than the phen method. For these reasons, the phen method is preferred and recommended. Its procedure is simpler, takes less
time, and avoids the errors found in the ferrozine method.
2008 Elsevier Ltd. All rights reserved.

1. INTRODUCTION 1998; Komadel and Stucki, 1988; Amonette et al., 1998)

Quantitative chemical analysis of clay minerals to reveal
the oxidation state of structural Fe is an essential aspect of
characterizing the eects of Fe oxidation state on important
properties of soils, sediments, and clay minerals. A variety
of methods have been employed and examined (Amonette
et al., 1994, 1998), but the colorimetric methods have be-
come the most widely used because of their simplicity,
accuracy, and precision. Among colorimetric methods, the
phenanthroline method (phen method, Stucki and
Anderson, 1981; Stucki, 1981; Amonette and Templeton,
*
Corresponding author.
E-mail address: jstucki@illinois.edu (J.W. Stucki).
has been commonly used for the analysis of silicate minerals
and the ferrozine method (Stookey, 1970; citations exceed
1400, over 100 of which are in the present journal) has been
widely used to analyze microbiologically modied soils and
sediments (Lovley and Phillips, 1986b; citations exceed 400,
over 28 of which are in the present journal). The phen meth-
od was developed primarily in the context of silicates,
whereas the ferrozine method was adopted for the realms
of aqueous, biological, and non-silicate mineral systems.
In recent years, however, the ferrozine method has been
increasingly applied, largely without adaptation, to systems
containing silicate minerals.
The phen method was designed for mineral systems and
is robust in eecting complete dissolution of Fe from the

