Abstract
Biogenic (e.g. phytochelatins, porphyrins, DOM)
as well as anthropogenic (e.g. NTA, EDTA, phospho-
nates) chelators affect the mobility and cycling of heavy
metals in environmental waters. Since such chelators can
form strongly bound anionic heavy metal complexes that
are stable and highly mobile, anion-exchange chromatog-
raphy coupled to ICP–MS was investigated. A narrow
bore HPLC system was connected to a micro concentric
nebuliser for in-line sample introduction. A new chro-
matographic procedure based on a synthetic hydrophilic
quaternary ammonium anion exchanger in combination
with nitrate as a strong eluent anion, and gradient elution,
provided high separation selectivity and a large analytical
window. Low detection limits (nmol L
–1
) were achieved
by on-column matrix removal and sample preconcentra-
tion. This allowed the method to be successfully applied
to different environmental research areas. In ecotoxico-
logical studies of heavy metal effects on algae low con-
centrations of metal EDTA complexes were determined in
nutrient solutions without interference from high (buffer)
salt concentrations. In groundwater, infiltrated by a pol-
luted river, mobile metal EDTA species were observed. In
river water of different pollution levels beside CuEDTA
other anionic Cu-complexes were found in nmol L
–1
con-
centrations.
Keywords Gradient anion-exchange chromatography ·
Metal chelates · Metal speciation · Environmental water
Introduction
From anthropogenic as well as from biological activities
various complexing agents are released into the hydro-
sphere [1] where they, together with geochemical ligands,
constitute the metal complexation chemistry [2]. Heavy
metal speciation of this kind of chelator is of particular in-
terest since, at the same concentrations level as the metals,
strong binding chelators fully dominate metal speciation.
Among them, aminopolycarboxylates [1] were found,
such as the persistent EDTA [3] and phosphonates [4].
The mobility and (eco)toxicological effects of heavy met-
als depend on the chemical properties of the metal species
in surface interactions [5, 6] and exchange kinetics [7, 8]
which cannot be calculated from total concentrations [9].
Most stable and mobile chelators and their heavy met-
als complexes are present in the environment in anionic
form. This allows an ion-exchange procedure to be di-
rectly applied to analytes without derivatisation prior to
analysis. Because of the stronger ion-interaction forces,
an ion-exchange-based separation procedure can provide
outstanding performance regarding matrix-compatibility,
selectivity, and sensitivity with on column sample precon-
centration. Comparing different liquid chromatographic
methods it was concluded [10] that ion exchange is one of
the most useful chromatographic techniques for specia-
tion in environmental samples.
The potential of capillary electrophoresis (CE) was
also evaluated. The separation of some metal EDTA and
NTA complexes was reported [11, 12] that were detected
by UV and laser-induced fluorescence [13].
Anion-exchange chromatography was also reported for
separation of negatively charged metal complexes with
carbonate eluents at pH>9 detected by conductivity [14,
15] and UV–Vis [16, 17]. Suppression of this carbonate
eluent improved the signal-to-noise ratio in ESI–MS de-
tection [18] and allowed the determination of nano-molar
concentrations. However carbonate elutes anionic metal
complexes together with sample ions NO
3
–
and SO
4
2–
at a
pH where, e.g., Al, Fe, and Zn chelates are not stable. The
pH cannot be adjusted below 9 without substantial loss in
eluent strength that is proportional to the increase of the
weak eluent anion HCO
3
–
.
This work reports on the extended application of an-
ion-exchange chromatography in metal speciation that
Adrian A. Ammann
Speciation of heavy metals in environmental water
by ion chromatography coupled to ICP–MS
Anal Bioanal Chem (2002) 372 : 448–452
DOI 10.1007/s00216-001-1115-8
Received: 29 May 2001 / Revised: 30 July 2001 / Accepted: 25 August 2001 / Published online: 20 December 2001
SPECIAL ISSUE PAPER
A.A. Ammann (✉)
Swiss Federal Institute for Environmental Science
and Technology (EAWAG),
Postfach 611, 8600 Dübendorf, Switzerland
e-mail: adrian.ammann@eawag.ch
© Springer-Verlag 2001
covers the range of natural water pH 6–8 and has broad
selectivity to separate complexes of diverse chelators in
the same run. Coupling to an ICP–MS was used for ele-
ment identification and low detection limits were obtained
for heavy metals [19].
