Journal of Chromatographic Science 2013;51:391– 399
doi:10.1093/chromsci/bms153 Advance Access publication September 27, 2012
Article
Simultaneous Analysis of SbIII, SbV and TMSb by High Performance Liquid
Chromatography– Inductively Coupled Plasma – Mass Spectrometry Detection: Application
to Antimony Speciation in Soil Samples
Zhaofeng Ge1,2 and Chaoyang Wei1*
1
Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing 100101,
and 2University of Chinese Academy of Sciences, Beijing 100049
*Author to whom correspondence should be addressed. Email: [email protected]
Received 26 March 2012; revised 17 August 2012
This study was conducted to develop a method for the simultaneous separation and detection of antimonite (SbIII), antimonate
(SbV) and trimethyl antinmony (TMSb) species in soils, using ultrasonic-aided citric acid extraction and high-performance liquid chromatography – inductively coupled plasma – mass spectrometry
separation and detection. The extractions were performed using
various chemical solutions. The separation was conducted on a
PRP-X100 anion exchange column (25 cm 3 4.1 mm i.d., 10 mm)
using an isocratic elution program. The various factors of the
elution procedure, e.g., pH, elution concentration and retention
time, were optimized for the best separation of the three Sb
species. It was found that two consecutive extractions using
100 mmol/L citric acid at pH 2.03 resulted in the highest extraction
efficiency, 53%. The optimal elution procedure was obtained by
using 200 mmol/L ammonium tartrate with 4% methanol as the
mobile phase at pH 5.0. Under these conditions, the retention times
for SbIII, SbV and TMSb species were 6.8, 2.1 and 3.8 min with detection limits of 0.03, 0.02 and 0.05 mg/L, respectively. Spiked recoveries for SbIII, SbV and TMSb ranged from 88 to 118%. The
proposed method is reliable for antimony speciation in soil
samples.
Introduction
Antimony (Sb) is a toxic element (1) that has frequently been
studied in recent decades because it is a trace element that is
not essential to life (2). In fact, Sb has been intensively used in
various industrial products for a long time (3– 4). Sb has also
been emitted into the environment through human activities,
such as waste incineration, mining, smelting and the combustion of fossil fuels. Increasing mining and smelting activities
have resulted in elevated Sb levels in several locations, especially in southern China (5). The United States Environmental
Protection Agency (EPA) and the European Union consider Sb
and its compounds to be priority pollutants (6). However,
knowledge about the valence changes of Sb, the ecotoxicology
of Sb and the extent of Sb dispersion in the environment are
still quite limited (7–8).
The toxicity of Sb compounds varies with its speciation state
(9 –11). Regarding the antimony compounds, the International
Agency for Research on Cancer (IARC) assigns inhaled antimony trioxide (Sb2O3) and antimony trisulfide to carcinogenic
groups 2B and 3, respectively (12). In general, antimonite
(SbIII) is ten times more toxic than antimonate (SbV), and
inorganic Sb species are more toxic than organic species
(10–11). However, the higher toxicity of SbV than SbIII was
reported by Takayanagi (13), who found that in Red Seabream
(Pargus major), the 96-h lethal dose of SbIII for 50% of test
animals (LC50s) was 12.4 mg/L, compared to 0.93 mg/L for
SbV. To date, most studies have focused on determining Sb
concentrations in the environment. However, because the Sb
species vary in the environment (14), it is important to detect
the individual Sb species. In addition to the two inorganic Sb
species, SbIII and SbV, trimethylated Sb compounds (TMSb) have
also been found in the environment (15 –17). Currently, knowledge about the presence of organoantimony compounds in
nature is scarce (18). Although trimethylstiboxide, a compound
that is stable in water, can be reduced to trimethylstibane via
some bacteriological processes in soils, trimethylstiboxide can
also be formed by the oxidation of the trimethylstibane produced from Sb compounds via biomethylation (19). Therefore,
it is necessary to determine the levels of various Sb species in
the soil to understand the mechanisms of Sb speciation changes
in soils. However, determining Sb species in solid environmental
samples presents many problems and challenges. For example,
the extraction efficiency for Sb in most previous studies has
generally been lower than 10%, and sometimes the efficiency
has even been below 1% (15, 17). López-Garcı́a et al. (20)
achieved an extraction efficiency of 35 –40% for Sb, which is still
much lower than those for arsenic (As) (21).
Other significant factors impeding the progress of Sb speciation research are the analytical methods. At present, most of
the analytical techniques for the separation and detection of Sb
species are based on the line-coupling of high-performance
liquid chromatography (HPLC) to element-specific detectors,
such as hydride generation– atomic absorption spectrometry
(HG– AAS) (15, 22–23), inductively coupled plasma mass spectrometry (ICP –MS) (16– 17, 21, 24) or atomic emission (ICP –
AES) (15, 25), and hydride generation atomic fluorescence
spectrometry (HG –AFS) (3, 26–29). Among them, the most
frequently used element-specific detector for analysis of Sb
speciation is ICP– MS (21). The low levels of Sb in the samples
present a problem for the instrumentation because they are
sometimes lower than the detection limits of the analytical
instruments.
HPLC has widely been used for the analysis of Sb speciation
during the last decade. Most efforts have been attempts to determine inorganic SbIII and SbV in aqueous samples using an
anion exchange column (2, 30 –31). Organic Sb compounds, in-
# The Author [2012]. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
cluding monomethylantimony, dimethylantimony and trimethylantimony, were generally detected in soils (32). However,
these methods cannot be applied to complex environmental
media, such as soil or plant samples, in which organic Sb compounds may exist. Therefore, there is an urgent need to
develop an experimental method that can simultaneously
analyze inorganic and organic Sb species. However, only a
handful of analytical methods capable of the simultaneous separation and online determination of the two types of Sb species
have been reported to date.