0016-7037/$ - see front matter 2008 Elsevier Ltd. All rights reserved.
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5002 A.S. Anastacio et al. / Geochimica et Cosmochimica Acta 72 (2008) 50015008
mineral matrix and determining both Fe(II) and total Fe in
the same dilution. It is also eective for biological and
aqueous systems. Its primary disadvantage is that the
absorptivity of the tris-1,10-phenanthroline iron(II) com-
plex is about half that of the ferrozine Fe(II) complex, thus
making the phen method less sensitive than the ferrozine
method.
As mentioned above, a great strength of the phen meth-
od is that it measures both Fe(II) and total Fe in the same
solution. The measured chromophoric agent in both the
phen and ferrozine methods is the respective complex with
Fe(II). Ferric complexes with these reagents are colorless
and thus undetected, so total Fe can be determined only
if the Fe(III) is converted to Fe(II). In the phen method,
this reduction reaction is achieved photochemically or by
the addition of a reducing agent (such as hydroxylamine
hydrochloride). The basis for the photochemical reduc-
tionrst reported by David et al. (1972) and Wehry and
Ward (1971) and incorporated into the phen method by
Stucki and Anderson (1981) and Stucki (1981)briey is
that Fe(III) forms a binuclear bridged complex (tetra-
kis(1,10-phenathroline)-l-oxodiiron(III), with a formula
of [(phen)2Fe-O-Fe(phen)2]4+) with 1,10-phenanthroline
and is colorless. Upon exposure to UV light in the presence
of excess 1,10-phenanthroline, the binuclear complex is
photochemically and quantitatively reduced to the tris-
1,10-phenanthroline Fe(II) complex, which is the chromo-
phoric agent.
A clear model for the chemical mechanism involving the
photochemical reduction of Fe(III) in the ferrozine system
appears to be lacking from the literature, but a number of
citations mention carrying out the analyses in the dark or
under red safety lights (e.g., Macur et al., 1991; Majestic
et al., 2006). One should not be surprised if the Fe(III)fer-
rozine complex were photosensitive, however, because of
the well reported photoreactivity of Fe3+ complexes in
water (Kuma et al., 1992; Meunier et al., 2005; Shaked,
2008). No mention of photosensitivity was made by the ori-
ginal author of the method (Stookey, 1970) because it was
developed for a totally reduced system from which Fe(III)
was absent, but in a mixed-valent iron medium in which
both Fe(II) and Fe(III) are present, this phenomenon must
be taken into account.
The rst investigation in which microbiologically re-
duced Fe sediments were analyzed with ferrozine was con-
ducted by Srensen (1982), who extracted Fe(II) using a
medium consisting simply of 0.1% ferrozine solution in
50 mM HEPES buer adjusted to pH 7 with sodium
hydroxide, which probably only achieved complexation of
Fe2+ that was already in solution. Lovley and Phillips
(1986b) introduced the use of the original method with
0.5 N HCl to determine both Fe(II) and total Fe in extrac-
tions from sediments by preparing parallel ferrozine solu-
tions, with and without the addition of the reducing agent
hydroxylamine hydrochloride. The ferrozine method con-
tinued to evolve as a method for identifying the various
reactive fractions of Fe in sediments, based on extraction
protocols using ascorbic acid, acid ammonium oxalate,
dithionite, or HCl as extracting agents (Caneld, 1988;
Kostka and Luther, 1994). Caneld (1988) and Kostka
and Luther (1994) calibrated the methods needed to selec-
tively extract Fe(III) from amorphous and crystalline
matrices, non-structural Fe(II) in silicates, and acid-volatile
sulde (AVS) forms of Fe from salt marsh sediments and
mineral samples. In general, the more crystalline the Fe
mineral phase, the greater the concentrations of HCl re-
quired to eect Fe extraction, leading to a number of stud-
ies recommending 0.5, 1, 2, or even 3 N HCl (Caneld,
1988; Leventhal and Taylor, 1990; Jolivet et al., 1992; Wall-
mann et al., 1993; Zachara et al., 1998).
With regard to determining Fe(II) in silicate minerals,
Kostka and Luther (1994) successfully extracted Fe(II)
from chlorite (a phyllosilicate clay mineral) using 0.5 M
HCl, followed by ferrozine complexation. They recognized,
however, that total mineral dissolution by HFH2SO4 was
necessary for the measurement of total Fe by ferrozine; but,
unfortunately, they measured only total Fe in the HF
H2SO4 extract, leaving open the possibility that some
non-HCl-extractable Fe(II) could have been present in the
chlorite.