Experimental
Chemicals and samples
Nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid di-
sodium salt (EDTA), N-(2-hydroxyethyl)ethylenediaminetriacetic
acid (HED3A), and citric acid were purchased from Fluka or
Merck in the highest purity available. Ethylenediaminetriacetic
acid (ED3A) and ethylenediaminedisuccinate (EDDS) was a kind
gift from M. Bucheli-Witschel and T. Egli (EAWAG). Ethylenedi-
aminetetramethylenephosphonic acid (EDTMP) was obtained as
Dequest 2041 (Monsanto) from H. Felber (EMPA, St. Gallen).
Metal complexes stock solutions (1 mmol L
–1
) were prepared
by adding equimolar ligand solutions to the diluted AAS-standard
(J. T. Baker) solution followed by controlled neutralisation. After
a few days at room temperature the solutions are ready to use.
Fe-chelates were stored in the dark. Dilutions from stocks were
freshly prepared daily for concentrations >0.1 µmol L
–1
, or imme-
diately before use for lower concentrations.
Calibration curves were obtained by the method of standard ad-
ditions to the samples.
Samples were passed through prewashed filters (0.45 µm) in
the field and stored at 4 °C in the dark.
Chromatography
A totally PEEK system was used, consisting of a DX-500 (Dionex)
microbore gradient pump, a Rheodyne (9126) injection valve
(270 µL loop) and a low capacity (0.011 mequiv.) microbore anion
separator column (AS11, 250×2 mm, Dionex) with hydrophilic
quaternary alkanol ammonium anion exchange sites. The eluent
flow (440 µL min
–1
) was chosen as optimum for fast separation
and highest nebuliser mass transfer. Eluents were prepared CO
2
-
free and kept under He over pressure. Eluent pH was adjusted with
conc. NH
4
OH. Gradients were mixed linearly (0–8 min) from wa-
ter and 200 mmol L
–1
NH
4
NO
3
solution and the pH monitored sev-
eral times a day at the pump head drain valve after mixing the sol-
vent lines. Metals were removed on line from the eluents by pas-
sage through a column (50×4 mm) filled with strong cation ex-
changer 50W-X8 (Bio-Rad). The stability and reproducibility of
flow rate, evolving gradient, and nebulisation was observed on
m/z=30. Sample preconcentration was achieved either by use of
the sample loop or by injections of larger volumes (1–5 mL) on
AG11 (50×4 mm, Dionex) in the loop position. Method detection
limits depended on several parameters – the sample volume pre-
concentrated, the actual metal background in the eluent as well as
on the column and on the detected isotope sensitivity. However the
dominating parameter was the sample volume that can be precon-
centrated. It is limited by the combination of the capacity of the
preconcentrator column and the content of sample anions (Cl
–
,
NO
3
–
, CO
3
2–
, SO
4
2–
). Metal deposited on the column from lower
stability complexes (logK<9) [20] and metal remobilisation was
controlled as recommended by Hering [21] and Szpunar [22] for
size-exclusion chromatography. Only free chelator was able to
mobilise metal from the column but not chelator coordinated to a
metal, e.g. EDTA mobilised metal but CaEDTA did not.
Coupling and ICP–MS measurements
The column was connected to a micro concentric nebuliser
MCN-100 M2 (Cetac) mounted on Scott-type Ryton double pass
spray chamber. The ICP–MS (Elan 5000, Perkin–Elmer–Sciex)
sensitivity and ion optics were optimised by measuring a solution
of Li, Rh, and U (10 µg L
–1
) delivered by a peristaltic pump
(Gilson) at the chromatography flow rate. Other ICP–MS operat-
ing conditions were as follows: RF power: 1100 W, Ar gas flows
(L min
–1
): plasma (15), auxiliary: (1), nebuliser: (0.8–1.0).
For fast sequential detection dwell times (25–100 ms), the
number of replicates and the number of detected m/z values were
selected according to the chromatogram time needed to elute com-
ponents detected by at least 10 points/peak. Data were acquired in
the Graphic mode. Single-mass chromatograms and peak integrals
were obtained from Chromafile MSplus software (LabControl).
Element isotopes were detected at the m/z values of minimal inter-
ference (C: 12, Fe: 57, Co: 59, Ni: 60, Cu: 65, Zn: 66, Cd: 111 and
Pb: 208) or, for higher sensitivity, more abundant isotopes were
chosen (Cu: 63, Cd: 114), if chromatography showed no coelution
of species that contain interfering elements. During acquisition of
high signals (e.g. m/z 12 or 30) the detector had to be desensitised
(omni range 10–20) because detector overflow in one chro-
matogram caused a data cut off in all other chromatograms of the
same run during data transfer with Chromfile.
449
Table 1 Species characteris-
tics and measured retention
times (t
R
) in isocratic elution
Species distribution (%) was
calculated by Vminteqa [33]
for pH 7 and 8 (50 mmol L
–1
NH
4
NO
3
, free chelator
0.1 mmol L
–1
, metal species
1 µmol L
–1
). LogK (I=0.1,
25 °C) values taken from Ref.