The primary Sb species in the environment are inorganic
SbIII and SbV and organic TMSb. Ulrich (15) developed a
method with a duration of more than 12 min that could simultaneously separate three Sb species by HPLC–ICP –MS. This
method had a detection limit for SbIII of 3 mg/L. The major
challenge in this endeavor is simultaneously separating the
organic and inorganic Sb species in a single chromatographic
system. The elution and peak tailing also present significant difficulties (33). In general, SbV and TMSb are easily separated using
anion exchange columns (15, 21, 34), whereas the elution of
SbIII is comparatively problematic because of issues such as long
retention time, irreversible retention and severe peak tailing
(35). In attempts to resolve the problem of the elution of SbIII,
complex mobile phases, such as ethylenediamine-tetraacetic
acid (EDTA) (17, 22, 24), EDTA mixed with potassium hydrogen
phthalate (KHP) (24, 36) and tartrate (22 –23), have been used
as alternatives. However, SbIII still showed strong retention and
no signal could be detected. This failure might be due to the
rather complicated equilibrium partitions of agglomerations and
non-defined species in the studies (37), or because of aqueous
interactions between SbV and TMSb with complexes in addition
to those observed for SbIII (3). Furthermore, the optimal separation conditions for various Sb species depend on environmental conditions, such as pH. The elution of (CH3)3SbCl2 is
achieved only by using an alkaline mobile phases, such as carbonate buffer, phosphate buffer or potassium hydroxide,
whereas acidic conditions are more suitable for separating SbIII
and SbV (30, 35, 40). SbIII and SbV can be separated under isocratic conditions of EDTA as the mobile phase with pH 4.5;
however, these conditions are not suitable for the elution
of TMSb. In addition, SbV and TMSb can be separated under isocratic conditions using a solution of NH4OH and 1 mmol/L
EDTA with pH 11, but it is impossible to elute SbIII at this pH
(30). Therefore, a gradient elution procedure has been developed in recent years (3, 30, 35). This method has improved the
isolation effect by changing the mobile phases and pH values of
the mobile phases (28, 30, 35). Hansen and Pergantis (33)
presented a review on analytical techniques and methods for
Sb speciation, summarizing the gradient elution procedure.
However, gradient elution still presents some challenges, such
as the instability of Sb species, especially SbIII, and challenges in
the complex operation procedure. The complexing agents and
other eluent anions sometimes form complexes during chromatographic separation (37).
The instability of Sb species in the process of elution needs
to be considered. Because no certified reference material exists
for Sb species, only a few studies have reported on species
transformation during extraction from samples with added Sb.
Dodd et al. (18) studied the stability of the trimethyl antimony
standard during analysis (HG –GC –MS), and found no change
392 Ge and Wei
in the TMSb standard. Later, Craig et al. (38) evaluated the stability of TMSb in the process of extraction by sonication in a
mixture of methanol and water (9 þ 1) or 0.2 mol/L acetic
acid and found that the TMSb standard was stable in this
process. These studies demonstrate that the TMSb standard is
stable during the process of extraction and analysis. However,
the instability of SbIII is the primary problem in Sb speciation
studies. Some researchers have studied the complexation
effects of Sb compounds with acid on the speciation of Sb in
the environment. Guy et al. (25) reported complexation
between SbV and citric acid, whereas no complexation was
observed between SbIII and citric acid. In contrast, Ulrich et al.
(34) showed the complexation of SbIII, but not SbV, with citric
acid. Later, Zheng et al. (36) found that both SbIII and SbV
could readily form complexes with citric acid in an aqueous
solution at room temperature. The contradictory results from
various reports highlight the importance of complexation
between extraction reagents and Sb species in environmental
samples.
The speciation techniques for Sb are still far from perfect
with regard to the detection limit, the extraction efficiency,
and the overall recovery. The objective of the present study
was to develop a simple isocratic elution procedure with high
extraction efficiency for the simultaneous separation and determination of inorganic SbIII and SbV, and TMSb using an HLPC
separation system. The effect of mobile phase concentrations,
pH values and the organic modifier of the mobile phases on
the separation efficiency were assessed. These factors in the
chromatography system were optimized to achieve better separation during a shorter retention time. The extraction and speciation methodology was then applied to determine Sb species
in the soil samples collected in an old Sb mine in China.
Material and Methods
Chemicals and reagents
All chemicals and reagents used in this study were of analytical
grade or better. Deionized water (18.2 MV.cm) produced with
a Millipore system (Milford, MA) was used throughout. The
tubes and equipment were soaked for 24 h in 10% (v/v) nitric
acid and rinsed several times with deionized water before use.
A 100 mg/L stock standard solution of SbV was prepared by
dissolving the appropriate amount of potassium hexahydroxiantimoniate [KSb(OH)6; Sigma-Aldrich, St. Louis, MO) in deionized water, which was then stored in a polyethylene bottle in
the dark at 48C. Stock standard solutions of SbIII and TMSb
were prepared before each use by dissolving potassium antimonyl tartrate [K(SbO)C4H4O6.H2O; Chemical Reagent Company,
Beijing, China) and trimethylantimony dibromine (C3H9Br2Sb;
Sigma-Aldrich), respectively, in deionized water. C3H9Br2Sb
probably forms the positively charged trimethylantimony hydroxide when dissolved in water under neutral conditions (16).
According to previous studies, SbIII can be oxidised to SbV
within 30 min at a low concentration (36). However, diammonium tartrate can effectively prevent the quick oxidation of
SbIII at low levels in mg/L (3). Therefore, the working standard
solutions were prepared daily by appropriately diluting the
stock solutions in diammonium tartrate (250 mmol/L), which
ensures the invariability of the Sb species.
The mobile phases, including EDTA (10 mmol/L), phthalic
acid (2 mmol/L) with 2% acetone or methanol and ammonium
tartrate (200 mmol/L) at different pHs, were prepared at the
specified concentrations by dissolving the appropriate compounds. The mobile phases were filtered using a 0.22 mm membrane and degassed by sonication before use.
Water, 100 mmol/L EDTA ( pH 4.5), 30 and 100 mmol/L
citric acid ( pH 2.03), 100 mmol/L citric acid in 1% ascorbic
acid ( pH 2.0), and methanol–water (1 þ 1, 3 þ 7, 1 þ 3) were
tested as the extractants of Sb from soils. The pH values of the
extractants were adjusted to the specified values using hydrochloric acid or ammonia water.
