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12. Arsenic speciation in environmental matrices
Quality Assurance for Environmental Analysis Quevauviller/Maier/Griepink (editors) © 1995 Elsevier Science B.V. All rights reserved.
285
12. Arsenic speciation in environmental matrices B. AmianS F. Lagarde\ MJ.F.Leroy\ A. Lamotte^ C. Demesmay^, M. 011e\ M. Albeif, G. Rauref and J.F. Lopez-Sdnchez' 1. 2. 3.
Laboiatoiie de Chiime Min^fale et Analytique, URA 405 da CNRS, E.H.I.C.S.,1 me Blaise Pascal F-67008 Strasbourg C6dex, France S^vice Ceatral d'Analyse, VSR 59 da CNRS, Echangeur de Solaize, BP 22 F-69390 Vemaison, France Universitat de Barcelona, Dqiartament de Quimica Analitica, Avda Diagonal 647 0828 Barcelona, Spain
Arsenic is widely distributed in the environment because of its natural origin and its industrial production. Arsenic compounds are mainly used in agriculture to prepare insecticides, herbicides and fungicides. They are also used as cotton dessicants or wood preservatives and in medicine as bactericides or parasiticides. The natural presence of arsenic is essentially due to the emergence of groundwaters containing high concentrations of that element and to volcanism. Terrestrial crust contains about 3 mg.kg"^As. In sea water as well as in freshwater, arsenic is present at the /ig.kg*^ level. In soils, contents are in the range of 0.05 to 0.2 mg.kg"\ Marine organisms contain very high arsenic moieties (1-100 mg.kg^). Arsenic occurs in various organic and inorganic species with several oxidation states (-3, 0, +3 and +5). The compounds most commonly found are arsenite and arsenate ions (As(III) and As(V)), monomethylarsonic and dimethylarsinic acids (MMA and DMA), arsine, di- and trimethylarsine as well as other organoarsenical compounds such as arsenobetaine (Asbet), arsenocholine (Aschol), arsenolipids and arsenosugars. Corresponding formula are presented in Table 1. The toxicity of arsenic depends on its chemical form. Contrary to lead or mercury, inorganic species of arsenic are more toxic than organic compounds as shown in Table 2. It can be seen that the toxicity of As(III) and As(V) is to be compared to that of strychnine, which is known to be a violent poison. On the contrary, DMA and MMA are as toxic as aspirin. Asbet and Aschol are roughly not toxic.
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Arsenic spedation in environmental analysis
Table 1:
Chemical formula of some arsenic compounds
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OH
I
O = As - OH Arsenious acid (As(III))
CH3
O = As - OH
I
I
O = As - OH
OH Arsenic acid (As(V))
CH3
CH3
CH3
I
0 = As-CH3
I OH Monomethyiarsonic acid (MMA)
I
I
CH3 - As+ - CH2 - COO- CH3 - As"*" - CH2 - CH2 - OH, X"
I
CH3
CH3
OH Dimethylarsinic acid (DMA)
Arsenobetaine
Arsenocholine
OH
I O = As - C6H5
I OH Phenylarsonic acid
Table 2:
Lethal Dose 50 of some arsenic compounds (LD50: dose which is fatal to onehalf a population of experimental animals) Compounds Arsine Potassium arsenite Arsenic trioxide Calcium arsenate Phenylarsonic acid Monomethyiarsonic acid (MMA) Dimethylarsinic acid (DMA) Strychnine Aspirin Arsenobetaine (Asbet) Arsenocholine (Aschol)
LD<5o (mg.kg'l weight of rat) 3 14 20 20 50 700-1800 700 - 2600 16 1000-1600 > 10000 > 10000
1
Arsenic speciation in environmental analysis
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287
In the environment, arsenic and its compounds may be submitted to physical, chemical and biochemical transformations with or without modification of their oxidation states, and/or mineralization, adsorption and precipitation processes. Figure 1 describes all the complex phenomena which may occur in the different compartments.