[24]
Ligand/Complex Species Distribution t
R
[min] LogK-
charge (eluent, range
% (pH 7) % (pH 8) mmol L
–1
)
NTAH –2 99.9 98.6 4.0 (20)
MeNTA Me(NTA)
2
–1...–4 99–100 89–100 4.0–5.5 (20) 10–16
HED3AH –2 3.0 (20)
MeHED3A –1 2.0 (20) 13–20
ED3AH –2 3.0 (20)
MeED3A –1 2.0 (20)
EDTAH –3 93.4 99.3 3.5 (50)
MeEDTA –2 99–100 100 2.0 (50) 15–25
EDDSH –3 3.0 (50)
MeEDDS –2 2.0 (50) 12–16
Citr –3 92.7 99.2 4.0 (70)
MeCitr –2 – 4–6
EDTMP –5.4 4.7 (130)
MeEDTMP –5.0...–5.5 3.5–4.0 (130) 15–23
NH
4
+
99.5 95.6 –
NH
3
0.5 4.4 –
Results and discussion
The hydrophilic column used exhibited a high affinity for
chelating agents and its complexes. This afforded a strong
eluent anion in high concentrations which should still be
compatible with ion extraction from an ICP into a MS.
Ammonium nitrate was an optimal eluent because it is
thermally not stable (decomposing to gaseous compo-
nents) and has a high affinity [23] to hydrophilic anion ex-
changers. Two superior properties of this eluent should be
emphasised:
1. the pH can be adjusted from acidic to slightly basic
without impairing the eluent strength, and
2. the high concentration tolerated by the plasma enabled
the powerful gradient elution without additional space
charge effects [20].
The chromatographic behaviour of the chelators and their
metal complexes analysed (Table 1) represented several
aspects of complexation chemistry as well as environmen-
tal concern. The anionic character of the species varied
between –1 and –5.5 and the ligands represent a broad
range of equilibrium stability constants [24] (see Table 1).
The metals analysed play either a crucial role in metal
speciation (Fe, Zn, Cu [9]) or represent metals reacting
with different rate constants [25], e.g. Ni and Pb as slowly
and fast reacting metals, repectively.
Table 1 gives an overview of isocratic eluent concen-
trations needed to elute uncomplexed chelators and their
metal complexes within a reasonably short retention time
and within a peak that can be integrated. As can be con-
cluded from the broad range of isocratic concentrations
the eluent provided high selectivity. The affinity of the
column for free metals required a complex stability of
logK>10 for metal complexes in order to survive the chro-
matographic conditions. According to an established an-
ion-exchange separation mechanism [20] more highly
charged species required higher eluent concentrations of
about 32 mmol L
–1
NO
3
–
/charge unit. So a particular iso-
cratic eluent provided high selectivity but only a narrow
separation window. Only one or maybe two chelators and
their complexes can be separated in the same run. How-
ever in environmental waters several chelators including
partially degraded compounds [1] (e.g. loss of a carbo-
xylic group [26, 27, 28]) are more likely to occur. There-
fore gradient elution was investigated and successfully
separated, in the same run, the chelators and their com-
plexes given in Table 1 [20]. Gradients were also used for
chemical speciation in different environmental research
areas as described below.
Ecotoxicological investigations with algae
In ecotoxicological studies of heavy metal effects on al-
gae, buffered metals in nutrient solutions are used to
maintain a constant metal supply [29]. This is achieved by
metal complexation of sufficient stability. A nutrition me-
dia which used EDTA for metal stabilisation was analysed
(see Fig. 1). It contained the metal–EDTA species at low
concentrations (component (µmol L
–1
): Co (0.05), Cu
(0.1), Mn (1.0), Zn (0.1), Fe (0.9)), MoO
4
2–
(0.08) and
EDTA (20), beside high salt concentrations (mmol L
–1
),
(morpholinopropanesulfonic acid (MOPS) (10), CaCl
2
(0.5), MgSO
4
(0.15), NaHCO
3
(1.2), KHPO
4
(0.05),
NaNO
3
(1.0)). Molybdate cannot react with EDTA and re-
mained stable during incubation with algae. It therefore
could be used as an internal standard. The large amount of
organic buffer salt MOPS was well separated (peak 12C
in Fig. 1) and did not impair the separation and quantifica-
tion of MeEDTA species. During storage of the medium
the MeEDTA species remained stable. Cobalt clearly
showed ligand exchange that was already present after
mixing the medium, whereas after incubation with algae
most Cu-, Zn- and FeEDTA disappeared, as demonstrated
in Fig. 1B. Without any sample pretreatment (except fil-
tration) the large loop injection was sufficiently sensitive.