Instrumentation
Speciation analysis of Sb was performed using HPLC–ICP –MS).
The chromatographic system (Perkin Elmer, Series 200),
equipped with an auto-sampler and a quaternary pump, was
coupled via a peek capillary to a cross nebulizer of an ICP –MS
system (PerkinElmer Sciex, Concord, Ontario, CA). Separation
of SbIII, SbV and TMSb was conducted on a Hamilton PRP-X100
anion exchange column (250 4.1 mm i.d., 10 mm). An automatic sampler with a 200 mL loop was used for sample injection. The flow rate of the mobile phase was 1.2 mL/min.
Samples
Soil samples were collected in the mining area of Xikuangshan
in Hunan province, China. The soils have been heavily polluted
by mining and smelting activities in the area. The samples were
freeze-dried, ground into 100 mesh powders and stored at 48C
before analysis. An aliquot (100 mg) of the powder was transferred into a 50 mL flask for digestion. The digestion followed
the procedure described by Shi and Wang (39). Samples in
flasks were soaked with 5 mL aqua regia for 12 h, then heated
at 1108C on an electric hot plate for approximately 3 h until
the volumes of the sample solutions in the flasks were less
than 0.5 mL. After cooling to room temperature, the solutions
were immediately filtered into a 25 mL tube and brought up to
25 mL volume with deionized water. An aliquot (1 mL) of the
supernatant was transferred to a tube, and 1 mL of HCl, 2 mL of
thiourea (5%, m/v) and 2 mL of ascorbic acid (5%, m/v) were
added. Each sample was completed to 10 mL with deionized
water and left for 0.5 h before Sb detection using an HG –AFS
(Beijing Titan Co., China). The reductant used was 2% (m/v)
KBH4 stabilized in 0.5% (m/v) NaOH (43). The total Sb concentration in the soil sample was measured as 866 mg/kg. Spiked
samples for three detected Sb species were prepared by accurately adding the corresponding amounts of K(SbO)C4H4O6.H2O,
KSb(OH)6, and C3H9Br2Sb to an aliquot of the fine powder of
the soil samples, representing 5, 25 and 25 mg/L of SbIII, SbV and
TMSb, respectively.
Each extraction was performed by sonication for one hour
with its extractant. The extracted solutions were appropriately
diluted and then filtered through a 0.22 mm membrane before
injection. In this study, several extractants were assessed to
compare the extraction efficiencies, the shape of the peaks
and the recoveries of spiked samples. Based on the results of
the comparison, the extractions were performed with
100 mmol/L citric acid at pH 2.03 for the rest of the study.
Briefly, to extract Sb from soil, a 0.1000 g powdered soil sample
was weighed into a 25 mL centrifugal tube in duplicate and
extracted with 10 mL citric acid in an ultrasonic bath for one
hour, followed by centrifugation at 8,000 g at 48C for 10 min.
The supernatants were transferred to glass tubes and diluted to
45 mL with the mobile phase solutions, and then filtered
through a 0.22 mm membrane before injection into the chromatographic system for the detection of Sb species. To
examine the effect of the number of extractions on the extraction efficiencies for different Sb species, extractions were performed one, two or three times for each sample, and the
resulting extractions were combined for measurements.
Results and Discussion
Optimization of chromatographic parameters
Several mobile phases were tested for their suitability for separating SbIII, SbV, and TMSb. After evaluating the mobile phases,
the methanol ratio and the adjusted pH value, the best results
were obtained with ammonium tartrate with appropriate
methanol at the lower pH value. The first peak was assigned to
SbV, followed by, in order, TMSb and SbIII (Figure 1).
Generally, SbV is found as Sb(OH)2
6 when present in aqueous
solution, and TMSbBr2 is most likely in the positively charged
trimethylantimony hydroxide form (44). Because an anion is
eluted before a cation in an anion exchange column, the peak
of SbV appears prior to that of TMSb (Figure 1). SbIII is in the
form of Sb(OH)3 in water and can quickly be oxidized to SbV;
however, this oxidation can be suppressed by adding diammonium tartrate (3). In this study, diammonium tartrate was used
Figure 1. Effect of ammonium tartrate concentration on the chromatograms of Sb
species in aqueous solutions. The tested levels of SbIII, SbV and TMSb were 25, 5
and 25 mg/L, respectively. The mobile phase was ammonium tartrate solution under
various concentrations at pH5.0, with: no methanol (designated with “a”) or 4%
methanol, using the isocratic elution procedure. The flow rate of the mobile phases
was 1.2 mL/min.
Simultaneous Analysis of SbIII, SbV and TMSb by High Performance Liquid Chromatography– Inductively Coupled Plasma – Mass Spectrometry Detection 393
as the stabilizing agent. Therefore, according to the elution
order, SbIII most likely combined with diammonium tartrate
and formed a positive ion.
Effect of mobile phase concentration on the chromatograms
of Sb species
The influence of the mobile phase concentration was tested
within a range of 100 to 300 mmol/L in 50 mmol/L steps, with
4% methanol as the organic modifier at pH 5.0. A significant
effect was observed regarding the concentration of the mobile
phases on the retention time of SbIII. The retention time
decreased with the increasing concentration of ammonium tartrate in solution (Figure 1), which is consistent with the results
of Miravet et al. (3). As the condition with the best combination of short retention, small peak broadening and high resolution, a mobile phase concentration of 200 mmol/L was
chosen for further investigation.
Effect of methanol ratio on the chromatograms of Sb species
The ratio of methanol to mobile phases is very important for
the separation of different Sb species by anion exchange chromatography. Organic solvents have significant effects on anion
chromatography, such as retention time, peak height, half-peak
width, shape of the peak and resolution of each peak.
Methanol was the best choice due to its low viscosity in
aqueous solution (41). In this study, at different methanol
ratios, the retention time and peak shapes of SbV remained
quite stable (Figure 2). This stability might be because SbV has
one negative charge in water solution as Sb(OH)2
6 (15) and
stays relatively stable in various methanol ratios. However, this
stability was not observed for TMSb and SbIII; the retention
time and peak shapes varied in different methanol ratios. The
Figure 2. Effect of methanol ratios on chromatograms of Sb species in aqueous
solutions. The tested levels of SbIII, SbV and TMSb were 25, 5 and 25 mg/L,
respectively; the mobile phase was 200 mmol/L ammonium tartrate solution with various
ratios of methanol at pH5.0. The flow rate of the mobile phases was 1.2 mL/min.