NATURAL AND ARTIFICIAL SOURCES : ATMOSPHERE, ADSORPTION ON ATMOSPHERIC PARTICULES
As (inorganic) oxidation 4
BIOSPHERE
(CH3)2AsH, (CH3)3As •
-(CH3)3As0
(CH3)3As'^CH2C00-
(FISHES, MAMMAL. ,
(BACTERIA, FUNGI)
(CH3)2AsOCHjCOOH
t
•
(CH-;)2AsO(OH) -*-arsenosonbosides*-(CH3)2AsOCH2CH20H (ALGAE) I (CH3)AsO(OH)2 f (CH3)3As^CH2CH20H,XAs(ni) • MICROORGANISMS MICROORGANISMS A As(V) As(iin -
HYDROSPHERE
^
r X | # . Fe(llI).As(V) COMinJIXES | - ^ ^ Fe(III) Asdll)
-
(A.B,C,D) LITOSPHERE
- - ik- •
aerobic medium
anaerobic medium
- | " B ~ | - Fe(Un Aadll)
aerobic medium
"^ FeAsS. AS2S3 -tc ! • — ^
Figure 1:
Bio-geochemical cycle of arsenic A: deposition and co-precipitation, B: dissolution, C: diffusion and reaction with sulfides, D: adsorption
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Arsenic spedation in environmental ana^is
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Speciation of arsenic has already been studied but has generally been performed on calibrant solutions. A Measurements and Testing Programme (BCR/EC) project concerning arsenic speciation in marine organisms is in progress. The aim is to prepare certified materials (mussels and tuna fish tissues) for sixarsenic species: As(III), As(V), MMA, DMA, Asbet and Aschol. Different interlaboratory studies have been conducted and have shown the necessity of validating an analytical methodology. 12.1
Critical review of existing methods
The first methodology developed for arsenic speciation was based on the hydride generation technique [1]. Since then, other methods have been developed and detection limits have considerably decreased. Nowadays, it is possible to analyse samples containing only some tens ng.g'^As of each individual species. Most of the techniques described in the literature are coupling methods using the hydride generation technique or liquid chromatography (LC) as separation methods and atomic absorption spectrometry (AAS), or inductively coupled plasma emission spectroscopy (ICP/AES) as detection technique. Direct current plasma emission spectroscopy (DCP/AES), or mass spectrometry have also been used. In this part, we present the most commonly used techniques, their advantages and their drawbacks. 12.1.1 Hydride generation method 12.1,1.1 Hydride generation and separation This derivatization method was originally developed for selenium speciation [2] but has been successfully applied to the determination of arsenite and arsenate ions, monomethylarsonic and dimethylarsinic acids and trimethylarsenoxide. In an acidic medium, some arsenic compounds may be reduced to volatile arsines and carried by an inert gas flow to a specific detector. Using suitable conditions, it is possible to generate arsines quantitatively and/or selectively. Main parameters are pH, as well as the nature and concentration of the reducing agent and the acid. In order to avoid problems due to the hydride generation kinetics, a liquid nitrogen trap is placed after the reactor. After reduction of the arsenic compounds, hydrides are carried to the trap where they are condensed. After complete reaction, the trap is progressively heated and hydrides are volatilized according to their respective boiling points. This technique, also used for tin speciation, is known as the "cold trap method". Arsines formed are further separated by a gas chromatograph or analysed directly. Many authors have studied the selectivity of hydride generation as a function of reactional parameters in the case of arsenic [3-9]. Anderson and coworkers (1986) have shown that it was possible to selectively generate hydrides from As(III), As(V), MMA and DMA. Conditions described allow the rapid determination of As(III), DMA, As(III) + As(V) and total arsenic concentrations [8].
