Talanta

ELSEVIER . Talanta 44 (I 997) I58I- 1604

Analytical methodology for speciation of arsenic in

environmental and biological samples
Marcela Burguera *, Josh Luis Burguera
IVAIQUIM (Venezuelan Andean Institute for Chemical Research), Faculty of Sciences, University of Los Andes, P.O. Box 542,
Mkrida 5101 -A, Venezuela

Received 16 August 1996; received in revised form 18 February 1997; accepted 20 February 1997

Abstract

A literature search on the speciation of arsenic in environmental and biological samples shows an increasing interest
of many researchers in the subject. Because of the low level of arsenic species in real samples, many problems related
with its speciation remain unresolved: species instability during sampling, storage and sample treatment, incomplete
recovery of all species, matrix interferences, lack of appropriate certified reference materials and of sensitive analytical
methods, etc. These aspects are underlined in this paper. The continued development of new analytical procedures
pretending to solve some of these problems claim for an up-to-date knowledge of the recent publications. Therefore,
this paper pretends to review the latest publications on the chemical speciation of arsenic, emphasizing the increasing
activity in the development of accurate and precise analytical methods. In most of the cases, separation and
preconcentration is necessary, followed by element-specific detection for sensitivity improvement. Hydride generation
following separation procedures (e.g., ion-exchange or high performance liquid chromatography) coupled to atomic
absorption or atomic emission detectors proved to have sufficient sensitivity to monitor arsenic exposure, although
restricts the analysis to hydride-forming species. Modified procedures including some kind of heating in the presence
of highly oxidizing agents have proved successful to completely decompose the arsenic containing compounds to
arsenate and so to extend the range of compounds which can be determined by these methods. On-line arrangements
have the additional advantage of avoiding excessive sample handling, although some of them involve numerous steps
and others are too costly to be recommended for routine use. The analytical figures of merits, specially detection
limits are given for most of the methods in order to afford comparison and judge possible applicability. These studies,
which have been approached in many different ways, would lead to knowledge that are determinant in the
understanding of the cycle of this element in environment and of its physiological and toxicological behavior in the
living organisms. 0 1997 Elsevier Science B.V.

Keywords: Arsenic speciation; Environmental and biological samples; Analytical methodology

1. Introduction

The term ‘speciation’ in analytical chemistry,

* Corresponding author. refers to the determination of different oxidation

0039-9140/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved.

PII SOO39-9140(97)00064-7
1582 M. Burguera, J.L. Burguera / Talanta 44 (1997) 1.581- 1604
states of an element that prevail in a certain
specimen or to the identification and quantifica-
tion of the biologically active compounds to
which the element is bound. Sparingly, in the
investigation of toxic effects, the speciation of
small molecules (organometallic compounds) is of
concern, while for the study of the biological
functions, the determination of large molecules
has priority. This knowledge could help to explain
the mobility, storage, retention and toxicity of the
different species in different environments includ-
ing the human body. The planning of the entire
work, from sampling to the interpretation of data
claim for an interdisciplinary research. Such stud-
ies require knowledge in the field of trace element
analysis combined with experience in environmen-
tal and/or biochemical analysis [l].
Previously, the determination of total element
concentrations was considered to be sufficient for
clinical and environmental considerations. Al-
though the total concentration of an element is
still useful to know, and indeed is essential in
many analytical schemes, the determination of
species is an important task. The concentration of
a toxic species is more relevant in the setting of
environmental and biological standards than is
the total elemental concentration. The collection,
treatment and preservation of samples for quanti-
tative analysis of species require careful consider-
ation and planning. The nature of this task is very
different from procedures for ‘total’ element de-
termination, therefore analytical chemists are
faced with very difficult problems in the acquisi-
tion of accurate data. Obviously, any procedure
and apparatus adopted for sampling and the
preservation of samples between the time of col-
lection and the opportunity for analysis, should
not disturb the equilibrium established among the
species [2-41. The introduction of biological con-
tamination is equally undesirable, specially if the
samples have to be stored for long periods; the
presence of microorganisms and traces of oxygen
might accelerate the deterioration of the sample,
producing changes between species [4]. A holistic
approach, which takes into consideration the vari-
ous factors that influence the distribution of spe-
cies of a given element before, during and after
sampling is very much required. This is indeed
essential if the results of experiments are to
provide meaningful information that is indispens-
able for various studies. Speciation analysis in-
volves a complex scheme of operations designed
to simplify the analytical procedures to be imple-
mented, but all, consist, in a way or another, in a
separation step followed by the determination of
the element in the different fractions. Despite the
fact that the topic on speciation has been exten-
sively documented in recent years in numerous
publications, so far, there is no ideal method for
any element speciation that unequivocally ensure
high accuracy of the results. A closed look to the
available literature on this subject shows the need
for the availability of reference materials for qual-
ity control of particular species. Until now, most
of the reference materials available for trace ele-
ment determination in biological and environmen-
tal samples are only certified for the total element
content [5,6].
A very tempting case to study for speciation is
arsenic, which has an interesting behavior since:
(1) it has different valence states, [As(III)/As(V)],
interchangeable under certain conditions; (2) form
small organometallic molecules which are
biomarkers of exposure because, it is well known
that in the mechanism of detoxification, inorganic
arsenic is metabolized to monomethylarsonic acid
(MMAA) and dimethylarsinic acid (DMAA); (3)
is part of other organic compounds which remain
unchanged in the body, like arsenobetaine (AsB),
arsenocholine (AsC), arseno-sugars (ASS), arseno-
lipids (AsL), etc.; (4) form macromolecular
biomolecules, like those of arsenate bound to
transferrin and hemoglobin [7]. The increasing
interest in this topic is reflected in more than 500
papers, exhaustive reviews [8,9] and comprehen-
sive monographs [3,10-121 published in recent
years.
2. Concentration of arsenic species in biological
and environmental samples
Arsenic is widely distributed in biosphere, thus
the mobilization of the element by human activi-
ties (mining, smelting, glass making, refineries or
pesticide manufacture) is estimated to exceed the
 M. Burguera, J.L. Burguera /Talanta 44 (1997) 1.581-1604 1583

Table 1
Common arsenicals and their uses

Compound Formula Uses

Lead arsenate PbHAsO, insecticide

Cupric arsenite (Schoele’s green) Cu(AsO,), pigment
Cupric acetoarsenate (Paris green) Cu(C,H,O,)f3Cu(AsO,), insecticide
3-nitro-4-hydroxy phenylarsonic acid C,H,AsNO, animal growth
Arsenilic acid C,H,AsNO, animal growth and veterinary medicine
Arsenic oxides As,O, and As,O, glass industry
Metallic arsenic As metallurgy
Arsenamide C,,H,,AsNO,S, veterinary medicine
Potassium arsenite (Fowler’s solution) KASO, (1%) medicine
Arsenic sulfide As& fireworks
Gallium arsenide GaAs semiconductors
Diphenylchlorarsines (blue cross) (C,H,),AsCl chemical weapons
Alkyldichlorarsines (mustard gas) RAsCl, chemical weapons
Melarsoprol Medicine (sleeping sickness)

Carbarsone Medicine (intestinal amebiasis)