Over time the ferrozine method, with HCl as the extract-
ing agent, has become commonly used to assess the reduced
state of Fe in microbiologically active soils and sediments,
including the level of Fe(II) in the silicates. Since Kostka
and Luther (1994) made no claim of complete Fe(II) extrac-
tion from chlorite by this method and current practice lacks
the use of HFH2SO4 to determine total Fe(II) and total
Fe, its reliability as a quantitative tool for assessing the re-
dox state of Fe in soils and sediments containing silicate
minerals is in doubt. Such uncertainty has signicant conse-
quences because of the current widespread use of the meth-
od without critical assessment of its relevance for the
system(s) to which it is being applied.
The objectives of the present study were to document the
eects of photoreactivity and choice of extraction acid on
results from the ferrozine method applied to the measure-
ment of Fe(II) and total Fe in silicate minerals. Results will
be compared with the phen method under controlled, but
varied, lighting conditions and with or without the use of
HFH2SO4 in the extraction medium.
2. MATERIALS AND METHODS
The materials used in this study were standard reference
clay minerals of varying Fe(II) and total Fe contents and
soils with a mixture of clay minerals and Fe (oxy-
hydr)oxides. The clay minerals were: Upton, Wyoming,
montmorillonite (American Colloid Company), designated
UP; ferruginous smectite (sample SWa-1, Source Clays
Repository of The Clay Minerals Society), designated S1;
and Uley green nontronite (sample NAu-1, Source Clays
Repository of The Clay Minerals Society), designated N1.
The clay samples were homoionically saturated with Na+,
fractionated to <2-lm particle size, dialyzed by centrifuge
washing to a NaCl concentration of 5  104 M, and freeze
dried prior to use.
Two of these reference clay minerals, namely, UP and
S1, were also prepared in their chemically reduced state,
using the procedure of Stucki et al. (1984), which included
the addition of sodium dithionite to a citratebicarbonate-
 Limitations of ferrozine assay for Fe
buered aqueous suspension at 70 C. Following reduction,
the clays were washed four times with 5 mM NaCl solution
under inert atmosphere conditions, one time with high pur-
ity (18 MX cm) H2O, and freeze dried inside an inert-atmo-
sphere glove box.
One soil sample, designated CAB, was a Cambisol
(FAO classication, having an incipient B horizon) col-
lected near the city of Patos de Minas, State of Minas Ger-
ais, Brazil. The soil was dry sieved to separate the <2-mm
particle-size fraction and dispersed in NH4OH. The <2-
lm particle-size fraction was obtained by centrifugation,
then Na saturated and freeze dried.
The other soil samples were obtained from a saprolitic
prole at Oak Ridge National Laboratory (sampled and de-
scribed by Petrie et al., 2003 and North et al., 2004) and
contained a mixture of Fe(II) and Fe(III) in phyllosilicates
and Fe(III) in (oxyhydr)oxides (Stucki et al., 2007). Sub-
samples of this soil were suspended in H2O and incubated
anaerobically for 27 days with or without the addition of
either glucose or ethanol as a carbon source to stimulate
microbial growth (Akob et al., 2007). The sub-samples
identied as R1 and R2 received glucose, R4 and R5 re-
ceived ethanol, and R7 received neither. Samples were fro-
zen inside their septum-sealed bottles until ready for
analysis, at which time they were allowed to equilibrate at
room temperature. An aliquot was then removed with a
syringe needle and freeze dried under N2, except sample
R2 was air dried in normal atmosphere. Portions of the
dried samples were then submitted to analysis and desig-
nated set A. The remainder of some of the samples was left
in the freeze-dried state under ambient conditions for
approximately 1 year, then reanalyzed and designated set B.
All samples were analyzed by the phen and ferrozine
methods as published by Komadel and Stucki (1988) and
Lovley and Phillips (1986a,b), respectively. For the phen
method, 2030 mg of each sample was digested in a mixture
of 2 mL of 10 mass% 1,10-phenanthroline in ethanol,
12 mL of 3.6 N H2SO4, and 2 mL of 48% HF for 30 min
in a boiling water bath (sample temperature nominally at
80 C) in open 100-mL polypropylene centrifuge tubes.
The solution was cooled and 10 mL of 10 mass% solution
of H3BO3 was added to quench excess F ions, then diluted
quantitatively with H2O to approximately 100 mL. Two
milliliters of each solution was then diluted 1:10 with
1 mass% sodium citrate buer. All manipulations were