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Arsenic spedation in environmental ana^is
12.1.1.2
289
Detection
Whatever the detection technique used, the main advantages of the hydride generation technique are: analyte/matrix separation improvement of the introduction system: the quantity introduced to the detector is 50 to 100 higher than with pneumatic nebulizers analyte preconcentration large choice of detection techniques easy on-line and automatized use This method has, nevertheless, several drawbacks: quantification is possible only for compounds forming volatile hydrides (arsenobetaine and arsenocholine, for example, can not be converted into arsines) the presence of some elements (Ni, Co, Cu, ...)decreases the efficiency of hydride generation one must strictly control the reaction medium to obtain good results. The hydride generation technique was first coupled to flame atomic absorption spectrometry (FAAS) [10]. The analyte is removed from the matrix so that classical interferences observed in A AS are reduced or even eliminated. Nevertheless, FAAS is not sensitive enough to allow the determination of arsenic traces in environmental samples and flames have been progressively replaced by quartz furnaces [8,11] which offer better control of atomization and lead to more suitable detection limits. Using this technique, inorganic species (arsenite and arsenate), monomethylarsonic and dimethylarsinic acids have been quantified in waters, sediments and biological tissue extracts [3,8,9,12-14]with detection limits in the /xg.l'^ range. Atomic emission spectrometry is not often used because of its higher cost. Hydride generation has been only coupled with a direct current plasma system for the determination of arsenite, arsenate and total arsenic in waters and fishes [15]. The most sophisticated system has been developed by Kaise et al [16]. Hydrides are generated, condensed, volatilized, separated by gas chromatography and then analysed in a mass spectrometer. The most intense peaks correspond to m/z = 76 and 78 for ASH3, 90 for CH3ASH2 and (CH3)2AsH, 103 and 120 for (CH3)3As. 12.1.2 Liquid Chromatography coupled with specific detectors Liquid chromatography is particularly suitable for the determination of arsenic compounds because of the hydrophilic and ionic or ionizable character of these species. This technique presents several advantages when compared to the previous one. Indeed, no derivatization is needed before separation, several types of chromatography may be used and a large choice of stationary phase/mobile phase couples are available. Nevertheless, classical detectors such as UV/Vis or electrochemical detectors are not sensitive enough and a specific detector (absorption or emission spectrometry) has to be used.
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Arsenic spedation in environmental analysis
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12.1.2.1 Separation by ion-pair chromatography Separations have been performed using apolar stationary phases constituted of C18 silica [17-20] or polymeric resin [21-23]. The ion-pairing agent is generally tetrabutylammonium phosphate [17,18] or hydroxide [20-23]. Brickman et al [24] have achieved the separation in the presence of tetraheptylammonium nitrate whereas Larsen has investigated Cg-Ciz alkylammoniums as well as butanesulfonate [19]. Elution is achieved using an isocratic regime or a gradient step program. Analysis time is in the range of 10 to 40 minutes. 12.1.2.2 Separation by ion-exchange chromatography Silica based [18,20,25-27] or polymeric [19,29-35] stationary phases grafted with strong anion-exchange groups (quaternary ammoniums) as well as weak anion-exchange columns [28] have been investigated. Compounds are eluted using a phosphate, carbonate or acetate buffer as mobile phase. pH is in the 6-7 range. The addition of an organic modifier has been found to decrease hydrophobic interactions [23-25]. Analysis times are also in the range of 10 to 40 minutes. These separations have been performed on calibrant solutions and on natural samples: pesticides and herbicides residues [29],urine [28,30,36],biological samples [25,26,32] and waters [31]. Some authors have mentioned the degradation of silica ionexchange columns after few weeks of extensive use [27,30,33]. 12.1.2.3 Detection by atomic absorption spectrometry Coupling HPLC with FAAS does not present any major technological problems. Nevertheless, the low sensitivity of the system (detection limit for arsenic : 1 mg.l"^) due to the difficult introduction of the sample to the flame and to the attenuation of the arsenic radiation intensity by radicals formed in the flame, has considerably limited its application in the arsenic speciation field. Several studies using electrothermal atomic absorption spectrometry (ETAAS) as the detector have shown that sensitivity can be improved by a factor of 10 to 100 when compared to FAAS. Nevertheless, coupling a continuous separation technique (HPLC) to a sequential detector is not easy. Two types of interface have been used, leading to off-line and on-line coupling methods: in the first [37], a graphite furnace sampler is used as a fraction collector . Better resolution is obtained but analysis time is considerably increased, in the on-line method [38-40], effluent fractions are collected and periodically analysed. This technique requires large chromatographic peaks because 30 to 60 s are needed for each determination. HPLC-ETAAS has been developed for arsenic speciation using anion-exchange [38,39]and reverse phase liquid chromatography [24]. Mobile phases, generally more complex, lead to an important increase of the background noise and require the use of a powerful apparatus equipped with a Zeeman background correction system [41]. It has been used for arsenic speciation in waters [24], pesticides [29,39] and soils [24]. Detection limits are in the nanogramme range. However, elution of significant quantities of organic materials together with arsenic compounds disturbs the detection. Moreover, some species are able to be volatilized without atomization if large amounts of salts or carbonaceous compounds are present in the sample.