natural rate (from marine sedimentary rocks, over 60 mg 1- ‘. In some cases, these levels are
weathering volcanic rocks or fossil fuels) by three- two and even three orders of magnitude higher
fold; hence it might be anticipated that the distri- than those of most natural waters consumed by
bution of the element in aquatic ecosystems and man [15,22]. Contamination of water and air near
body fluids and tissues would be closely related to copper smelters in Argentina [15], Chile [16], USA
antropogenic activities [13,14]. As a result of the [17] and Sweden [18] as well as the contamination
extensive use of the numerous arsenic compounds of drinking water in Taiwan [19] and India [20-
(Table l), the element is found in a large variety 23] have been reported in numerous papers. In
of samples (fresh and sea waters, sediments, soils, such samples, As(II1) concentrations are normally
plants, marine organisms, body fluids and tissues, only a small proportion of the total dissolved
etc.), in variable amounts and in a great variety of arsenic [24]. In an attempt to protect aquatic life
species. as well as to establish a control over the implica-
In ground water, for instance, the levels of tions of arsenic concentrations for public health,
arsenic could exceed 20 mg 1~ ‘, while in drinking several international organizations [25-271, have
water the concentrations may vary from 0.05 to prescribed a permissible dissolved, total arsenic
1584 M. Burguera, J.L. Burguera / Talanta 44 (1997) 1.581- 1604
concentration in potable water between 0.008 and
0.05 mg l- ‘. Despite the fact that it was known
that different arsenicals exhibit very distinct toxi-
cological properties, all regulations and reports
related only to total arsenic and none differenti-
ates between the toxic impacts of the various
chemical species of arsenic [ 131. Ingestion of con-
taminated ground water (containing arsenic of
geological origin well above the maximum permis-
sible limit), by people from several districts of
West Bengal, India, resulted in excretion of inor-
ganic arsenic and its metabolites in a proportion
of more than 90%. It was reported that a daily
intake of 1500 ug of inorganic arsenic (equivalent
to drinking 1.5 1 of water with an inorganic
arsenic concentration of 1 mg 1~ ‘) could produce
signs of overt chronic arsenicism in some individ-
uals [22]. Many of the affected people from India,
usually drink more than 5 1 per day and they
showed serious signs of long-term arsenic expo-
sure [20-231.
The concentration of arsenic in sea water is
generally around 2 ug 1 - l with the trivalent spe-
cies being less than 10% of this level, while in
normal soils the total arsenic concentration usu-
ally ranges from 1 to 40 ug g _ ‘, with higher levels
in places where arsenicals are used for insect
control or on disused mines tips. Certain plants
growing on arsenic-enriched soils accumulate ex-
tremely high amounts of the element. Most hu-
man foods contain less than 0.5 ug g-i arsenic,
except those of marine origin which are more
richer in arsenic than others. In marine animals
and algae, because of biotransformation and ac-
cumulation, arsenic is present in organic forms
and its concentration typically ranges from 1 to
100 ug g-1. So, the total amount of arsenic
ingested daily would be influenced by the amount
and type of food included in the diet [28]. For
instance, in rivers and soils it is present as arsen-
ate, As(V), and arsenite, As(II1); both inorganic
species might be subjected to chemically and/or
microbiologically mediated oxidation/reduction
and methylation reactions, resulting in the trans-
formation of inorganic arsenic in organic deriva-
tives; simple methylated species (MMAA and
DMAA) or more complex compounds (AsB,
AsC, ASS, AsL, etc.) [29]. It is now certain that
phytoplankton take up arsenate from aquatic en-
vironments, reduce it to arsenite and methylate
the arsenite to produce MMAA and DMAA
which are then excreted. This represents a mecha-
nism of detoxification. Arsenobetaine, first re-
ported as a major constituent of total arsenic in
the tail muscle of the western rock lobster [30], the
most frequently encountered water-soluble form
of arsenic in higher aquatic organisms, is gener-
ally highly stable and is heavily bioaccumulated.
This compound is believed not to be metabolized;
after ingestion it is rapidly excreted unchanged in
the urine [31]. Some studies, have however,
demonstrated that arsenobetaine is unstable when
irradiated with ultraviolet light alone [32] or in
combination with an oxidizing agent [33]. Higher
organisms also contain trimethylarsine oxide
[29,34] and tetramethylarsonium ion [35,36].
There are also evidences of the presence of ar-
senocholine in seafood from polluted areas [37]
and in dogfish reference material [38], although in
lower concentrations than arsenobetaine. While
some authors believe that this compound is also
absorbed from the diet and it is partially trans-
formed in arsenobetaine and trimethylarsine oxide
[39], Shibata and Morita [36] demonstrated that
trimethylarsonium ion and not arsenocholine was
present in dog&h reference material. Undoubt-
edly, more work is necessary to solve these con-
troversies. Arseno-sugars are others arsenic
compounds in edible seaweed; there are evidences
that the ingestion of seaweed may increase the
urinary concentration of arsenic metabolites, one
of which is DMAA [40]. Also the existence of
arseno-lipids in marine and estuarine biota may
be explained by the esterification of arseno-sugars.
Although there is lack of evidence that the arseni-
cals present in foodstuff of marine origin do not
cause any hazard to human health, the transfor-
mation among species might be taken into ac-
count and for reasons of toxicological reassurance
should be determined by methods which can be
routinely applied. From these statements, it seems
clear that consumption of different kinds of food,
water or beverages, as well as the amounts in-
gested, will affect differently the concentration of
the different arsenic species in urine [34].
 M. Burguera, J.L. Burguera / Talanta 44 (1997) 1.581- 1604
Arsenic is also present in the air at trace
amounts, specially in the areas where its com-
pounds are industrially used or where coal is
burned. Its atmospheric concentration ranges
from about 0.01 to 0.1 ng m ~ 3 in clean areas such
as Antarctica [41] and up to 500 ng m - 3 near
certain industrial environments such as copper
smelters 1421.Particular attention must be paid to
the measurement of these species while maintain-
ing the initial As(III)/As(V) ratio during the sam-
ple collection, preparation and analysis steps. The
average atmospheric concentrations of As(II1)
and As(V) were about 1.6 f 1.4 and 5.4 + 3.3 ng
m -3, respectively, in urban area of Los Angeles.
Size-fractionated samples of atmospheric particu-
late matter were obtained employing a high-vol-
ume dichotomous virtual impactor as a sampler
which allowed for the collection of a sufficient
amount of sample so that both species could be
routinely detected by HGAAS technique. Fine
( < 2.5 urn aerodynamic diameter) and coarse ( >
2.5 urn) particles were collected employing differ-
ent size filters with emphasis on the first fraction
which is most efficiently collected in the lungs.
This would help assess the impact of the poten-
tially toxic species on human health. The data
obtained indicate that both As(II1) and As(V)
species are present in ambient aerosol and that
most of both species (about 75%) were found in
the fine particle fraction. The primary source of
arsenic in air is assumed to be As,O,, formed by
the sublimation of As,O, emitted from stationary
high-temperature combustion processes (e.g.,
glass furnaces, primary metallurgical processes
and fuel oil combustion) [43,44].
There are only limited studies on the arsenic
distribution among different organs and body
fluids. For example, the concentration of arsenic
in urine of subjects without known exposure to
arsenic is generally in the order of lo-20 ug l- ’
while in the blood averages O-12 ug l- ‘, but
intake of a meal of fish or shellfish may increase
the total arsenic concentration to more than 50-
fold, with no [45] or only two-fold [46] increase in
excretion of methylated arsenic species. This in-
validates the use of total urinary arsenic as an
indicator of exposure to inorganic arsenic. In
these cases it is advantageous to supplement the
1585
analysis with a more selective speciation. The
results obtained by the application of such a
method might be of interest if a person is sus-
pected of being unable to methylate inorganic
arsenic or of having a reduced ability to do so.
One of the first studies of this kind determined the
arsenic species (As(III), As(V), MMAA and
DMAA) in urine of volunteers after ingestion of
arsenite-rich wine (50 ug As), arsenate-rich drink-
ing water (200 ug As) and crab meat (340 g)
containing organo-arsenic compounds (about
2000 ug) [47]. Resulted that 10% of the arsenite
was excreted as As(III), but the majority was
methylated and excreted as MMAA and DMAA,
5 h after ingestion. Most of the arsenate was
rapidly excreted without noticeable changes and
only a small proportion was methylated, while the
organic arsenic from the crab meat was quickly
excreted without any change. Exposure to As,O,
in different industrial situations, results in in-
creased urine arsenic concentrations, being inor-
ganic arsenic, MMAA and DMAA the only
relevant chemical forms; DMAA represented 60-
70% of the total urinary arsenic elimination when
comparing with the results from a reference popu-
lation [45]. However, ingestion of seafood, even in
such low amounts as 100 g, brings about a sharp
increase in the total arsenic excretion; this is only
confined to organic forms others than MMAA
and DMAA. The time course of elimination of
this dietary arsenic from the organism varies ac-
cording to the load, but does not take more than
35-48 h. These results provide a simple tool to
differentiate increases in urinary arsenic due to
occupational or environmental exposure to inor-
ganic arsenic from increases due to dietary intake
of organic arsenic.
Some organic arsenicals have been extensively
used as herbicides; it is likely to be present, to-
gether with inorganic arsenic species in soil sam-
ples. Extraction methods followed by
spectroscopic detection have been applied with
certain degree of success [48,49]. However, the
differences between the soil types as well as the
tedious clean-up of the arsenic residues are re-
sponsible for the low recoveries obtained.
The major problems in speciation studies are
the immense hazards that exist due to contamina-
1586 M. Burguera, J.L. Burguera /Talanta 44 (1997) 1581-1604