394 Ge and Wei
retention time of TMSb decreased with increasing methanol
ratios. Without methanol, the peaks of TMSb and SbIII were coincident and showed broadening and tailing, whereas with
methanol, the tailings disappeared and the two species flowed
out separately. Methanol as an organic modifier might depress
the non-ionogenic interactions between the Sb compound and
the analytical column (15). Organic solvents create more
complex ion exchange behavior in the mobile phase. Two opposing effects of organic solvents on chromatography behavior
may exist. On one hand, organic solvents can reduce the dielectric constant of the mobile phase to enhance its combining
force with the positive ions, but they also reduce the elution
ability of the mobile phase (42). The mechanisms of the effects
that organic solvents have on chromatographic behavior have
yet to be fully resolved. Because of its short retention time, the
shape of the peak and the resolution, 4% methanol was chosen
as an organic modifier of the mobile phases for further
investigation.
Effect of pH on the retention time
The pH value is another important factor in separating different
Sb species by anion exchange chromatography. In this study,
the retention times for various Sb species were tested with pH
values ranging from 3.5 to 6.5 in 0.5 intervals.
Among the three Sb species, the retention time of SbV was
the shortest, and that of SbIII was the longest between pH 3.5
and 6.5 (Figure 3). When the pH value was lower than 5.0, the
peaks of TMSb and SbIII overlapped, while at pH 5.0, the three
peaks were completely separated. At pH 6.5, the peaks of TMSb
and SbIII nearly coincided again (Figure 4). Consequently, in
this study, pH 5.0 was determined to be the most suitable for
separating the three Sb species, with an elution time of approximately 8.6 min.
The structure of a chemical determines its properties, such
as mobility and affinity. The stability of the retention time for
SbV indicated that SbV existed as a stable ionic form, which was
Figure 3. Change of retention times with pH for SbIII, SbV and TMSb in aqueous
solutions. The tested levels of SbIII, SbV and TMSb were 25, 5 and 25 mg/L,
respectively; the mobile phase was 200 mmol/L ammonium tartrate solution with 4%
methanol at various pH values. The flow rate of the mobile phases was 1.2 mL/min.
Table I
Summary of the Optimal Conditions for the HPLC –ICP –MS System
HPLC (PerkinElmer, Series 200)
Column
Mobile phases
Flow rate (mL/min)
Injection volume (mL)
ICP –MS (PerkinElmer Sciex)
Nebulizer gas flow (L/min)
Auxiliary gas flow (L/min)
Plasma gas flow (L/min)
ICP RF power (W)
Column pressure (psi)
Nebulizer type
Dwell time (ms)
Isotopes monitored
Hamilton PRP-X100(25 cm 4.1mm i.d., 10 mm)
200 mmol/L ammonium tartrate þ 4% methanol, pH 5.0
1.2
25
0.87
1.2
15
1,300
980
Cross
20
121 Sb
Figure 4. Effect of pH on the chromatograms of Sb species in aqueous solutions.
The tested levels of SbIII, SbV and TMSb were 25, 5 and 25 mg/L, respectively;
the mobile phase was 200 mmol/L ammonium tartrate solution with 4% methanol
at various pH values. The flow rate of the mobile phases was 1.2 mL/min.
in accordance with the generally accepted fact that SbV is
represented as Sb(OH)2
6 in the environment (44). Over the
range of the tested pH values, the change of the retention time
of TMSb exhibited two different trends. At pHs less than 5.0,
the retention time decreased, whereas at pHs greater than 5.0,
it increased. This result might reflect the changing affinity of
TMSb in the anion exchange column. The change at pH 5.0
may suggest that substantial changes happened to chemical
structure of TMSb, creating a compound with the mobile
phases in the anion exchange column. This result is also in
agreement with Morgan et al. (46), who found that TMSb in solution was either in the form of trimethylantimony dihydroxide
or a dimer of two trimethylantimonyhydroxide molecules. The
effect of pH on the retention times of SbIII was much more
complicated, indicating that SbIII incorporated into the mobile
phases in a more unstable way than SbV and TMSb. This result
underscores the importance of adjusting the pH of the mobile
phases to obtain reasonable results for Sb speciation.
The flow rate of the mobile phases and the injection volume
were studied. The optimal chromatographic parameters are
summarized in Table I. Simultaneous separation of the three Sb
species was successfully obtained under the optimized separation and detection conditions. The retention times for SbIII,
SbV, and TMSb were 3.8, 2.1 and 6.8 min, respectively (Figure 5).
Method validation
Linear range
The normalization method using the peak area from the ICP –
MS signal under the optimum conditions described previously
Figure 5. Chromatogram of the separation of SbIII, SbV and TMSb in aqueous
solutions (25, 5 and 25 mg/L, respectively) using the Hamilton PRP-X100 column at
the optimal conditions as described previously.
was used to quantify the tested SbIII, SbV and TMSb. In this
study, linearity was obtained in the range of 0.1 to 50 mg/L for
all three tested Sb species (Table II). Among the three Sb
species, the signal intensity of SbV was the most sensitive
(Figure 5); therefore, the slopes of the standard curves of the
three Sb species were different, with the slope of SbV being
the steepest, followed by SbIII and then TMSb.
Detection limits
In this study, the detection limits (LODs) were calculated as 3
s/m, where m was the slope of the calibration curve and s was
the standard deviation of 10 blank injections.
The LODs for SbIII, SbV and TMSb were 0.03, 0.02 and
0.05 mg/L, respectively. The LODs of SbIII and TMSb were
slightly higher than that of SbV, which might be due to the
strong retention of SbIII and TMSb on the column, leading to
broader peaks.