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12.1.2.4 Detection by inductively coupled plasma atomic emission spectrometry The compatibility of flow rates used in HPLC and ICP/AES (generally 1 ml.min^) allows a very easy coupling between these two techniques. A PTFE tube of adequate internal diameter is connected from the exit of the column to the nebulizer of the apparatus. This coupling method has been developed for arsenic speciation with ion-exchange [18,26, 32,42-44] and ion-pair liquid chromatography [17,21,45].Detection limits are some tens of nanogrammes for ion-exchange chromatography or some hundreds of nanogrammes for ionpair liquid chromatography. Some attempts to decrease the detection limits by improving the introduction system have been realized but no conclusive results have been obtained. The detection limits obtained allow the determination of the most abundant arsenic compound found in marine food, ie, arsenobetaine, but are generally not low enough to reach the other concentrations. 12.1.2.5 LC procedures involving hydride generation Matrix interferences observed in LC-AAS may be suppressed and detection limits improved by a post-column derivatization: for example the reduction of arsenic species into volatile arsine increases the quantity of analyte introduced in the atomization cell. Hydrides are carried by an inert gas current into a heated quartz cell where they are atomized and detected. Detection limits are in the range of few hundreds of picogrammes As, which are particularly suited for arsenic determination in environmental samples. ICP-AES detection may also be used after hydride generation in a simple coupling configuration: a PTFE tube is connected from the column to the entrance of the reduction coil, the nebulizer of the ICP/AES is removed and the exit of the gas-liquid separator is directly connected to the plasma. This modification may be realized by the user himself but some hydride generation kits are commercially available. The use of hydride generation for arsenic speciation increases sensitivity (some nanogrammes) but restricts the application of the technique to species which are able to form hydrides ie. As(III) , As(V), MMA and DMA. Some authors have proposed a photolitic reaction to convert Asbet, MMA and DMA in As(V) [46,47]. Nevertheless, the long period of time (hours) required for quantitative photolysis of Asbet makes it unsuitable for "on-line" measurements. The UV irradiation with addition of peroxodisulfate has been used for Asbet, DMA, MMA and As(V) determination in a LC-UV-HG-ICP system [48]. Recently, Rauret et al have studied the photo-oxidation of Asbet and Aschol by UV irradiation in persulfate media in order to convert these inert compounds in simple molecules able to produce volatile hydrides which could be determined by LC-UV-HGICP/AES [49]. The experimental conditions such as the photoreactor design, use of persulfate in different media, irradiation time and the effect of the power lamp have been optimized and it was shown that all the arsenic species of environmental interest are able to be converted into As(V) and consequentiy to be quantified by the LC-UV-HG-ICP/AES technique. Detection limits are very close to those obtained with HG-ICP/AES detection.