done in a dark room with red photographic lamps (using
regular red light bulbs) to avoid photochemical reduction
of the binuclear Fe(III)-phen complex to the Fe(II)phen
complex. The Fe(II) complex was then measured by UV
visible transmission spectroscopy at 510 nm. Total Fe was
measured in the same solutions after exposing them to
UV light for 2 h (Komadel and Stucki, 1988), which com-
pletely converted the Fe(III) to Fe(II).
The conventional ferrozine method (CFHCl) consisted
of extraction of Fe from the sample for 1 h in 0.5 N HCl at
room temperature. A 0.1-mL aliquot of each sample extract
was pipetted into 20 mL of the Fe(II) buer solution, which
was previously prepared by dissolving 0.2 g of ferrozine in
HEPES buer adjusted to pH 7 with NaOH solution. A
second 0.1-mL aliquot was also transferred to 20 mL of
5003
the total Fe buer, which was prepared by adding 5 g of
hydroxylamine hydrochloride to 500 mL of the Fe(II) buf-
fer. The absorbance of the resulting solutions was measured
at 562 nm.
The phen method served as the reference point to which
results from the ferrozine method were compared. It was se-
lected because of extensive previous studies which estab-
lished its reliability and limitations (Stucki, 1981; Stucki
and Anderson, 1981; Komadel and Stucki, 1988; Amonette
et al., 1994, 1998). Several hypotheses were tested regarding
the performance of the ferrozine method.
Hypothesis 1: Replacing HCl with HFH2SO4 will en-
hance the amount of Fe(II) and total Fe detected in the
sample extract, and thereby eect a more reliable assay.
Hypothesis 2: FerrozineFe(III) complexes are photo-
chemically reduced by light, thus causing erroneously high
assays for Fe(II) in mixed-valent solutions that are unpro-
tected from the light.
Hypothesis 3: Photochemical reduction may substitute
for hydroxylamine hydrochloride as the reducing agent in
total Fe determinations by the ferrozine method.
To test these hypotheses, the following variations in the
ferrozine method were implemented under controlled light
conditions:
1. The conventional ferrozine method as described above
(identied here as procedure CFHCl) was carried out
under normal laboratory lighting conditions.
2. The extractant solution used in the ferrozine method was
changed from HCl to HFH2SO4, the same mixture used
in the phen method (identied here as procedure CF
HF).
3. The foregoing methods were also repeated under dier-
ing light conditions. Samples were exposed to either
room light (abbreviated R) or UV light (the same used
in the phen method and abbreviated U) for 0 min
(abbreviated D for dark), 4 h (abbreviated 4R or
4U), or 24 h (abbreviated 24R or 24U).
3. RESULTS AND DISCUSSION
3.1. Total Fe
Total Fe results from the phen and variations on the fer-
rozine method (Fig. 1; Table 1) revealed a very poor corre-
lation between the methods. Linear regression analysis of
the plot of total Fe determined by the CFHCl method vs.
the phen method gave an R2 value of 0.38 and accounted
for only about 21%, on average, of the total Fe measured
by the phen method (based on the regression equation
CFHCl = 0.21 phen 0.05). Replacing HCl with HF
H2SO4 as the digesting or extracting acid improved the fer-
rozine results dramatically (Fig. 1), increasing the R2 to 0.99
and accounting for about 89% of the total Fe measured by
phen (CFHF = 0.89 phen 0.04). These results were ob-
tained from reference clay samples in both their oxidized
and reduced states and from the soil samples.
The eect of light on the total Fe content measured by
the CFHF method (Fig. 2) was less dramatic than the
5004 A.S. Anastacio et al. / Geochimica et Cosmochimica Acta 72 (2008) 50015008
Fig. 1. Comparison of methods for determining total Fe in soil and
clay mineral samples. Phen, phenanthroline reference method; CF
HCl, conventional ferrozine method dissolving with HCl; CFHF,
same as CFHCl except HCl was replaced by HFH2SO4.
dierence between HCl and HFH2SO4 digestion, but re-
vealed a detectable degradation eect on the Fe(II)ferro-
zine complex. When carried out in the dark (CFHF-D),
results from the conventional ferrozine method were virtu-
ally indistinguishable from those carried out under normal
room light (CFHF); however, when the Fe(II)ferrozine
complex was exposed to light for extended periods of time
(424 h), it appeared to degrade increasingly as the total Fe
content increased. The extent of degradation was greater in
room light than in UV light. A condent explanation for
this phenomenon will require further understanding of the