Simultaneous Analysis of SbIII, SbV and TMSb by High Performance Liquid Chromatography– Inductively Coupled Plasma – Mass Spectrometry Detection 395
Table II
Standard Surves for SbIII, SbV and TMSb*
Sb species
III
Sb
SbV
TMSb
Standard curves
R2
Linear range (mg/L)
Y ¼ 1945x – 13631
Y ¼ 3361x þ 766
Y ¼ 1295x þ 4806
0.9964
0.9944
0.9980
1 –50
1 –25
1 –50
*Note: Each of the standard curves was a regression line of five points.
Table III
LODs (mg/L) for SbIII, SbV and TMSb*
SbIII
SbV
TMSb
Reference
0.8
—
3.0
1.7
0.3
0.014
0.07
0.03
0.5
0.005
0.1
0.14
0.1
0.012
0.07
0.02
0.6
0.005
—
—
—
0.009
1.0
0.05
Lintschinger et al. (17)
Lintschinger et al. (16)
Ulrich (15)
Lindemann et al. (23)
Zheng et al. (24)
Krachler and Emons (21)
Quiroz et al. (45)
This study
*Note: Dashes indicate species not considered in the chromatographic investigation.
Previous studies have primarily focused on inorganic Sb
species, with the LODs often higher than 0.1 mg/L or even up
to 3 mg/L (15, 23– 24). In recent years, simultaneous analysis of
organic and inorganic Sb species has received increasing attention. Researchers have paid more attention to improving LODs,
and HG –AFS has become a satisfactory alternative detection
system because of its low cost. Potin-Gautier et al. (28) developed methodologies for determining SbIII, SbV and TMSb in
sediment reference samples with a post-column photooxidation step and HG –AFS as the detection system, which
were able to separate three Sb species within 6 min, but LODs
for SbIII, SbV and TMSb were very high: 30 mg/L for SbIII and
TMSb, 40 mg/L for SbV. De Gregori et al. (35) presented an improvement on the simultaneous separation the three Sb species
with HPLC–HG –AFS detection. The detection limits for SbIII,
SbV and TMSb were 0.07, 0.13 and 0.13 mg/L, respectively, but
the method was only applied to sea water samples, and never
to a highly complicated matrix such as soil or sediment
samples. In recent years, Quiroz et al. (45) reported an
improved methodology using post-column pre-reduction HG –
AFS with L-cysteine as reduction reagent. This method was
applied to the analysis of Sb species in soil samples and LODs
of SbIII and SbV were improved from 0.1 and 0.3 mg/L to 0.07
and 0.07 mg/L. LODs for TMSb was still high at 1.0 mg/L.
Therefore, the LODs were not improved for each species. In
this study, the LODs for simultaneous analysis of SbIII, SbV and
TMSb with the ICP –MS detection system were improved to
0.03, 0.02 and 0.05 mg/L, respectively. LODs were generally
one order of magnitude lower than the literature has previously
reported (Table III).
Precision
Precision was established in terms of repeatability. Repeatability
was calculated as the percent relative standard deviation (RSD)
from six peak area measurements of the standard solution containing the three Sb species at concentrations of 5, 10 and
10 mg/L for SbV, SbIII, and TMSb, respectively. The repeatability
was good, at less than 6% RSD.
396 Ge and Wei
Extraction efficiency and recovery studies
One of the problems for Sb speciation is the lack of certified
reference materials, which makes validating the analytical
methods challenging for Sb speciation. However, spiked recovery studies allow evaluation of the proposed methodology (47).
In addition, extraction efficiency is another parameter used for
evaluation.
The extraction efficiency was obtained by first adding the
concentrations of three detected Sb species, then dividing by
the total Sb concentration measured in the soils (839 mg/kg).
The spiked recovery was defined as detected Sb species as a
percentage of the spiked concentrations.
Consecutive extraction times directly affect the length of the
extraction procedure, thereby affecting the stability of the Sb
forms during the extraction process. Therefore, in this study,
each sample was extracted one, two or three times.
Supernatants of each extraction were combined for the detection of Sb to compare the effect of the number of extractions
on the extraction efficiency.
The extraction efficiencies of two extractions were obviously
higher than those of one extraction (Figure 6). However, no
further improvement in the average extraction efficiency was
observed with three extractions. The average extraction efficiency of two extractions reached 53%. Decreased spiked recoveries of SbIII and TMSb were found in three extractions,
whereas increased spiked recovery of SbV was observed. This
result might be because of speciation transformation among
the Sb species. The spiked recoveries for the three tested Sb
species ranged from 88 to 118%.
The extraction efficiencies of different extractants for Sb
have been reported. Generally the extraction efficiencies have
been shown to be lower than 1% when using water (17),
water–methanol or acetic acid (15). Zheng et al. (40)
improved the extraction efficiency of Sb to 10% by using EDTA
or phosphate as the extractants. Later, Zheng et al. (36)
Figure 6. Extraction efficiencies and spiked recoveries for SbIII, SbV and TMSb as a
function of the number of citric acid extractions. Extractions were combined for each
detection. The tested levels of SbIII, SbV and TMSb were 25, 5 and 25 mg/L,
respectively; the mobile phase was 200 mmol/L ammonium tartrate with 4%
methanol at pH5.0 using isocratic elution procedure. The flow rate of the mobile
phases was 1.2 mL/min.
Figure 7. Chromatogram of soil samples in citric acid extracts from the Xikuangshan
mine area. Analysis was performed using HPLC –ICP–MS with an ammonium
tartrate isocratic elution program and a citric acid extract.
Table IV
Extraction Efficiency of Antimony Species
Extraction efficiency (%)
Extractant
Reference
,0.1
0.5
,20
10.7
16.1
26.9
0.7-37
62
55
Methanol – water or acetic acid
Water
Water
Water
Phosphate
EDTA
Water
Citric acid
Citric acid
Ulrich (15)
Lintschinger et al. (17)
Lindemann et al. (48)
Zheng et al. (40)
Zheng et al. (40)
Zheng et al. (40)
Koch et al. (49)
De Gregori et al. (29)
This study
improved the extraction efficiency of Sb up to 35% using
microwave-assisted extraction under the conditions of
26 mmol/L citric acid with a microwave power of 100 W for
20 min, but they also noted that the sum of the Sb species
determined with the HPLC–ICP –MS method was only 21% of
the total concentration of Sb in the Sb-containing sample, indicating that there were hidden Sb species in the citrate extractions. In recent years, the extraction efficiency of Sb from
marine biota was improved up to 62% using the 100 mmol/L
citric acid as the extractant with a gradient elution procedure.