lion and losses of the element in the preparative distinguish between several forms of arsenic in
steps of the sample for analysis. As these proce- different samples.
dures require the use of various materials and
reagents, highly pure materials and chemicals
have to be made commercially available in order 3. Analytical methodology for arsenic speciation
to exclude errors due to contamination. Large
discrepancies are noted when comparing the data The numerous arsenic forms (Table 2) present
reported in the literature by different workers. in the environment and living organisms show
The variations among workers may be due to two large differences in their metabolism and toxicity
reasons: differences among the populations stud- [61]. Therefore, speciation studies are of critical
ied or inadequacy of the analytical methodology importance. Also the arsenic species occur at very
applied to the preparation of the samples for low concentrations so that reliable results are
analysis. Although the first cause must be seri- achieved only by means of very sensitive methods
ously taken into account, the latter seems more of analysis.
convincing; many researchers have experienced
difficulties in the quantitative extraction of arsenic Table 2
from the samples [13] while others claimed losses Arsenicals of environmental and biological importance
of volatile arsenic compounds during dry-ashing
Compound Formula
or heating with mineral acids [50]. To avoid these
difficulties, investigators have also used nitric/sul- Arsenious acid; arsen- HAsO,
furic acids, both without catalysts [49] or in the ites: AsfIII)
\I

presence of strong oxidizing agents like V,O, Arsenic acid; arsen- H&O,
[51,521, W&O, [511, WP,, WL K2CW7, ates; As(V)
Monomethylarsonic
KMnO,, V,O,, alone or in a mixture [53], claim-
acid; (MMAA)”
ing full recovery for all the species. Ultraviolet Dimethylarsinic acid;
[33,39,53-561 or microwave [57-591 radiation and (DMAA)”
base hydrolysis 1601 of the sample in highly oxidiz- Trimethilarsineoxide; (CH,),As+O-
ing solutions have proved successful to completely (TMAO)
decompose the arsenic containing compounds to Tetramethylarsonium (CH,L&’
ion
arsenate. As photo-oxidation could result in the Arsenobetaine; (AsB) (CH,),As+CH,COOH
loss of potentially significant speciation informa- Arsenobetaine; (AsC) (CH,),As+CH,CH,OH
tion, less severe photolysis must be applied so that Arsenolipids; (AsL) (CH,),As+CH,CHOHCOOH
the presence of methylated arsenic compounds in (trimethylarsoniumlactate)
Arsenosugars, (Ass)”
marine waters could be detected in aqueous artifi-
cial samples [33,54], serum of uremic patients [55]
or marine waters [56]. Also, to avoid the use of
CH3
strongly reactive media resulted from digestion
procedures, investigators developed methods for O=A,
0--&---x
rapid solid-phase extraction of the non-toxic ar- ClHJ
0
senic compounds from the matrix and the deter- HO OH
mination of the other arsenic species in the
remaining portion [40,47].
Unless convincing evidence is given on the ac- a Compounds forming gaseous species: As(III) and As(V) form
curacy tests, the data obtained by any procedure ASH, (B.P. = -55’C); MMAA forms monomethylarsine
(CH,AsH, with B.P. = 2°C); DMAA forms dimethylarsine
could be dubious. Also it will be possible to reach
((CH,),AsH with B.P. = 36°C); TMAO form trimethylarsine
solid conclusions only when sufficiently sensitive
((CH,),As).
and selective analytical methods are developed. b R, = OH, R, = OH (Giant clams and algae); R, = OH, R, =
These methods must be capable to accurately SO?H (Macroalgae); R, = NH,, R, = SO,H (Brown algae).
 M. Burguera, J.L. Burguera / Talanta 44 (1997) 1581-1604 1581

Arsenic (III) and (V) are the most often deter- selective determination of arsenic, are usually ap-
mined species in environmental waters, soils and plicable only to inorganic arsenic. They are based
sediments, while organic arsenic species are com- on the formation of colored complexes with cer-
mon constituents of biological tissues and fluids. tain ligands. For example, arsenic(II1) forms a
Usually the concentration of these species is at colored complex with silver diethylditiocarbamate
low ranges and necessitates a preconcentration [62-641 while As(V) forms arseno-molybdenum
step because most of the available detectors still blue complex [65,66]. Based on the pH control
lack the sensitivity for direct determination. and on the reduction characteristics of the boro-
Moreover, these detectors are also deficient in hydride ion, the former method was modified so
selectivity, thus a separation step is required prior that arsine was generated at pH 4.5 [63] or 6 [64]
to determination. The most popular analytical from As(II1) and at lower pH from As(V) [63,64].
methods used for arsenic speciation are based on The resultant gaseous compound was quantita-
a combination of a powerful separation process tively absorbed in silver diethylditiocarbamate so-
with an adequate element-specific detector. The lution and the molecular absorbance of the red
methods frequently used for separation and pre- complex was measured at 525 nm. This procedure
concentration are solvent extraction, precipitation is appropriate to determine 0.1 mg l_ ’ of arsenic,
and coprecipitation, ion-exchange chromatogra- but it is susceptible to interferences from both,
phy (IEC), gas chromatography (GC) and high trace elements and methylated species. The latter
performance liquid chromatography (HPLC).
method consists in the use of an oxidizing agent
Numerous instrumental methods have been de-
like permanganate [65] or iodate [66] to convert
veloped either for the determination of total ar-
all arsenic present in the sample to As(V) used to
senic or its methylated and inorganic forms in
form the blue compound and then measure the
soils and sediments, water, air and biological
absorbance at 865 nm. As little as 0.005 mg 1~ ’ of
fluids and tissues. Among them, the most com-
each species can be detected. Selectivity is
monly used include ultraviolet spectrometry [62-
achieved by on-line separation of the main inter-
671, electrochemical methods (EQ) [68-741,
ference phosphate, arsenate and silicate using a
atomic absorption spectrometry (AAS) mainly
strong anion-exchange microcolumn located in
coupled to hydride generation (HG-AAS) [75-
the aspiration line of the FI system [65].
1111, in continuous flows or flow injection systems
Also As(II1) reacts quantitatively with potas-
(FI), atomic emission spectrometry (AES), gener-
ally with inductively coupled plasma (ICP-AES) sium iodate in the presence of sulfuric acid releas-
[l 11~ 1301, ICP-mass spectrometry (ICI?-MS) ing an equivalent amount of iodine which imparts
[l 1 1- 114,124- 13011, electrothermal-AAS in a pink color to a carbon tetrachloride extract [67].
graphite furnace (ETAAS) [ 13 1~ 1461, X-ray spec- The absorbance is measured at 520 nm. The last
trometry [ 149,150], neutron activation analysis procedure is more sensitive than the previously
(NAA) [ 150,15 11, atomic fluorescence spectrome- two already mentioned above; the amount of
try (AFS) [ 152,153], capillary electrophoresis arsenic detected is 0.002 mg 1- ‘. Besides that the
[155- 1571, etc. On-line combinations of some sep- spectrophotometric procedures are less sensitive
aration techniques (e.g., HPLC or IEC) with the than other alternatives, they only apply to inor-
most sensitive detection methods (AAS or ICP- ganic species, require long reaction times, and the
AES) seem to be highly successful and lower procedures involve reduction, oxidation, chelation
detection limits are obtained, specially if HG is and sometimes extraction and evaporation steps
performed before detection. which might disturb the equilibrium between spe-
cies in the original sample leading to erroneous
3.1. Spectrophotometric and electroanalytical results and interpretations.
methods Among the electrochemical methods, differen-
tial pulse polarography seems to offer possibilities
The spectrophotometric methods used for the for quantifying arsenic in both oxidation states.
1588 M. Burpera, J.L. Burpera
The procedure is based on the fact that only
As(II1) is electroactive and directly determined in
perchloric or hydrochloric acids as supporting
electrolytes [68,69], while total inorganic arsenic
was determined after reduction with aqueous sul-
fur dioxide. As(V) was evaluated by difference.
Sulfur dioxide was selected as reducing agent
because it reduces As(V) rapidly and quantita-
tively and the excess reagent is readily removed
from the reaction mixture. At low arsenic concen-
trations, which is really the case for environmen-
tal and clinical samples, the procedure is subjected
to interferences from some elements present in the
matrix {Fe(II) and Cu(II)}, therefore a separation
step might be necessary; distillation of As(II1) as
AsCl, in concentrated HCl has proved appropri-
ate [70]. Determination of methylated species is
also possible by voltametric methods after separa-
tion by ion-exchange chromatography [68]. The
detection limits of 18 and 8 ug l- ’ obtained for
MMAA and DMAA, respectively, are far from
being sufficiently low so that the procedure be
applied for the direct determination of the arseni-
cals in natural waters. Additionally, before ob-
taining the analytical signal and after the
separation of the species, there are other steps like
digestion of each fraction in hot, concentrated
perchloric acid, reduction with sulfur dioxide and
removal of the excess reagent, each being a pow-
erful source of errors. As As(V) is the stable
oxidation state in natural waters and in biological
digests, its reduction, at very low concentrations
to the trivalent state is difficult and the use of
reducing agents like iodide, hydrazine, Cu(I), etc.,
interfere with the stripping process. Therefore,
oxidation of As(II1) which takes place sponta-
neously in the presence of dissolved oxygen is
preferred since some investigations on gold-coated
platinum-fiber electrodes have shown that As(V)
can also be reduced to elemental arsenic provided
that extremely low reduction potentials are used
( - 1.60 V versus Ag/AgCl electrode) [71]. So, as
As(II1) is easily reduced at a gold electrode, it
ought to be possible to determine As(II1) in the
presence of As(V) by suitable choice of the elec-
trolysis potential. Some voltametric procedures
for the determination of As(II1) and As(V) have
been considered in an expert system developed
using KES (knowledge engineering system) [72].
/ Talanta 44 (1997) 1581-1604
However, given all the difficulties mentioned
above, specially those concerning interferences
due to complex matrixes or to the presence of
certain reagents, the electrochemical methods are
not used for routine analysis in the speciation and
determination of arsenic in complex matrices sam-
ples.
A combination of electrochemical and spec-
trophotometrical detection for As(II1) and As(V)
following ion chromatographic separation, respec-
tively, permitted the sequential determination of
arsenite and arsenate in water samples [73]. The
detection limits were found to be 2.9 and 13 ug
l- ’ while the recoveries for spiked waste water
samples were in the ranges 97.5-104% and 93.5-
103% for As(II1) and As(V), respectively. In a
recent work, Boucher et al. [74] succeeded to
differentiate six arsenic species (As(III), As(V),
MMAA, DMAA, AsB and AsC) by ion-pair re-
versed-phase liquid chromatography coupled with
amperometric and ultraviolet detection. Arseno-
betaine and As(II1) were not separated and only
As(II1) is amperometrically active. The limits of
detection ranged from 0.1 uM for As(II1) to 1.O
uM for As(V), the method being adequate for the
detection of typical arsenical concentrations of
20-50 ug I- ’ in normal urine.
3.2. Hydride generation-atomic absorption
spectrometry.
The most widely accepted procedures for the
analysis of arsenic at ug l- ’ level exploit the
reduction of some arsenic compounds to gaseous
arsines (Table 2). The gas is then thermally de-
composed to give elemental arsenic for atomic
detectors like AAS, or ICP-AES. Early methods
for arsine generation involved the dissolution of
metals (Zn, Mg, Al) in mineral acids to form
nascent hydrogen which reacts with As(II1) to
form ASH,. Other hydride ion precursors that
have found use in generating hydrides are TiCl,
[75], SnCl, in concentrated HCl [76] or Al in basic
medium known as Fleitmann reaction [77,78]. The
effective reaction with NaBH, gained acceptance
and is now almost universally used for the genera-
tion of hydrides ever since its introduced into
analytical chemistry [79]. It is well known that
 M. Burguera, J.L. Burguera /Talanta 44 (1997) 1581-1604 1589