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12.1.2.6 HPLC coupled to ICP-MS This coupling method has been used for the speciation of arsenic with ion exchange, ionpair, or gel permeation chromatography [22,28,33,34,50-57]. Detection limits are very low (20-200 pg As) and the detector is linear over a wide dynamic range. This technique has been successfully applied to the determination of arsenic compounds in seafood products and sediments [50, 52]. 12.2
Means of validation
Some reference materials certified for total arsenic concentration are commercially available. In the environmental field and more particularly concerning the biological tissues and sediments CRM's, the Measurements and Testing Programme (BCR) of the Commission of the European Communities provides 7 materials: -
CRM CRM CRM CRM CRM CRM CRM
185 (bovine liver) : 186 (pig kidney) : 278 (mussel tissue) : 422 (cod muscle) : 277 (estuarine sediment): 280 (lake sediment) : 320 (river sediment) :
2 4 + 3 fig.g^ 63 ± 9 /xg.g' 5.9 ± 0.2/xg.g' 21.1 ± 0.5iLtg.g' 47.3 ± 1.6/xg.g'^ 51.0 ± 2.4^g.g' 76.7 ± 3A fig.g'
The National Research Council Canada (NRCC) has also prepared two fish tissues certified for their total arsenic content: - DORM-1 (dogfish muscle) : - DOLT-1 (dogfish liver) :
17.7 ± 2.1 fig.g' 10.1 ± lAfxg.g'
Finally, the National Institute of Standards and Technology (NIST) produced two sediments: - SRM 1646 (estuarine sediment) : - SRM 2704 (buffalo river sediment) :
11.6 ± 1.3/xg.g"^ 23.4 ± 0.8/ig.g"'
Speciation studies have been conducted on DORM-1 CRM [50,54,57]and have shown that 91 to 96 % of the arsenic extracted in the aqueous phase is present as Asbet. Nevertheless, no reference material certified for arsenic species is available. A BCR project which aims to elaborate reference materials of this type is now in progress. Pilot laboratories are the Laboratoire de Chimie Minerale et Analytique (Strasbourg, F) and the Service Central d'Analyse (CNRS, Vemaison, F). At the beginning, four materials (soil, sediment, fish and mussel tissues) and six arsenic species (As(III), As(V), DMA, MMA, Asbet and Aschol) were proposed. Nevertheless, the idea of certifying soils and sediments was abandonned because of the difficulty of collecting the appropriate materials.
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293
Moreover, the easy interconversion between As(III) and As(V) has led the organizers to restrict the quantification to As(III)+As(V), Asbet, Aschol, DMA and MMA. Intercomparison studies involving 10 to 20 laboratories have been organized using the classical step by step approach: 123456-
Preparation of the calibrants Study of pure calibrants or mixtures of pure calibrant solutions Study of mixtures of calibrants containing interfering compounds Study of clean extracts Study of raw extracts Study of real samples
Each step involves one or two intercomparison exercises and stability studies of the solutions distributed to the participants. With respect to calibrants, As(III), As(V), DMA and MMA are commercially available and can be used to prepare calibrant solutions. On the contrary, Asbet and Aschol have to be synthesized. The preparation has been achieved at the Laboratoire des Materiaux Organiques (Solaize, France) and characterized by elemental analysis, mass spectrometry and inductively coupled plasma atomic emission spectrometry. Calibrant solutions have been prepared in freshly boiled deionized water and their stability studied at 4 °C,25 °Cand 40 °C.It has been proved that solutions are stable at least for six months if kept in the dark at 4 °C. If storage conditions are not respected, oxidation of As(III) into As(V) and degradation of methylated species may occur. Stability studies performed for steps 3, 4 and 5 have shown that solutions which do not contain inorganic arsenic are stable at -40 °C and 4 °C in the dark. In the presence of mineral arsenic, interconversion between As(III) and As(V) is observed, this phenomenon depending on the temperature and on the sample composition. The different intercomparison studies have helped the participants to improve their methods of determination and have allowed the identification of several sources of error (problem of calibration, column washing and pre-conditioning, sample treatment, presence of chlorides for ICP-MS detection etc.) which have been solved. The 6th step has been started in the second half of 1994 and the certification campaign of mussels and fish tissues has been organized at the end of 1994. 12.3
Description of a validated technique
Sample treatment and arsenic speciation procedures described in this paragraph are the methods used in laboratories 1 (Laboratoire de Chimie Minerale et Analytique, Strasbourg), 2 (Service Central d'Analyse, Vemaison) and 3 (Universitat de Barcelona, E). These procedures have been established for the six more commonly found arsenic species in sediments and seafood products: As(III), As(V), Asbet, Aschol, DMA and MMA.