Likewise, De Gregori et al. noted the presence of unknown
species that either did not elute from the column or were not
detected (29). In this study, an unknown Sb peak was found
during the soil sample chromatography (Figure 7); this might
be the hidden Sb species described by Zheng et al. (36),
which was eluted by the mobile phase as ammonium tartrate
solution. The extraction efficiencies in this study were lower
than those obtained by De Gregori et al. (29), which might be
attributed to matrix differences between marine biota (29) and
soils in this study. Compared to previous studies, the extraction
efficiency in this study was improved (Table IV). However, the
extraction efficiency is still distinctly inferior to those for
arsenic (21).
Application
The extraction procedure with two repetitions was suitable for
the speciation analysis of Sb species in soil samples (Figure 7).
The first and third peaks could be assigned to SbV and SbIII, respectively; however, the second peak could not be assigned to
TMSb. According to the results of X-ray absorption fine structure (XAFS) detection (unpublished data), the possibility of the
existence of TMSb in the soils was excluded. On the other
hand, because TMSb should be stable during extraction and
analysis (18, 38), the additional test with a TMSb-spiked soil
sample also clearly showed that only the spiked soil sample
demonstrated a TMSb peak. In addition, because the recovery
of spiked TMSb was fairly high (105%), the possible existence
of TMSb in the soil sample (Figure 8) was excluded. Therefore,
Figure 8. Chromatography of the Sb-contaminated soil sample in citric extracts with TMSb: non-spiked (A); spiked (B). The spiked TMSb was 22 mg/kg.
Simultaneous Analysis of SbIII, SbV and TMSb by High Performance Liquid Chromatography– Inductively Coupled Plasma – Mass Spectrometry Detection 397
an unknown Sb species might exist in the soils from the
Xikuangshan Sb mine. The consistency of elution order and
times for the three Sb species between the standard and the
soil sample indicated that the optimal condition obtained for
Sb speciation in the aqueous standard is applicable to citric
acid soil extracts.
Previous studies in this mining area focused on inorganic
SbIII and SbV, and no unknown Sb species has been reported
(50– 51). SbV has been shown to be the dominant valence in
soil (51), which agrees with the results from other studies (45,
52 –53). The primary Sb species in this study could not be confirmed due to the problem of quantifying unknown Sb species.
Significant concentrations of the SbIII species were detected in
the soils from the Xikuangshan Sb mine in this study, which is
consistent with the results of the authors’ X-ray absorption
near-edge structure (XANES) study (unpublished data). The
results were also in agreement with those of Dula
Amarasiriwardena (unpublished data for Sb speciation in
Xikuanghsan soils; personal communication). The results were
not in agreement with other reports, in which only low concentrations of SbIII species were detected in soils from Sb
mines (45, 52 –54). These results may indicate that Sb species
in the soils of the Xikuangshan Sb mine are quite different
from those near other mines, which deserves further study.
Similar unknown Sb species were found in Pteris vittata, an
arsenic hyperaccumulating plant (30), which raises the question of whether it is the same Sb species that this study
detected in the soil samples. If they are the same, does the
plant directly take up this Sb species or does the plant take up
inorganic Sb but transform it into the other unknown species?
These questions are interesting and deserve further investigation to completely elucidate the Sb speciation in the soil
samples of the Xikuangshan mine.
Conclusions
The use of a PRP-X100 anion exchange column and an isocratic elution consisting of ammonium tartrate and 4% methanol
permitted a reliable and straightforward simultaneous separation of SbIII, SbV, and TMSb species in soil samples. This separation relied on the experimental conditions for the separation
of Sb species by HPLC and their detection by ICP –MS, as optimized in this study. The optimized results provide generally
shorter retention times, small peak broadening, good separation and lower LODs than the few previous studies on Sb speciation. Improved extraction efficiencies were obtained with
citric acid. In addition to SbIII and SbV, an unknown Sb species
was found in the soil from the Xikuanghsan Sb mine. Further
investigations will be conducted on the application of the optimized method to Sb speciation on additional environmental
samples.
Acknowledgments
This study was financially supported by the National Natural
Science Foundation of China (40971264; 10979052).
398 Ge and Wei
References
1. Kuroda, K., Endo, G., Okamoto, A., Yoo, Y.S., Horiguchi, S.;
Genotoxicity of beryllium, gallium and antimony in short-term
assays; Mutation Research, (1991); 264(4): 163–170.
2. Amereih, S., Meisel, T., Kahr, E., Wegscheider, W.; Speciation analysis
of inorganic antimony in soil using HPLC-ID-ICP-MS; Analytical
and Bioanalytical Chemistry, (2005); 383(7-8): 1052– 1059
3. Miravet, R., López-Sánchez, J.F., Rubio, R.; New considerations about
the separation and quantification of antimony species by ion chromatography –hydride generation atomic fluorescence spectrometry; Journal of Chromatography A, (2004); 1052(1-2): 121–129.
4. Liu, B.J., Wu, F.C., Li, X.L., Fu, Z.Y., Deng, Q.L., Mo, C.L., et al.;
Arsenic, antimony and bismuth in human hair from potentially
exposed individuals in the vicinity of abtimony mines in southwest
China; Microchemical Journal, (2010); 97(1): 20– 24.
5. He, M.C., Wang, X.Q., Wu, F.C., Fu, Z.Y.; Antimony pollution in
China; Science of the Total Environment, (2012); 421-422: 41 –50.
6. Srogi, K.; Mercury content of hair in different populations relative
to fish consumption; Reviews of Environmental Contamination
and Toxicology, (2007); 189: 107– 130.