trivalent and pentavalent arsenic show different lected either in a container under pressure to-
behavior in the generation process, although it gether with the hydrogen gas produced in the
was claimed that some procedures only measure reaction or in a cold trap for preconcentration
the total content of As in the sample. However, it when high sensitivity is required. Then, are di-
was demonstrated that even at high concentra- rectly swept into the atomizer or are released via
tions of borohydride and at optimized acid con- a chromatographic column which separates the
centrations, the response obtained from As(V) is products [81-851. In such systems, peak height
10% lower than that of As(II1) [80]. The process and shape are critically dependent on some fac-
of formation of arsine from As(V) suggests that tors which are difficult to control. However, each
there are two steps in the reaction: the reduction instrumental setup can be optimized either to
of As(V) to As(II1) and the subsequent formation yield a high hydride concentration to attain a high
of ASH,. The rates of the redox reaction that sensitivity or to provide the appropriate condi-
involve electrons transfer are rather slow and pH tions to eliminate or at least to suppress the effect
dependent, therefore it appeared that it might be of varying matrix components, A strict control of
possible to differentiate between the two species the experimental conditions permits the volatiliza-
provided that the first stage of the reaction is tion of hydrides from As(II1) and from the methy-
slower than the second at high pH values. There- lated species in a single sample aliquot due to the
fore, differentiation is made possible using pH-se- differences in their boiling points (Table 2). The
lective arsine generation technique, in which, for arsines, together with water vapors, carbon diox-
As(V) strongly acidic solutions are required ide and any other vapor generated during the
(pH I l), while for As(II1) hydride formation oc- reduction reaction are freezed-trapped and se-
curs in solutions up to pH 5 [81-871. MMAA and quentially volatilized for elemental determination
DMAA are also reduced to the corresponding by AAS. These features have been described by
hydrides at pH 1. Furthermore, it is not only the several researchers [81-871. The use of liquid ni-
pH that affects the speciation, but other factors trogen traps has greatly improved the sensitivity
such as kinetics and complexation are also in- of the technique since the volatile arsines are
volved [88,89]. Several researchers, each using preconcentrated over a period of 5 - 10 min from a
their own procedure, came to different conclu- large volume of sample and ultimately passed as a
sions concerning the redox stability of aqueous ‘plug’ of analyte to the detector. Temperature-
solutions containing arsenic species and the time controlled selective volatilization from the trap
scale on which spontaneous changes in species makes it possible to differentiate between the dif-
distribution occurred [83,88]. ferent arsenic species, but the other frozen gasses
There are two major operation modes of hy- are also released. To prevent freezing of the water
dride generation systems, batch and continuous vapors in the cold trap, some hygroscopic materi-
modes; both involve reduction of an acidic sample als are used to dry the gases. Calcium chloride
followed by the transport of the hydride to the was mostly used but after several determinations
atomizer. In the batch mode, acidified sample and become wet and thus impermeable to argon. This
reducing reagent are injected into a reaction vessel often causes a back-pressure which can blew off
and any hydride produced is purged and carried the system [90]. Frequent replacement is therefore
by a stream of an inert gas. while in continuous necessary and loss of arsines is reported. Van
systems, acidified sample solution and reductant Elteren et al. [82] interfaced a novel drying system
are continuously delivered for the production of composed by a commercially available hygro-
hydride and the gas is separated from the liquid scopic ion exchange membrane and separates the
waste in a gas-liquid separator (GPS) also with organic compounds without losses of the arsines.
the aid of a carrier gas. The last mode has two The separation of the trapped compounds is car-
versions: continuous flow and FI. ried out under non-isothermic conditions and the
The hydrides generated in batch systems are low-boiling gases volatilize out of the trap in
either directly delivered to the detector or col- lo-30 s. A slow heating rate is optimum since at
1590 M. Burguera, J.L. Burpera
high heating rates the separation can become
poorer and thermal decomposition might take
place with subsequent loss in sensitivity [91].
However, the heating rate is greater than 100°C
min - ’ in any case, since the trap is heated from
- 195 to approximately 60°C in l-2 min. Apart
from being less precise than the selective reduc-
tion procedures, these approaches have other
problems associated, like the molecular rearrange-
ment during HG and incomplete collection of the
hydrides. Insufficient selectivity during the reduc-
tion step is another drawback; at pH 5 only
As(II1) is reduced but MMAA and DMAA to a
certain degree as well [82]. The complexity of the
system makes on site speciation impossible which
implies storage of sample prior to analysis; specia-
tion alteration has to be faced then, in particular
for As(II1). The generally applied cold trap is
U-shaped and made of glass; the relatively high
thermal resistance of glass makes cooling and
especially heating a slow process at room temper-
ature. In order to achieve better trapping, the tube
was half-filled with glass beads, although it was
reported that arsines are irreversibly captured by
this material [83,92]. Better, more efficient and
more reproducible arsines separation was
achieved when using the U-tube packed with a
gas chromatographic support. The use of a
Teflon-lined stainless steel U-tube with a gas chro-
matographic solid phase permits faster cooling
and heating [82-851. Some kind of electrical heat-
ing was also applied to volatilize the species.
These systems are also subjected to interference
effects and their precision and recovery are
strongly dependent on the experimental condi-
tions such as flow of carrier gas, rate of thermal
desorption, type of absorbing phase and size of
column. Most of these difficulties have been over-
come by using an automated and on-line HG-
cryogenic trapping followed by MW irradiation to
release the hydrides for AAS detection [93].
The use of continuous systems allow minimum
sample handling, being less likely to suffer from
the problems of contamination or alteration of
arsenic speciation. Also continuous flow methods,
mainly flow injection, appear to be more tolerant
to the presence of nitric acid and to be less prone
to transition elements interferences. This effect, at
/ Talanta 44 (1997) 1581-1604
least in part, is due to kinetic discrimination; the
reduction of the element to its hydride is com-
pleted before reduction of the transition metal
ion. The kinetic discrimination in flow systems is
responsible for the pronounced difference in sensi-
tivity between the two oxidation states (at least
one order of magnitude), so that a pre-reduction
step is mandatory. In some procedures, As(V) is
reduced to As(II1) prior to HG by the use of
reducing reagents like: KI [94], thiosulphate [95],
or mixtures of KI-ascorbic acid [95] and L-Cys-
teine [96-991 to achieve pre-reduction for the
determination of total arsenic. L-Cysteine was
found to be clearly superior as reductant and
releaser since provides greater freedom from inter-
ference and much better stability of solutions with
low concentrations of analyte. The reagent con-
sumption and particularly the acid concentration
are significantly lower, which permits the change
from use of highly corrosive solutions to solutions
of low acidity and low toxicity. This is of advan-
tage for the operator and for the instrumentation,
it reduces the cost per determination and pro-
duces less hazardous waste. However, controlling
the acid nature and concentration, no prereduc-
tion is necessary to improve the kinetics of hy-
dride from As(V) [93,100,101]. Such procedures
were used to determine As(III), As(V), MMAA
and DMAA [93,100], as well as total arsenic and
inorganic As(II1) and As(V) by either FI or con-
tinuous flow HG-AAS in water samples [loll.
Although simpler to operate and easier to auto-
mate, these systems are limited in their ability to
obtain a correct speciation of As. In the presence
of MMAA and/or DMAA, As(II1) or As(V) will
be overestimated. The error will be function of the
pH values chosen for the selective volatilization of
the inorganic arsenic species and of the amount of
organic arsenicals present.
Brannon and Patrick [IO21 inserted a H,S04
trap between the hydride generator and the AAS
to separate alkylarsines from inorganic arsines.
While ASH, passed through, the alkylarsines were
trapped. However efficient, this system does not
allow for the identification of the organic arseni-
cals present. Improved sensitivity was obtained
for all the arsine forming species after IEC separa-
tion [33,103-1051. The fractions containing ar-
 M. Burguera, J.L. Burguera / Talantu 44 (1997) 1581-1604
senic species were submitted to hydride generation
in batch [33], in a FI system [102,103] or in an
on-line arrangement [104]. Although the detection
limits, in all cases, are ‘in the range of the ng
levels, the methods are only applicable to studies
related to arsenic exposure and its monitoring,
Determination of ultra-trace amounts of As(II1)
(detection limit = 0.003 ug 1 - ‘)was recently
achieved by FI-HGAAS prior on-line preconcen-
tration by coprecipitation with lanthanum hy-
droxide or hafnium hydroxide [ 1061.
During the last decade HPLC methods have
been reported to be capable of separating the
most commonly found arsenic species in samples
of different types. They offer the advantage of
minimizing chemical interferences and rearrange-
ment reactions. However, the time required for
the overall analysis is in the range lo&300 min,
limiting their usefulness for routine applications.
Furthermore, due to the high dilution factors
always associated with conventional chromato-
graphic separations, the most sensitive and spe-
cific detection techniques such as HG coupled to
Flame-AA& ICP-AES or ETAAS are needed to
compensate for dilution. Also the eluents typically
contain high concentration of salts which quickly
build up on the nebulizer, ICP torch and/or sam-
pling cones of the ICP-MS interface, leading to
instrumental drift. HG following HPLC separa-
tion, eliminates this problem because the arsenic is
converted to its gaseous hydride which is sepa-
rated without the associated dissolved solids._ In
the application of coupled techniques care must
be taken in the design of the interface, so that the
flow rate of a gas or liquid through the chromato-
graphic column must be matched or adjusted in
some manner to the gas or liquid uptake rate of a
particular detector [2].
Several systems have been developed, based on
post-column HG for the determination of the four
arsine-forming species in natural waters [107],
brawn algae [108], urine [109,1 lo], etc. Hakala
and Pyy [109] used an HPLC coupled to a contin-
uous flow system for HG and tetrabuthylammo-
nium ion in phosphate buffer as the ion-pairing
agent in the Cl8 reversed-phase column to deter-
mine arsenic metabolites in urine. Matrix interfer-
ences from urine samples with different salt
1591
content occurring in the separation of arsenic
species can be reduced by matching the samples
with the mobile phase and by using a relatively
low injection volume. The detection limits were
1.0, 1.6, 1.2 and 4.7 ug 1-l for As(III), As(V),
MMAA and DMAA, respectively. An automated
and direct HPLC-FI-HG-AAS method was devel-
oped and statistically validated for the assessment
of inorganic arsenic and its metabolites in urine
[l lo]. The detection limits of this procedure were
compared with those obtained by directly cou-
pling the HPLC column to an ICP spectrometer.
The limit of detection of the FI-AAS system is
approximately 35 times lower than that of the
ICP-AES system, but still not low enough to be
able to detect arsenic compounds in urine from
non-exposed persons. Therefore, these methods
are not applicable to the analysis of urine from
normal persons, but could be useful in analysis
for arsenic species in urine from highly exposed
persons, or persons suspected of being unable to,
or having a reduced ability to methylate inorganic
arsenic compounds. The major limitation in using
HG as a part of the HPLC separation-element
specific detector is that many environmentally and
biologically important organoarsenic compounds
such as AsB, AsC, tetramethylarsonium ion, ASS,
etc., do not form volatile arsines and would not
be detected. To solve this problem, an appropriate
decomposition procedure is required.
An on-line HPLC-microwave-oven (MWO) ox-
idation with persulphate and HGAAS detection
has been successfully applied for the determina-
tion of arsenite, arsenate, MMAA, DMAA, AsB
and AsC in environmental samples [55]. An an-
ionic cartridge located before the HPLC anionic
column, quantitatively retained arsenite, arsenate,
MMAA and DMAA. The AsB and AsC which
passed through, were separated in the HPLC
column, eluted and, each fraction, microwave ir-
radiated after mixing with persulfate for their
oxidation to arsenate. The conversion efficiency
was reported to be closed to 100%. The solution
from the MWO was cooled in an ice bath and
mixed downstream with acid and borohydride to
form the arsine which was swept into an AAS
atomization cell. To determine the anionic species,
the same procedure was followed, except that the
1592 M. Burguera, J.L. Burguera / Talanta 44 (1997) 1581- 1604
anionic cartridge was removed, and water was
introduced instead of persulfate. The detection
limits were between 0.3 and 0.9 ng (for 100 ul
sample). A similar procedure was developed by Le
et al. [ll l] except that they used a dual system to
avoid removal of the cartridge during the proce-
dure. Comparable resolution was obtained by us-
ing HPLC-separation-MW-digestion with
HGAAS or ICP-MS detection. Complete separa-
tion of five arsenicals was achieved on a reversed
phase Cl8 column by using sodium heptanesul-
fonate as ion pair reagent and the detection limits
were for example 10 ug 1- ’ for arsenite, DMAA
and AsB, 15 ug 1 - ’ for MMAA and 20 ug 1 - ’ for
arsenate.
3.3. Inductively coupled plasma-atomic emission
spectrometry
The detection limits achieved in an ICP-AES
system are far too high to allow detection of the
low concentration of arsenic species present in
urine of normal persons. Additionally, when ICP-
AES is used for detection, a severe interference by
background ions strongly degrades the detection
limits. The single 75As isotope has an interference
from 40Ar35C1species which has the same mass-to-
charge ratio as that of monoisotopic arsenic [112-
1141. The extent of this interference is such that
the direct determination of the analyte is impossi-
ble. In the determination of total arsenic this
interference has been eliminated by the addition
of nitrogen to the argon carrier gas [115]. Nitro-
gen addition was found to reduce analyte sensitiv-
ity, which made the technique unsuitable for
analysis at the required levels. A substantial in-
crease in sensitivity has been reported when using
HPLC in combination with HG-ICP-AES
[I 12,116,117]. In the HG step, the concentration
of the HCl used is relatively high; as a result, the
conventional GLS produces a ‘residual aerosol’
which is transported to the torch and converted
into the interferent. Therefore, HCl should be
replaced by mild HNO, and L-Cysteine be used as
reductant and releasing agent. In that way, the
interference from Ar +Cl is avoided and, based
on the kinetical reduction, simultaneous determi-
nation of As(II1) and As(V) in waters was possi-
ble without interferences from transition elements
[99]. The detection limit for As(V) was 3.4 ug 1~ ’
and for As(lI1) 0.7 ug 1~ ‘. Also the effect of the
‘residual aerosol’ can be eliminated by using mi-
croporous membranes as GLS [ 118,119]. Some
workers suggested that selectivity could be im-
proved by an initial separation ,of the analyte
from the matrix (e.g., solvent extraction) followed
by the generation of the hydride directly from
non-aqueous media and the detection of the ana-
lyte by ICP-AES [120,121]. There are some ad-
vantages of these applications: (i) a
straightforward combination of liquid-liquid ex-
traction with HG to overcome matrix interfer-
ences and (ii) the opportunity to determine
hydride forming elements in organic samples and
solvents directly, without resorting to lengthy re-
extraction procedures. The introduction of or-
ganic solvents into ICP presents particular
difficulties and therefore, the use of miscelles and
other organized media with a hybrid aqueous/or-
ganic character, could prove advantageous. Ar-
sine can be generated with sodium borohydride
from a didodecyldimethylammonium bromide
(DDBA) vesicular medium [122]. It was found
that the analytical performance of this vesicles-en-
hanced method is superior to the HG from
aqueous media. No speciation studies have been
reported, although the detection limits were im-
proved by a factor of two compared with those
observed when arsine was generated from
aqueous medium, and greater tolerance to inter-
ferences was observed [121,122].
However, the most direct method for dealing
with this interference is to use a high resolution
mass spectrometer. Since the advent of the combi-
nation of ICP with MS, exceptionally low detec-
tion limits have been achieved
[99,111-114,124-1271. Coupling HPLC to ICP-
MS, the simultaneous determination of up to six
compounds was possible in about 10 min with
detection limits in the range few picograms. These
values are sufficiently low to study the chemical
species at their naturally occurring concentration
levels. Also the speciation of arsenosugars in
marine reference materials by HPLC-ICP-MS has
been recently reported [127].
 M. Burguera, J.L. Burguera /Talanta 44 (1997) 1581-1604
The cost of an existing commercial ICP-MS
instrument makes it one of the most expensive
HPLC detector and it may not be realistic to use
it in routine analysis or’ for environmental moni-
toring. Unless a previous separation technique is
used prior to the detection by ICP-AES, the use
of HG is mandatory, limiting the method, as in
the case of HGAAS, to the speciation of arsine-
forming compounds, thus leaving valuable infor-
mation behind. So, coupling IEC with ICP-AES
simplified the process and overcame the interfer-
ence of Ar + Cl -, as it does when coupled to
ICP-MS [128]. Optimizing the two systems and
using an ultrasonic nebulizer, it was possible to
reach detection limits in the range of ng 1- ’ for
As(V) and MMAA [ 1291. The procedure was
completely automated for sample retention, elu-
tion and regeneration of the resin.
3.4. Atomic absorption spectrometry with
electrothermal atomization (ETAAS)
Although the detection of the separated species
using on-line techniques, like those described
above is ideal, ETAAS is still advantageous be-
cause of its sensitivity (two orders of magnitude
higher when compared with flame-AAS) and ease
of operation. Furthermore, the instrumentation is
available in many laboratories. However, this
technique has proved limited potential for specia-
tion, apart from being prone to errors owing to
pre-atomization losses and to the fact that differ-
ent arsenic compounds produce different ETAAS
responses. Larsen found that using conventional
STPF furnace program, calibration for all species
was possible with standards prepared from only
one calibrant, e.g., arsenate, but the operation of
the instrument is relatively slow [131]. He studied
the conditions under which ETAAS gives equal
and optimum sensitivities for all the arsenic spe-
cies under study. To improve sample throughput,
a fast furnace program that includes the use of a
chemical modifier, shorten the time needed for
analysis and makes calibration of all species possi-
ble using two arsenic species as calibrants, i.e.,
one for the quaternary arsonium compounds and
one for the other species. However, systematic
investigations on the behavior of arsenic species in
1593
graphite furnace are lacking. Krivan and Arpad-
jan [132] studied the influence of different ma-
trices (HCl, NaCl, HNO, and urine) and of
various chemical modifiers (W, Pd and a mixture
of W + Pd + citric acid) on the behavior of
As(II1) and As(V) in the graphite furnace by
means of an 75As radiotracer. Different treat-
ments of the pyrocoated tubes showed to ther-
mally stabilize the arsenic species. Such is the case
of Ce(IV) [133] and tungsten-carbide coated tubes
[ 1341 which improved the responses from arsenite,
arsenate, MMAA, DMAA. No pronounced stabi-
lizing effect was observed for AsB and AsC with-
out the addition of palladium chloride to the
treated tubes [134]. Aqueous and methanolic solu-
tions of these species were studied because they
are the main components of the various extraction
systems and HPLC effluents. Attention was di-
rected to a detailed investigation of the pyrolisis
stage, which is the most critical step for the
analysis of highly volatile species. An attempt to
separate in situ As(II1) from AsB in W-treated
tubes failed because, the authors claim, of the
presence of significant amounts of AsB under the
conditions used for As(II1) determination.
Nevertheless some of the arsenic species occur
in some samples at such low concentrations that
even ETAAS cannot provide sufficient sensitivity,
so that preconcentration procedures have to be
combined in order to achieve the required detec-
tion power. Attempts to preconcentrate water
samples by evaporation before injection onto the
furnace have resulted in losses of arsenic and in
redox reactions affecting the ratio of species.
Many separation-preconcentration schemes, fol-
lowed by ETAAS detection, include thin-layer
chromatography [ 135,136], or coordination of the
species to organic molecules and extraction in
organic solvents [ 137- 1411, precipitation or co-
precipitation [ 1421, adsorption on surfaces like
those of ion-exchange resins [143,144] or HPLC
columns [145,146].
Extraction as a means of separation and pre-
concentration of species has been applied most
often to solids such as soils, sediments, particulate
matter filtered from water and air or biological
tissues. A number of extractants have been used
by many researchers as a means of selectively
1594 M. Burguera, J.L. Burguera / Talanta 44 (1997) 1581-1604