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12J.1 Sample treatment The sample preparation is one of the most difficult steps for trace element speciation in natural samples. The entire procedure has to be achieved in such a way that no loss or contamination or change in the speciation happens. 12.3.1.1 Analysis of seafood materials Fresh material is collected, washed with sea water and stored at O^'C. In the case of shellfish, shells are opened by freezing at -25 °C. After that, products are lyophilized so that the moisture content does not exceed 8-9 %, The powder obtained is homogeneized, pulverized and filtered on a 125 ^m sieve. Further treatment applied by Lab.l to this type of sample is based on a water/methanol extraction followed by a clean-up with ether as shown in Figure 2. Methanol/water evaporation should not be taken to dryness because of the possibility of volatilization and/or degradation. In the extraction step, the final volume indicated corresponds to the minimal volume required to take the sample out from the flask . It may be reduced if possible but practically can not be lower than 5 ml. Indeed, the solution becomes too viscous and further purification can not be achieved. Filtration may be replaced by centrifugation in the extraction step. Use of an ultrasonic bath has been prefered to mechanical stirring because sample powder is more easily dispersed that way. In order to check that the procedure does not induce any transformation of the species of interest and leads to 100 % recovery, a codfish powder was: first spiked with calibrant solutions of the six species of interest and then extracted and purified (I) secondly extracted, purified and spiked (II). Speciation was then performed using LC-ICP/AES (for Asbet and Aschol) and LC-HGQFAAS coupling techniques. The same results were obtained by procedures I and II. Moreover, total arsenic concentration independently determined by EDXRF (Energy Dispersive X-Ray Fluorescence) on the starting powder was found equal to the sum of Asbet, DMA and As(V) concentrations, the only As species detected in the sample. Finally, concentrations found for each compound in procedures I and II corresponded to the sum: concentration in the sample + concentration of the spike. 12.3.1.2 Sediment analysis In sediments, the most commonly found arsenic species are As(III), As(V), DMA and MMA. Extraction may be performed by classical acid attack in beaker or tubes or by microwave digestion after homogenization, pulverization and filtration on a 125 fxra sieve. The advantages of microwave digestion are both the reduction of analysis times and the possibility of automatization.
Arsenic spedation in environmental analysis
[Ch.l2
SAMPLE POWDER
295
1000 mg.