7. Telford, K., Maher, W., Krikowa, F., Foster, S., Ellwood, M.J., Ashley,
P.M., et al.; Bioaccumulation of antimony and arsenic in a highly
contaminated stream adjacent to the Hillgrove Mine, NSW,
Australia; Environmental Chemistry, (2009); 6(2): 133– 143.
8. Tighe, M., Ashley, P., Lockwood, P., Wilson, S.; Soil, water and
pasture enrichment of antimony and arsenic within a coastal floodplain system; Science of the Total Environment, (2005); 347(1-3):
175–186.
9. Kentner, M., Leinemann, M., Schaller, K.H., Weltle, D., Lehnert, G.;
External and internal exposure in starter battery production;
International Archives of Occupational and Environmental
Health, (1995); 67(2): 119–123.
10. Filella, M., Belzile, N., Chen, Y.W.; Antimony in the environment: A
review focused on natural waters I. Occurrence; Earth-Science
Reviews, (2002); 57(1/2): 125– 176.
11. Gebel, T.; Arsenic and antimony: comparative approach on mechanistic toxicology; ChemicoBiological Interactions, 1997; 107(3):
131– 144.
12. Youn-Joo, A., Minjin, K.; Effect of antimony on the microbial
growth and the activities of soil enzymes; Chemosphere, (2009);
74(5): 654– 659.
13. Takayanagi, K.; Acute toxicity of waterborne Se(IV), Se(VI), Sb(III),
and Sb(V) on Red Seabream (Pargus major); Bulletin of
Environmental Contamination and Toxicology, (2001); 66(6):
808– 813
14. Nash, M.J., Maskall, J.E., Hill, S.J.; Methodologies for determination
of antimony in terrestrial environmental samples; Journal of
Environmental Monitoring, (2000); 2(2): 7 –109.
15. Ulrich, N.; Speciation of antimony(III), antimony(V) and trimethylstiboxide by ion chromatography with inductively coupled plasma
atomic emission spectrometric and mass spectrometric detection;
Analytica Chimica Acta, (1998); 359(3): 245– 253.
16. Lintschinger, J., Schramel, O., Kettrup, A.; The analysis of antimony
species by using ESI-MS and HPLC-ICP-MS; Fresenius’ Journal of
Analytical Chemistry, (1998); 361(2): 96 –102.
17. Lintschinger, J., Koch, I., Serves, S., Feldmann, J., Cullen, W.R.;
Determination of antimony species with high-performance liquid
chromatography using element specific detection; Fresenius’
Journal of Analytical Chemistry, (1997); 359(6): 484– 491.
18. Dodd, M., Pergantis, S.A., Cullen, W.R., Li, H.; Antimony speciation in
freshwate -r plant extracts by using hydride generation gas chromatography mass spectrometry; Analyst, (1996); 121(2): 223– 228.
19. Gurleyuk, H., VanFleetStalder, V., Chasteen, T.G.; Confirmation of the
biomethylation of antimony compounds; Applied Organometallic
Chemistry, (1997); 11(6): 471– 483.
20. López-Garcı́a, I., Sánchez-Merlos, M., Hernández-Córdoba, M.;
Arsenic and antimony determination in soils and sediments by
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
graphite furnace atomic absorption spectrometry with slurry sampling; Spectrochimica Acta Part B: Atomic Spectroscopy, (1997);
52(4): 437– 443.
Krachler, M., Emons, H.; Speciation analysis of antimony by highperformance liquid chromatography inductively coupled plasma
mass spectrometry using ultrasonic nebulization; Analytica
Chimica Acta, (2001); 429(1): 125–133.
Krachler, M., Emons, H.; Potential of high performance liquid chromatography coupled to flow injection hydride generation atomic
absorption spectrometry for the speciation of inorganic and
organic antimony compounds; Journal of Analytical Atomic
Spectrometry, (2000); 15(3): 281–285.
Lindemann, T., Prange, A., Dannecker, W., Neidhart, B.;
Simultaneous determination of arsenic, selenium and antimony
species using HPLC/ICP-MS; Fresenius’ Journal of Analytical
Chemistry, (1999); 364(5): 462–466.
Zheng, J., Shibata, Y., Furuta, N.; Antimony speciation in environmental samples by using high-performance liquid chromatography
coupled to inductively coupled plasma mass spectrometry;
Analytical Sciences, (2000); 16(1): 75– 80.
Guy, A., Jones, P., Hill, S.J.; Identification and chromatographic separation of antimony species with a-hydroxy acids; Analyst, (1998);
123: 1513– 1518.
Ferreira, H.S., Ferreira, S.L.C., Cervera, M.L., Guardia, M.D.L.;
Development of a nonchromatographic method for the speciation
analysis of inorganic antimony in mushroom samples by hydride
generation atomic fluorescence spectrometry; Spectrochimica Acta
Part B, (2009); 64(6): 597– 600.
Miravet, R., López-Sánchez, J.F., Rubio, R.; Leachability and analytical
speciation ofantimony in coal fly ash; Analytica Chimica Acta,
(2006); 576(2): 200– 206.
Potin-Gautier, M., Pannier, F., Quiroz, W., Pinochet, H., de Gregori, I.;
Antimony speciation analysis in sediment reference materials using
high-performance liquid chromatography coupled to hydride generation atomic fluorescence spectrometry; Analytica Chimica Acta,
(2005); 553(1-2): 214– 222.
De Gregori, I., Quiroz, W., Pinochet, H., Pannier, F., Potin-Gautier, M.;
Speciation analysis of antimony in marine biota by HPLC-(UV)HG-AFS: Extraction procedures and stability of antimony species;
Talanta, (2007); 73 (3): 458– 465.
Müller, K., Daus, B., Mattusch, J., Stärk, H.J., Wennrich, R.;
Simultaneous determination of inorganic and organic antimony
species by using anion exchange phases for HPLC–ICP-MS and
their application to plant extracts of Pteris vittata; Talanta,
(2009); 78(3): 820– 826.
Amereih, S., Meisel, T., Scholger, R., Wegscheider, W.; Antimony speciation in soil samples along two Austrian motorways by HPLC-ICP-MS;
Journal of Environmental Monitoring, (2005); 7(12): 1200–1206.