removing the species from the bulk of the sample species and prevents their shift during the analy-
and also as a means of preconcentration prior to sis. One of the main advantages of the on-line
the quantification by ETAAS. Since As(V) cannot technique is that preconcentration takes place in a
be extracted from acidic media, only As(II1) could closed system, avoiding any exposure of the sam-
form the ammonium tetramethylenedithiocarba- ple to atmosphere. Because of this greatly reduced
mate complex which is extracted into chloroform risk of contamination, very low and reproducible
and then, is selectively quantified. Prior to the blank values can be obtained. Sample loading,
extraction, however, As(V) can be reduced with column washing, elution and sample transfer are
potassium iodide, thiosulfate, L-Cysteine, or mix- operations on-line which leads to enhanced preci-
tures of hydrogensulfite-thiosulfdte and iodide- sion and accuracy compared with manual batch
ascorbic acid. In this case, both oxidation states procedures for sample treatment. Equilibrium
are determined simultaneously and the As(V) con- needs not be attained because of the exact timing
centration is quantified by difference. Ammonium of all process. The strict reproducibility of the
pyrrolidinedithiocarbamate (APDC) and o,o-di- process excludes interferences due to kinetic ef-
ethylditiophosphate have been also used as chelat- fects. The total time for sample treatment fits well
ing agents for the extraction of As(II1) into into the running time of a standard graphite
chloroform followed by back-extraction into an furnace program.
aqueous phase [ 1371. A reliable analytical procedure aimed to sepa-
As(II1) is also separated by solid-liquid extrac- rate arsenic species in waste and potable waters: it
tion with sodium diethylditiocarbamate (NaDDC) involves a separation step on an extraction chro-
into methyl isobuthyl ketone (MIBK). Aliquots of matographic column filled with a support
the organic phase are determined by ETAAS us- modified with dioctyltin dichloride [140]. This ar-
ing palladium in MIBK as chemical modifier. rangement selectively extracts As(V) which is
Total arsenic is determined using slurry sampling eluted from the column with HCl. The effluate
and Pd-Mg(NO,), to stabilize the arsenic [138]. containing As(II1) and the eluate containing
Extraction of arsenite with set-butyl dithiophos- As(V) are then determined by ETAAS in the
phate followed by the determination of arsenic in presence of a mixed modifier of Pd, W and citric
the extract by ETAAS [139] is another mean of acid.
inorganic arsenic speciation. These procedures are It can be concluded that the extraction proce-
time consuming because they involve multi-step dures mentioned above apply only to inorganic
procedures and usually permit only a moderate arsenic species. A recent work [141], however,
preconcentration because there is a limited ratio confirmed the suitability of the application of an
of organic to aqueous phase that can be reliably extractive procedure to the methylated arsenic
handled. The on-line combination of liquid-liq- species. Only inorganic arsenic, MMAA and
uid extraction principles with sorption on a solid DMAA were quantitatively extracted in toluene
phase column material permits the use of a wide from acidified urine samples, then were stripped
selection of functional groups. Rapid and com- from the organic solvent with diluted nitric acid
plete elution prevents extensive dispersion of the and detected by ETAAS in the range lo-50 ug
sample, such as in chromatographic systems. The 1-l. None of the other known organic arsenicals
use of a flow injection on-line separation of were extracted. The results were in good agree-
As(III)-diethylditiocarbamate complex sorbed on ment when compared with those obtained by
a Cl8 reversed phase packing (bonded silica with HPLC-ICP-MS and the extractive procedure was
octadecyl functional groups), reduction of As(V) recommended for the evaluation of occupational
with a sulfite-thiosulfate-iodide solution and pre- exposure to inorganic arsenic.
concentration for ETAAS allows the determina- Chen et al. [142] determined As(V) and As(III)
tion of As(II1) and total arsenic in a short time species in environmental samples by ETAAS after
[89]. The fast complexation and sorption (approx. coprecipitation with zirconium hydroxide and
100 ms) maintain the natural equilibrium between were able to differentiate between As(II1) and
 M. Burguera, J.L. Burguera / Talanta 44 (1997) 1581-1604 1595