METHANOL / WATER (1: 1) - (5 x 10 mL)
;r
ULTRASONIC BATH
RE-EXTRACTION
-N SOLID V^
FILTRATION
f LIQUID 1
EVAPORATION
WATER r EXTRACT
^ E T H E R (5 X 10 mU
fsEPARATION
ORGANIC PHASE
J
\ '
i
- ( RE-EXTRACTION J
AQUEOUS PHASE
PURIFIED EXTRACT
Figure 2: Extraction/Clean-up procedure for arsenic speciation in seafood products
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Arsenic spedation in environmental analysis
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Two procedures have been developed by laboratory 2 for sediment analysis [58]: Digestion with a mixture of hydrochloric and nitric acids: 7 ml HCl and 3 ml HNO3 are added to 200 mg powder sample and introduced into a microwave digestion apparatus (Microdigest A 300 type, Prolabo, power: 0-200 W) using 20 % of maximum power for 5 minutes and then 25 % maximal power for 10 minutes. The same quantity of HCI/HNO3 mixture, 1 ml H2O2 and 5 ml water are successively added to the solution and heated at 30 % for 10 minutes, 20 % for 5 minutes and 25 % for 5 minutes respectively. It was observed by HPLC-ICPMS that, under those conditions, As(III) is totally converted into As(V) but DMA and MMA are preserved. This procedure allows the determination of mineral arsenic, DMA and MMA. Digestion with orthophosphoric acid: The best extraction yields are obtained using orthophosphoric acid 0.3 mol.l'^ at pH 1.3 stirring the solution (100 mg powder and 15 ml acid) for 10 minutes in the same microwave system as previously described. In those conditions, As(III) oxidation is slight. DMA and MMA can quantified. 12,3.2 Arsenic spedation The separation technique used by laboratories 1, 2 and 3 is liquid chromatography. Lab. 1 performed the separation using an ion-pair reverse system followed by ICP/AES and HGQFAAS techniques whereas labs. 2 and 3 used ion-exchange chromatography with ICP-MS and UV-HG-ICP/AES detections respectively. Arsenic was measured at a wavelength of 193.7 nm. 12.3.2,1 Procedures used: Laboratory 1 Determination of arsenobetaine by ICP/AES The first approach is based on the direct connection between the chromatographic system and the atomic emission spectrophotometer. The interface between both is made of a PTFE tube (250 jLcm, length: as short as possible) which is connected from the exit of the column to the nebulizer of the apparatus. Taking into account the poor sensitivity of the technique (see paragraph 12.1), only arsenobetaine can be determined this way in seafood products. 200 yil of the purified extract is injected onto a Hamilton PRP-1 ion-pair column using a solution of tetrabutylammonium phosphate (TBAP, 0.5mmol.r\pH 9.5) as a mobile phase. The eluate is directly introduced into the ICP/AES nebulizer and analysed. Determination of As(III), As(V), DMA and MMA by LC-HG-QFAAS Chromatographic conditions were modified to separate the four arsenic species forming hydrides in one run. This time, 200 yl of the sample was injected onto the same column but with a mixture of TBAP (10 mM) and TBAOH (Tetrabutylammoniumhydroxide, lOmmol.l"') at pH 6.15 as a mobile phase. The pH of the mobile phase as well as buffer concentration have been optimized. In the derivatization phase, the type of acid, its concentration and borohydride concentration have been considered. H2SO4 (0.5mol.r\l ml.min"^) and NaBH4
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297
(1 % in NaOH 0.1 %, 1 ml.min*^) were added to the eluate and the solution was introduced into the gas-liquid separator of the hydride generation system (Perkin Elmer FIAS 400, shown in Figure 3a). The separator includes a membrane which prevents the liquid entering the quartz atomization cell. Nevertheless, the mobile phase used becomes very effervescent when acid and borohydride are added and membrane is often wet if no precaution is taken; one solution consists of inserting a spring into the separator. The argon flow rate was set at a value of about 75 ml.minVhich leads to the best compromise between reproducibility and sensitivity. Cell temperature was about 900 ^'Cand the slit width was set at 0.7 nm. An EDL lamp (20 mA) was used. 123,2,2 Procedures used: Laboratory 2 Determination of As(III), As(V), DMA and MMA by LC-HG-QFAAS The same method as Lab. 1 has been used. Only the LC conditions and hydride generation system (FIAS 200, Perkin Elmer) were different. 