exact photoreactive nature of Feferrozine complexes.
3.2. Fe(II)
The most important use of the conventional ferrozine
method (CFHCl) is to measure the Fe(II) content of a
mixed mineral system. In the present study, however, values
for Fe(II) found by this method were erratic (Fig. 3, Table
2), both surpassing and falling short of results from the phen
method. Total Fe analyses found that HCl produces incom-
plete dissolution of the sample, which could explain why
some of the Fe(II) values obtained by CFHCl were less
than those obtained by phen. It fails, however, to explain
why the Fe(II) results would be greater than found by phen.
A plausible hypothesis is that some of the Fe dissolved by
HCl is present as Fe(III), which then complexes with ferro-
zine and is subsequently reduced photochemically to
Feferrozine3 2 if exposed to light. This hypothesis was
tested by carrying out the CFHCl analysis away from nor-
mal light, using the same red-lamp system as in the phen
method. Soil samples R4A and R7A were among those that
were found by the conventional (no protection from light)
CFHCl method to have a greater amount of Fe(II) than re-
ported by phen. When kept under the red-lamp system,
however, the values were less than found by phen (Fig. 4,
Table 2). This was a consistent result observed throughout
this batch of samples. One must conclude, therefore, that
the conventional ferrozine method is light sensitive and
the values greater than obtained with phen (Fig. 4, Table
2) are artifacts due to photochemical reduction of ferric
Fe in the light.
Regarding the eectiveness of HCl as an extractant or
solvent for removing Fe(II) from soils and sediments, fur-
ther experiments were undertaken to compare results using
HFH2SO4 in the sample digestion solution, which is
known to fully dissolve silicate minerals. The HFH2SO4
released a much larger amount of Fe(II) to solution and
yielded Fe(II) contents that were consistently much greater
than when using HCl to digest the sample (compare Figs. 3
and 5). The method produced Fe(II) contents that were, on
average, about 77% of the values reported by the phen
method (CFHF = 0.77 Phen + 0.01, R2 = 0.90).
To further test the hypothesis that results for Fe(II)
using ferrozine are aected by light, the CFHF experi-
ments were repeated without protecting the samples from
the light. The result (Fig. 5) was a signicant, although scat-
tered, increase in the amount of Fe(II) reported by the fer-
rozine method compared to the phen method. This
observation further attests to the photoreactivity of the
Fe(III) complex in analyte solutions.
A signicant, but unsuccessful, eort was exerted to de-
velop a revised ferrozine method which would account for
the aforementioned deciencies. The strategy was to modify
the much simpler phen method by replacing 1,10-phenan-
throline with ferrozine. Unfortunately, the adaptation of
the method was more complicated than anticipated because
of the high absorptivity of ferrozine, which required a very
small amount of sample to be used. This meant that sample
digestion had to take place in the absence of ferrozine, then
a small (< 1 mL) aliquot of the digestate transferred to the
ferrozine solution. The extra steps in the procedure more
than negated the original advantage envisioned by adapta-
tion of the phen method. Hence, if ferrozine is the required
or desired chromophoric complexing agent, the conven-
tional ferrozine method must be modied by replacing
HCl with HFH2SO4 and carrying out the analyses in the
dark or under protective red lamps. These modications
will greatly improve the reliability of the method but fail
to reduce the number of steps required in the analytical
procedure.
4. SUMMARY AND CONCLUSIONS
In view of the foregoing observations, results from stud-
ies using ferrozine to measure Fe(II) in mixed-valent Fe
mineral systems, and especially in silicates, likely contain
signicant errors and should be viewed cautiously. The con-
ventional ferrozine method has three principal deciencies
when applied to mineral systems: (1) it underestimates the
total Fe content in silicate clay minerals; (2) it unpredict-
ably overestimates and underestimates the Fe(II) content;
Table 1
Results for Total Fe analysis of soil and clay mineral samples by the phenanthroline (phen) method and variations of the ferrozine methoda
Sample Phen CFHCl CFHCl-D CFHF CFHF-D CFHF-4U CFHF-4R CFHF-24U CFHF-24R
(mmol/g) (mmol/g) (mmol/g) (mmol/g) (mmol/g) (mmol/g) (mmol/g) (mmol/g)
Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD
Total Fe
S1A 2.85 0.08 0.5 0.06