As(V) by using different temperature programs in As(V) to As(III), while DMAA is not coprecipi-
the graphite furnace. tated at all. This implies that MMAA interferes in
Pacey and Ford [143] and Grabinski [144] used the determination of As(V) [150]. The precipitates
the strong affinity of arsenic species for certain were collected successively on membrane filters
ion-exchange resins to completely separate four and arsenic determined by NAA of the filters and
arsenic species and measure the analyte content in subsequent y-spectrometry.
each fraction by ETAAS. These systems gave Extraction and preconcentration of arsenic spe-
better detection limits and/or shorter analysis cies prior to irradiation were deemed necessary in
times than the extraction procedures. NAA methodology, because firstly, the oxidation
The fairly long time required for the analysis of states of arsenic in the original samples irradiated
a series of collected fractions may be inconvenient cannot be distinguished after radiochemical sepa-
for practical analytical work. More work is ration and secondly the use of large volumes of
needed in this direction to fully exploit the poten- samples for irradiation without preconcentration
tiality of ETAAS for speciation. Probably cou- is unsuitable due to the small concentration factor
pling ETAAS directly with HPLC might be the involved. The separation step is also ideal to
answer; interfacing a stepwise operational proce- create an interference-free environment in the ma-
dure (ETAAS) with a continuously flowing trix during the activation period. The selective
effluent from the HPLC column is a real analyti- extraction of the ammonium te-
cal challenge. Although the work reported until tramethylenedithiocarbamate complex of As(II1)
now [145,146], do not give experimental details on into chloroform and the detection of arsenic by
the instrumental arrangement, making this type of NAA after back-extraction was reported by
coupling it is not impossible [147,148]. Yusuf et al. [151]. Reduction of As(V) to As(II1)
was achieved with thiosulfate. Marine samples
3.5, Other methods were subjected to microwave radiation choosing a
suitable program to prevent the change in oxida-
Apart from the procedures described above, tion states of arsenic species during the heating
and which are massively studied, there are scarce process.
data in the literature about other potential tech- Contrary to AAS, AFS has not been widely
niques, like neutron activation analysis, mass used in speciation studies because of its low sensi-
spectrometry, X-ray crystallography, NMR and tivity when HG is coupled to introduce the ana-
IR spectroscopy, etc., which can be used in ar- lyte in the diffusion flames normally employed as
senic speciation. This is probably because the atom cells for this technique. However, the detec-
amount of sample per analysis for some of them, tion limits have been recently improved, because
as well as the availability of sophisticated and high-intensity excitation sources e.g., boosted-dis-
costly instruments in many laboratories are the charge hollow cathode lamps, have become com-
limiting factors. mercially available [152]. When coupled with
Coprecipitation with dibenzildithiocarbamat- HPLC, the fractions could be efficiently nebulized
e(DBDTC) and hydrated iron(II1) oxide for into the flame, and so, AFS becomes a powerful
As(II1) and As(V), respectively, followed by the tool for arsenic speciation, achieving detection
determination of arsenic by X-ray spectrometry limits as low as 35, 50, 20 and 20 ng for As(III),
was reported [149,150]. Detection limits as low as As(V), DMAA and MMAA, respectively, in 250
0.02 ug 1-l have been obtained. These procedures ul of volume injected [153]. The analytical poten-
are also limited to the inorganic species only, tial of such hybrid techniques has been recently
although investigations on the behavior of some illustrated on coupling vesicle-mediated HPLC to
other arsenic species, e.g., MMAA and DMAA, low-power argon microwave-induced plasma
showed that MMAA is coprecipitated quantita- (MIP) for mercury and arsenic speciation. A HG
tively with DBDTC when a strong reducing mix- technique was used as interface between the exit
ture of iodide and thiosulfate was used to reduce of the HPLC column and the MIP held in a
Table 3
Analytical details of arsenic speciation in different samples