The method was ideally suited to observation of a bunch
of single anionic metal species and its interaction with al-
gae. The dynamic and exact reaction path is under further
investigation.
450
Fig. 1 Gradient (20–100 mmol L
–1
) anion-exchange chromato-
grams of algae nutrient media containing EDTA-buffered heavy
metals. A. Stability of the MeEDTA complexes after 30 days at
4 °C in the dark. B. The same media filtered after incubation with
algae at room temperature and with illumination
Mobility of MeEDTA from river water into groundwater
Metal EDTA species could be observed at the well inves-
tigated site Glattfelden [30] where the polluted River Glatt
[31] is infiltrating permanently (~0.5 m
3
day
–1
m
–2
) into the
top groundwater layer. Samples were taken in the mixing
zone (16 m from river bank and at 6 m depth). In the river
water 30–90 nmol L
–1
EDTA was usually determined [13].
Figure 2 depicts chromatograms from preconcentrated
samples (2 mL) and shows metal EDTA species identified
and quantified by standard addition (NiEDTA 6 nmol L
–1
,
CuEDTA 16 nmol L
–1
, and FeEDTA 19 nmol L
–1
). This
provided direct information on metal species that has pre-
viously been attempted by determination by EDTA-species
calculation[9].
Cu species in river water
Cu species were determined in a river at four different lo-
cations that represent catchment areas with different pol-
lution levels [32]. Due to low nanomolar concentrations,
river water samples had to be preconcentrated (5 mL).
Measuring both Cu isotopes (
63
Cu and
65
Cu), the occur-
rence of Cu in the species was verified by the maintained
ratio according to their natural abundance. The relative
pollution level at the four sites was reflected by the dis-
solved copper concentration ([Cu]
d
, Fig. 3) which was the
upper limit for the sum of all Cu complexes. At the station
Necker, after running through an almost unpopulated
alpine catchment area, no Cu species was detected. Fur-
ther downstream increasing [CuEDTA
2–
] was detected at
all other locations. At the station Andelfingen, after the
contributions from industrial and agriculture areas, the
highest [CuEDTA
2–
] (=2 nmol L
–1
) was determined; also
present were other yet unidentified Cu complexes – one at
t
R=
2.6 min bearing approx. one charge unit less and an-
451
Fig. 2
Gradient (10–200 mmol L
–1
) anion-exchange chromato-
grams of groundwater polluted by infiltrating river water.
A. Metal EDTA complexes observed in a groundwater sample.
B. The same groundwater after spiking with Cu- and NiEDTA
(10 nmol L
–1
)
Fig. 3 Cu species in the river Thur at sites representing different
pollution levels indicated by increasing dissolved copper concen-
trations ([Cu]
d
) [32]. Filtered samples were used to determine
[Cu]
d
by ICP–MS. Gradient (20–200 mmol L
–1
) elution separated
three Cu species. At t
R
=5.3 min CuEDTA
2–
was identified and
quantified by standard addition to the sample from Andelfingen
452
other (t
R
=5.5 min) of about the same anionic charge as
CuEDTA
2–
. Based on the quantification of Cu the three
Cu-species detected accounted together for 40% of [Cu]
d
.
This is in good agreement with investigations done by
other methods at this site [32]. For example, size fraction-
ation of trace metals by ultra filtration found Cu-binding
ligands in the fractions <10 kD and voltammetric mea-
surements including ligand titration by Cu
2+
observed an
over all conditional stability constant of logK=14.6 for the
Cu-binding ligands.
Conclusions
The use of on column preconcentration and matrix
removal, a very efficient feature of anion exchange gradi-
ent separation, in combination with sensitive on line de-
tection by ICP–MS, provided detection limits of around
10 nmol L
–1
for large-loop injection and 1 nmol L
–1
for in-
jection on a preconcentrator column. The eluent, which
was highly compatible with the ICP, provided a large se-
lectivity for the elution of several anionic metal species
within a few minutes. The eluent pH can be adjusted in a
broad range without compromising eluent strength. Based
on all these features, the procedure presented is likely to
become the method of choice for the search and behaviour
of anionic metal complexes in different matrices and re-
search areas. It can provide accurate concentrations of
single strongly bound metal species, information that is
complementary to voltammetric methods.
Acknowledgements Thanks are due to D. Kistler and R. Schö-
nenberger for sampling the river and groundwater, to B. Wagner
for preparation of the algae medium, and to D. Weirich and L. Sigg
for helpful discussions.
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index.htm