Limitations of ferrozine assay for Fe

UPA 0.46 0.008 0.06 0.01
N1A 3.1 0.2
R1A 0.72 0.03
R4A 0.75 0.03 0.33 0.01 0.3 0.005 0.72 0.02 0.73 0.02
R5A 0.769 0.004
R7A 0.799 0.007 0.29 0.01 0.27 0.02 0.756 0.003 0.761 0.004
R2A 1.05 0.04
UPB 0.432 0.006 0.01 0.02 0.39 0.08 0.34 0.002 0.35 0.01 0.12 0.02 0.40 0.01 0.19 0.02
N1B 3.94 0.08 0.03 0.03 3.5 0.1 2.7 0.2 0.5 0.3 3.2 0.2 0.6 0.3
CAB 2.08 0.02 0.13 0.07 1.72 0.06 0.99 0.06 0.69 0.03 1.2 0.06 0.33 0.01
R1B 0.9 0.2 0.05 0.04 0.62 0.02 0.75 0.08 0.65 0.06 0.24 0.02 0.66 0.05 0.36 0.02
R4B 0.78 0.02 0.02 0.02 0.64 0.06 0.65 0.04 0.66 0.05 0.201 0.005 0.66 0.04 0.32 0.002
R5B 0.79 0.01 0.02 0.02 0.62 0.03 0.63 0.01 0.66 0.01 0.20 0.01 0.66 0.06 0.33 0.03
R7B 0.84 0.02 0.02 0.01 0.69 0.01 0.66 0.05 0.64 0.09 0.17 0.02 0.7 0.1 0.30 0.03
R2B 0.9 0.2 0.03 0.01 1.01 0.03 0.77 0.04 0.21 0.04 0.70 0.05 0.31 0.09
S1R 2.84 0.04 1.14 0
UPR 0.50 0.03 0.2 0.08
a
CFHCl: conventional ferrozine; CFHCl-D: CFHCl method carried out in the dark, under red lamps; CFHF: CFHCL method carried out with HFH2SO4 replacing HCl as the digesting
solution; CFHF-D: the same as CFHF, except carried out in the dark, under red lamps; CFHF-4U: the same as CFHF-D, except subsequently exposed to UV light for 4 h; CFHF-4R: the
same as CFHF-D, except subsequently exposed to room light for 4 h; CFHF-24U: the same as CFHF-D, except subsequently exposed to UV light for 24 h; CFHF-24R: the same as CFHF-
D, except subsequently exposed to room light for 24 h.

5005
5006 A.S. Anastacio et al. / Geochimica et Cosmochimica Acta 72 (2008) 50015008

Fig. 2. Eect of light exposure on total Fe analysis of soil and clay

mineral samples. (CFHF, conventional ferrozine method, except
HCl was replaced by HFH2SO4; CFHF-D, same as CFHF,
except analysis performed in the dark; CFHF-4U or -4R, same as
CFHF except exposed to UV or room light for 4 h; CFHF-24U
or -24R, same as CFHF-4U or -4R except 24 h).
and (3) it is much more tedious and time consuming than
the phen method. The underestimation of total Fe is due
to HCl being unable to dissolve the silicate clay minerals
Fig. 3. Comparison of results for analysis of soil and clay mineral
samples for Fe(II) using the phen and conventional ferrozine
methods.
completely, leaving some Fe (both ferrous and ferric) in
the solid phase and thus unavailable for complexation with
ferrozine. Substituting HFH2SO4 for HCl brings the re-
sults largely into line with the phen method.
The overestimation of Fe(II) content in a mixed-valent
Fe mineral system occurs because the Fe(III)ferrozine