Duester, L., Diaz-Bone, R.A., Kosters, J., Hirner, A.V.; Methylated
arsenic, antimony and tin species in soils; Journal of
Environmental Monitoring, (2005); 7(12): 1186–1193.
Hansen, H. R., Pergantis, S. A.; Analytical techniques and methods
used for antimony speciation analysis in biological matrices; Journal
of Analytical Atomic Spectrometry, (2008); 23(10): 1328– 1340.
Ulrich, N., Shaked, P., Zilberstein, D.; Speciation of antimony(III)
and antimony(V) in cell extracts by anion chromatography/inductively coupled plasma mass spectrometry; Fresenius’ Journal of
Analytical Chemistry, (2000); 368(1): 62– 66.
De Gregori, I., Quiroz, W., Pinochet, H., Pannier, F., Potin-Gautier, M.;
Simultaneous speciation analysis of Sb(III), Sb(V) and
(CH3)(3)SbCl(2)by high performance liquid chromatographyhydride generation-atomic fluorescence spectrometry detection
(HPLC-HG-AFS): Application to antimony speciation in sea water;
Journal of Chromatography A, (2005); 1091(1-2): 94– 101.
Zheng, J., Iijima, A., Furuta, N.; Complexation effect of antimony
compounds with citric acid and its application to the speciation of
antimony(III) and antimony(V) using HPLC-ICP-MS; Journal of
Analytical Atomic Spectrometry, (2001); 16(8): 812– 818.
37. Ulrich, N.; Study of ion chromatographic behavior of inorganic and
organic antimony species by using inductively coupled plasma
mass spectrometric (ICP-MS) detection; Fresenius’ Journal of
Analytical Chemistry, (1998); 360(1-2): 797– 800.
38. Craig, P.J., Forster, S.N., Jenkins, R.O., Miller, D.; An analytical
method for the detection of methylantimony species in environmental matrices: methylantimony levels in some UK plant material;
Analyst, (1999); 124(8): 1243–1248.
39. Shi, J.M., Wang, W.; Detecting arsenic and antimony using by
Hydride
Generation-Atomic Florescence Spectrometry; NonFerrous Mining and Metallurgy (in Chinese), (2011); 26(4): 51–52.
40. Zheng, J., Ohata, M., Furuta, N.; Studies on the speciation of inorganic and organic antimony compounds in airborne particulate
matter by HPLC-ICP-MS; Analyst, (2000); 125(6): 1025– 1028.
41. Zhu, Y., Zhu, L.Z., Xu, S.J.; Effects of organic solvents on some
anions chromatography; Journal of Hangzhou University
(Natural Science), (1991); 18(4): 438– 444.
42. Dean, T.H., Jezorek, J.R.; Demonstration of simultaneous anionexchange and reversed-phasebehavior on a strong anion-exchange
column. Journal of Chromatography A, (2004); 1028(2):
239– 245.
43. Wei, C.Y., Deng, Q.J., Wu, F.C., Fu, Z.Y., Xu, L.B.; Arsenic, antimony,
and bismuth uptake and accumulation by plants in an old antimony
Mine, China; Biological Trace Element Research, (2011); 144(1-3):
1150–1158.
44. Takaoka, M., Fukutani, S., Yamamoto, T., Horiuchi, M., Satta, N.,
Takeda, N., et al.; Determination of chemical form of antimony in
contaminated soil around a smelter using X-ray absorption fine
structure; Analytical Sciences, (2005); 21(7): 769–773.
45. Quiroz, W., Olivares, D., Bravo, M., Feldmann, J., Raab, A.; Antimony
speciation in soils: Improving the detection limits using postcolumn pre-reduction HG-AFS (HPLC/pre-reduction/HG-AFS);
Talanta, (2011); 84(2): 593– 598.
46. Morgan, G.T., Davies, G.R.; Proceedings of the Royal Society A,
(1926); 110: 523– 534.
47. Careri, M., Mangia, A.; Validation and qualification: the fitness for
purpose of mass spectrometry-based analytical methods and analytical systems; Analytical and Bioanalytical Chemistry, (2006);
386(1): 38– 45.
48. Lindemann, T., Prange, A., Dannecker, W., Neidhart, B.; Stability
studies of arsenic, selenium, antimony and tellurium species in
water, urine, fish and soil extracts using HPLC/ICP-MS; Fresenius’
Journal of Analytical Chemistry, (2000); 368: 214–220.
49. Koch, I., Wang, L., Feldmaw, J., Andrews, P., Reimer, K.J., Cullen,
W.R.; Antimony species in environmental samples; International
Journal of Environmental Analytical Chemistry, (2000); 77:
111– 113.
50. Liu, F.Y., Chris Le, X., McKnight-Whitford, A., Xia, Y.L., Wu, F.C.,
Elswick, E., et al.; Antimony speciation and contamination of waters
in the Xikuangshan antimony mining and smelting area, China;
Environmental Geochemistry and Health, (2010); 32(5):
401– 413.
51. Okkenhaug, G., Zhu, Y.G., Luo, L., Lei, M., Li, X., Mulder, J.;
Distribution, speciation and availability of antimony (Sb) in soils and
terrestrial plants from an active Sb mining area; Environmental
Pollution, (2011); 159(10): 2427–2434.
52. Lintschinger, J., Michalke, B., Schulte-Hostede, S., Schramel, P.;
Studies on speciation of antimony in soil contaminated by industrial
activity; International Journal of Environmental Analytical
Chemistry, (1998); 72(1): 11– 25.
53. Steely, S., Amarasiriwardena, D., Xing, B.; An investigation of inorganic antimony species and antimony associated with soil humic
acid molar mass fractions in contaminated soils; Environmental
Pollution, (2007); 48(2): 590– 598.
54. Ure, A.M., Thomas, R.,d, Littlejohn, D.; Ammonium acetate extracts
and their analysis for the speciation of metal ions in soils and sediments; International Journal of Environmental Analytical
Chemistry, (1993); 5: 65 –84.
Simultaneous Analysis of SbIII, SbV and TMSb by High Performance Liquid Chromatography– Inductively Coupled Plasma – Mass Spectrometry Detection 399