As species (samples) Matrix Measurement technique Some details of the proce- D.L. (ug IF’)* Ref.
dure

As(III), AsT Waters, sediments, syn- UV-vis Red complex with diethyldi- 100 [62-641
thetic mixtures tiocarbamate (2 = 525 nm)
Also arsine absorbed on lig-
and solution
As(V), AsT Waters UV-vis Molybdenum blue complex 5 [65,66,70]
(A = 865 nm) Use of oxidiz-
ing agents.
As(III) Polluted water UV-vis Indirect determination. Reac-
tion with iodate in H$O,-I,
give a pink color to a Ccl,
extract. (2 = 520 nm)
As(III), AsT Synthetic mixtures, SRM EQ In voltametric methods only N.I. 168,691
As(II1) is electroactive using
HCIO, or HCl as supporting
electrolytes (As(V) is reduced
with SO,).
MMAA, DMAA Synthetic mixtures EQ Differential pulse polarogra- 18, 8 m
c phy applied after separation
by IEC
As(III), AsT Seawater, urine EQ stripping In the presence of As(V) by 0.1 [7 1,721
suitable choice of the elec-
trolysis potential. (60 s elec-
trolysis)
As(III), As(V) Waters EQ + UV-vis Following IEC 2.9, I3 [731
As(III), As(V), MMAA, Urine EQ + UV-vis Coupled to ion-pair reversed 0.1-l PM [741
DMAA, AsB, AsC phase liquid chromatography
Soluble As Soils HG-AAS Continuous flow HG-AAS. 0.90 all 1641
Selectivity based on pH se-
lective reduction
As(III), As(V) Sediment interstitial wa- HG-AAS As(II1) coprecipitated with N.I. K21
ters dibenzyldithiocarbamate in
methanol and As(V) with
sodium molybdate and te-
traphenylphosphonium chlo-
ride. (The filters were
digested)
As(III), As(V) Waters FI-HG-AAS with CT An electrically heated Al 0.25 [771
(Fleitmann reaction) column was used to generate
arsine in alkaline medium.
Table 3 (continued)

As species (samples) Matrix Measurement technique Some details of the proce- D.L. (ug I-‘)* Ref.
dure