Table 2
Results for Fe(II) analysis of soil and clay mineral samples by the phenanthroline (phen) method and variations of the ferrozine method (see
footnote to Table 1 for abbreviations)
Sample Phen (mmol/g) CFHCl CFHCl-D CFHF (mmol/g) CFHF-D
(mmol/g) (mmol/g) (mmol/g)
Mean SD Mean SD Mean SD Mean SD Mean SD
Fe(II)
S1A 0.018 0.004 0.26 0.08
UPA 0.063 0.002 0.050 0.005
N1A 0.016 0.003
R1A 0.159 0.006 0.098 0.005
R4A 0.161 0.005 0.20 0.03 0.086 0.006 0.152 0.007
R5A 0.148 0 0.082 0.009
R7A 0.092 0.001 0.16 0.1 0.046 0.007 0.116 0.008
R2A 0.134 0.003
UPB 0.0351 0.0005 0.022 0.005 0.063 0.006 0.0300 0.0009
N1B 0.005 0.001 0.01 0.01 0.057 0.001
CAB 0.049 0.002 0.04 0.02 0.061 0.004
R1B 0.13 0.02 0.017 0.006 0.16 0.03 0.12 0.02
R4B 0.14 0.01 0.017 0.007 0.136 0.009 0.110 0.005
R5B 0.13 0.02 0.010 0.005 0.11 0.01 0.090 0.006
R7B 0.093 0.003 0.02 0.04 0.10 0.03 0.08 0.01
R2B 0.122 0.002 0.011 0.008 0.11 0.02
S1R 1.0 0.2 0.46 0
UPR 0.31 0.04 0.03 0.02
 Limitations of ferrozine assay for Fe 5007

Fig. 4. Eect of light exposure on Fe(II) analysis. Phen, phenan-

throline method; CFHCl, conventional ferrozine method dissolv-
ing with HCl; CFHCl-D, same as CFHCl except samples kept in
the dark.

complex, which contributes no absorption at 562 nm, can

be photochemically reduced to the colorimetrically deter-
mined moiety Feferrozine3 2 . The original protocol
makes no mention of protecting analyte solutions from
the light, so overestimation of Fe(II) content will occur
and probably has often occurred without being reported.
Compared to the phen method, the ferrozine procedure
is tedious and time consuming, and separate solutions must
be prepared for Fe(II) and total Fe. In the phen method,
these two values are obtained from the same diluted solu-
tion. The ferrozine procedure states that each sample must
be done in duplicate, one set of the duplicates for Fe2+ mea-
surement and another for total iron measurement. Two dif-
ferent ferrozine buers have to be prepared, one with and
one without the reducing agent, as well as the standard
solution. In order to keep within the standard curve, dilu-
tions of small volumes (100 lL) must be performed with
high accuracy and precision. In all, the method requires
twice the time as the phen method. In view of all these fac-
tors, the phen method is recommended over the ferrozine
method. However, if ferrozine is the required or desired
Fig. 5. (a) Comparison of the phen and CFHF-D methods for
chromophoric complexing agent for systems containing
analyzing soil and clay mineral samples for Fe(II). (Phen, phenan-
mixed-valent Fe species and/or silicate minerals, the con- throline method; CFHF-D, conventional ferrozine method,
ventional ferrozine method must be modied by replacing except HCl was replaced by HFH2SO4 as the dissolving acid
HCl with HFH2SO4 and carrying out the analyses in the and the samples were kept in the dark during analysis). (b)
dark or under protective red lamps. Comparison of the phen and CFHF methods for analyzing soil
and clay mineral samples for Fe(II). (Phen = phenanthroline
ACKNOWLEDGMENTS method; CFHF, conventional ferrozine method, except HCl was
replaced by HFH2SO4 as the dissolving acid). Linear regression
This study was supported in part by the University of Illinois, equation and R2 were based on 8 points excluding the outlier at
College of Agricultural, Consumer, and Environmental Sciences, 0.19 on the vertical scale.
5008 A.S. Anastacio et al. / Geochimica et Cosmochimica Acta 72 (2008) 50015008
Oce of Research; The Academy of Applied Science; The Council
for International Exchange of Scholars Fulbright Foundation (fel-
lowship to J.D.F.); the National Science Foundation, Division of
Petrology and Geochemistry, Grant No. EAR 01-26308; and the
Environmental Remediation Science Program (ERSP), Biological
and Environmental Research (BER), U.S. Department of Energy,
Grant No. DE-FG02-07ER64374. The authors thank Dr. Joel E.
Kostka, Florida State University, for supplying the Oak Ridge soil
samples.
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Associate editor: David J. Vaughan