MMAA, DMAA Sediment interstitial waters HG-AAS with CT ASH, generated in continuous 0.05-O. 13 WI
flow(released on progressive
worming at room temperature)
As(llI), As(V), MMAA, Interstitial water HG-AAS with CT ASH, generated in continuous 0.01990.061 WI
DMAA. flow using pH selective proce-
dure, trapped in liquid nitro-
gen (released on progressive
worming at room temperature)
As(III), As(V), MMAA, Waters HG-AAS with CT Arsines generated ‘in batch’ 1 ng each (up to 70 ml sam- [91]
DMAA, by pH selective procedure and ple)
trapped in liquid nitrogen (re-
leased on electric heating)
Inorganic As, MMAA, Waters HG-AAS with CT, GC Arsines generated ‘in batch’ 0.01 each P51
DMAA separation by pH selective procedure and
trapped in a GC column in
liquid nitrogen (released on
progressive heating up to
1SOOC)
As(III), As(V), MMAA, Soil extract. water HG-AAS with CT and Arsines generated ‘in batch’ 0.026 1841
DMAA, GC separation by pH selective procedure and
trapped in a GC column in
liquid nitrogen (released on
electric heating) (Flame-in-
tube burner for AAS)
As(III), As(V), MMAA, Urine HG-AAS with CT and Arsines generated by FIA, N.I.
DMAA GC separation cryogenically trapped in a GC
column and released on heat-
ing in water bath)
As(III), As(V), MMAA, Urine IEC-FIA-HG-AAS Separation on a GC column. 2 each
DMAA Determination of As in the
fractions by FIA-HG-AAS
As(III), As(V), MMAA, Urine, waters, synthetic fishHPLC-HG-AAS On-line HPLC, microwave ox- 446 [57,581
DMAA, AsB and AsC and sediment extracts idation of all species to As(V),
HG-AAS detection. The
column retains the anionic
species, while AsB and AsC
are determined after MW-
K&O, decomposition.
As(III), As(V), MMAA, Synthetic mixtures HPLC-HG-AAS HPLC-persulfate-mediated 0.3 [541
DMAA, AsB and AsC, o- photo oxidation with a low
and p-arsanilate, phenylar- power UV-irradiation stage
sonate, tetramethylarsonium prior to HG-AAS
Table 3 (continued)

As species (samples) Matrix Measurement technique Some details of the proce- D.L. (ug l-r)* Ref.
dure

As(III), As(V), MMAA, Urine HPLC-HG-AAS Coupled techniques 1.0, 1.6, 1.2, 4.7 I1091
DMAA
As(III), As(V), MMAA, HPLC-HG-AAS HPLC- Coupled techniques with corn- 10 for As(lII), DMAA,AsB [ill]
DMAA, AsB ICP-MS parable results. 15 for MMAA 20 for As(V)
As(III), AsT Marine sediments ETAAS As(II1) was chelated with 25-44 ug kg’ 11381
sodium diethylditiocarbamate
and extracted in MIBK
As(III), AsT Bottled mineral water ETAAS Complexed As(III) extracted N.I. [l371
in organic solvents. As(V)
must be reduced.
As(HI), AsT Synthetic mixtures, sea wa- ETAAS on-line FI-on-line separation and pre- 0.32, 0.43 ng [89]
ters concentration. As(III)-NaD-
DTC complex is separated in
a Cl8 reversed phase column
As(III), As(V), MMAA, Waters IEC-ETAAS Separation on a dual column. 10 each [I441
DMAA Determination of As in the
fractions by ETAAS
Total As, As(V), MMAA, Synthetic mixtures IEC-ETAAS As is determined in collected 4.0, 0.4, 2.0, 0.02 [l431
DMAA fractions
As(III), As(V) Waters HG-ICP As(V) was reduced by L-Cys- 0.7, 3.4, respectively 1991
teine (use of a co-concentric
generator without GLS)
As(V), MMAA SRM IEC-ICP On-line preconcentration with 0.015 ~1291
ultrasonic nebulization
As(lII), As(V), MMAA, Synthetic fish extracts HPLC-HG-ICP On-line coupled techniques. 3.5, 9.2, 3.8, 21.3 (isocratic [116]
DMAA, elution)
2.7, 11.4, 9.2, 9.4 (gradient [117]
elution)
As(III), As(V) Coal fly ash HPLC-ICP-MS Water and diluted acid ex- N.I. ]l261
tracts from the solid
Table 3 (continued)

As species (samples) Matrix Measurement technique Some details of the proce- D.L. (ug I-‘)* Ref.
dure
2
As(III),
As(V), MMAA, Urine HPLC-ICP-MS Directly coupled techniques N.I. 11411
DMAA, AsB
As(III), As(V), MMAA, Waters HPLC-ICP-MS Directly coupled techniques 1.o-3.0 [llgl
DMAA, AsB and AsC
As(III), As(V), MMAA, Simulated fish and sedi- HPLC-ICP-MS Coupled techniques 10-30 pg (20 ul) [1131
DMAA, AsB and AsC ment extracts
As(III), As(V), MMAA, Waters HPLC-HG-ICP-MS Coupled techniques 0.011 PO.051 11141
DMAA
As(III), AS(V) SRM NAA After sequential coprecipita- 0.02 for both u 501
tion of As(II1) with diben-
zyldithiocarbamate
As(III), AS(V) Waters, marine species NAA Preconcentration of As(II1) N.I. [1511
and As(V) by solvent extrac-
tion
As(III), As(V), MMAA, HPLC-AFS Coupled techniques using ul- 35, 50, 20, 20 ng 11531
DMAA trasonic nebulization
As(III), AsT HG-direct current plasma Modified hydride generator in-O.51 1901
terfaced to a redesigned tube
nebulizer
As(III), As(V), MMAA, Tap, sea water and urine HPLC-HG-MIP Coupled techniques 1-6 [1541
DMAA

* Unless otherwise stated. 1= wavelength; D.L. = detection Limit; N.I. = not indicated; AsT = total arsenic; MMAA = monomethylarsonic acid; DMAA = dimethy-
larsinic acid; AsB = arsenobetaine; AsC = arsenocholine; and other abbreviation as in Fig. 1.
1600 M. Burpera, J.L. Bur.guera/Talanta 44 (1997) 1581-1604

surfatron at reduced pressure. Enhancements in

the emission signals of about 100% were found in
miscelles of cetyltrimethylammonium bromide
and the detection limits for the arsenic species
investigated (arseneous, arsenic, monomethylar-
sonic and dimethylarsinic acids) were in the range
l-6 ng l- ‘. This novel methodology has been
successfully applied for arsenic speciation in tap
and sea waters and in human urine [154].
Also direct current plasma (DCP) atomic emis-
sion spectrometry coupled to a modified HG sys-
tem proved successful for the determination of
As(II1) and As(V) [90]. Both species could be
determined as total arsenic without the need for
any prereduction step.
Fig. 1. Procedures for arsenic speciation in different matrixes.
Mohan et al. [59] evaluated the capability of (UV-vis. = spectrophotometry; EQ = electrochemical methods;
HG-DC helium emission spectrometry as an ana- AAS = atomic absorption spectrometry; ICP-AES = induc-
lytical tool in the speciation of arsenic in soils tively coupled plasma atomic emission spectrometry; ICP-
treated with arsenical herbicides. Due to differ- MS = inductively coupled plasma mass spectrometry;
HG = hydride generation; FAAS = flame atomic absorption
ences in soil type and samples pore size, the
spectrometry; CT = cryogenic trapping; CC = gas chromatog-
method only permitted semicuantitative specia- raphy; HPLC = high performance liquid chromatography;
tion. IEC = ion exchange chromatography; ETAAS = electrother-
The separation power of capillary electrophore- mal atomic absorption spectrometry; NAA = neutron activa-
sis has been widely demonstrated, most of the tion analysis; DCP = direct current plasma; AFS = atomic
fluorescence spectrometry; MIP = microwave induced
studies being focused on the optimization of sepa-
plasma;and CE = capillary electrophoresis).
ration parameters for complex mixtures of com-
pounds of biochemical interest. Little effort has
been devoted to quantitative studies and less to
arsenic speciation. Lopez-Sanchez et al. [155] sep- and speciation of arsenic are diverse. A schematic
arate arsenite, arsenate, MMAA, DMAA and diagram summarizing the different techniques and
phenilarsonic acid in a fused silica capillary filled some of the most relevant information, such as
with a phosphate buffer and detected the signals samples type, procedure and detection limits, are
with an on-column UV detector operated at 190 given in Fig. 1 and Table 3, respectively. Each
nm. Capillary electrophoresis with conductivity approach possesses both advantages and disad-
[156] or AAS [157] detection was also used to vantages that must be considered with respect to
speciate arsenic and selenium compounds in wa- the scope of the study and also the laboratory
ter. The samples were injected electrokinetically at facilities available. Adequate quantitative determi-
25 mbar for 12 s on to a pretreated fused silica nation of complex matrices require the establish-
column. The separation was carried out at 25 KV ment of efficient separation and preconcentration
with triton X-100 and cyclohexil-amine-ethanesul- processes, good recovery in clean-up procedures
fonic acid at pH = 9.4. The detection limit was 40 and precision and accuracy controls. All these
ug I- ’ for As(V). problems can be partly solved by performing spe-
ciation studies with in vivo and in vitro labeled
radioactive compounds followed by gamma or
4. Conclusion beta measurements. This would greatly eliminate
the hazard of distorted results due to exogenous
From this survey it can be concluded that the amounts of the element; which are not radioactive
analytical techniques available for the detection and will escape detection.
 M. Burguera, J.L. Burguera ralanta 44 (1997) 1581-1604 1601

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1983.
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