100 jLil of sample solution was injected onto a Hamilton PRP X-100 anion exchange column and a gradient elution was performed using a flow rate of 1 ml.min'^ As mobile phases solution A (NH4)2P04/(NH4)2HP04, 10 moLl\ pH 6.2) and solution B ((NH4)2HP04, 100 mol.r\pH 8) were used. The following gradient program was performed : 100 % A for 3.4 min, decreasing to 50 % A in 0.1 min and maintained for 3 min. 100 % A was reached again in 0.1 min and maintained for 7 min. This mobile phase did not become effervescent when acid and reductant were added. H2SO4 1 mol.r^(1.3ml.min'^) and NaBH4 (1 % in NaOH 0.1 %, 1.3 ml.min"^) were used as reactants for hydride generation. Reaction takes place in a PTFE tube (25 cm length, 0.5 mm i.d.).Detection limits were similar to those obtained by Lab.l. Determination of all the species by HPLC-ICP-MS [51] LC conditions were adapted to the specific detector used. Separation was performed using the mobile phases described previously but adding 2 % CH3CN to enhance the sensitivity of the method. NH4* counter ions are prefered to Na* ions because signals observed in ICPMS are greatly affected by the presence of alkaline ions. Parameters of the ICP-MS apparatus (VG Plasma Quad 2) were optimized each day with a solution of 20 ng.ml'^As in buffer A in order to reach the highest possible signal. Coupling between LC and ICPMS system was achieved with a simple PTFE connection as previously described. As was determined at m/z 75. The signal was recorded on a Shimadzu CR3 A integrator connected to the analog output of the electron multiplier (pulse counting mode). A resistance-capacity filter was used in order to lower the background noise. High concentrations of easily ionizable cations such as Na* or K^ could interfere with Aschol determination because of their similar retention times. Chloride ions, which may combine with argon to produce an interfering peak at m/z 75 in ICP-MS, cannot disturb arsenic speciation in this system since they elute later than the arsenic compounds of interest.
298
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quartz
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fiiniacc
M intcrfecc
acid borohydride peristaltic pump
peristaltic pump gas-liquid separator
HonochroiiMtor
Potassium Persulphate Plasma Torch
^/W-^
/////////// LC Pump
Figure 3:
UV Photoreactor
Schematic of the (A) LC-HG-QFAAS and (B) LC-UV-HG-ICP/AES systems used for arsenic speciation
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123.2,3 Procedures used: Laboratory 3 Determination by HPLC-UV-HG-ICP/AES As already said, ICP/AES allows the detection of Asbet if its concentration is high enough (for example, in seafood products) and HG-ICP/AES is limited to the quantification of species which are able to form hydrides. In order to determine all the species on line and in one experiment. Lab.3 has developed a method in which they are all converted into As(V) in a photo-reactor located just after the LC chromatograph. The As(V) obtained is introduced into an hydride generation system and determined by ICP/AES. Working conditions have been optimized in order to obtain the best detection limits. The signal is filtered by using a Fourier Transform in order to better evaluate the peak height. A photoreactor was developed to be easily coupled on line between the exit of the HPLC column and the entrance to the reduction chamber. The length and the internal diameter of the PTFE coil and flow rate, were considered in order to minimize the dispersion of the chromatographic peaks, since this depends on L, r and, according to the equation: Tcr^L ay^ = k
(j) 24 Dj^
r : internal radius of the capillary (|): liquid flow rate Djn : molecular diffusion coefficient for analyte in solvent k : proportionality factor which depends on the flow profile in the capillary The variance can be minimized when the length of the tube is increased and its internal diameter is reduced. Thus, 0.35 mm, the narrowest easily available PTFE tube was chosen. The optimal length of the tube was determined by the time necessary to complete the photooxidation reaction. This photo-oxidation permits "on line" coupling between chromatographic separation and hydride generation. The eluate is introduced in the photo-reactor connected to the outlet of the column with addition of 3 % K2S208in3 % NaOH (0.2 ml.min"^) as photo-oxidation reagent. The vapour emerging from the photo-reactor is introduced into the hydride generation system. As hydride generation system, HCl 8 mol.l' (1 ml.min^) and NaBH4 1 % in NaOH 0.5 % (1 ml.min"^) are used. The resulting solution reaches the gas-liquid separator and then the plasma torch (see Figure 3b). Figure 4 presents some chromatograms obtained by the arsenic speciation methods developed in the three laboratories. Detection limits and reproducibility of the techniques are shown in Tables 3 and 4.
300
Arsenic spedation in environmental analysis
[Ch.l2
AS(tll)
AS (III) AsBat
AsChol 10 15 Time (min)
A»(V)
DMA MMA
A8(V)
J IJ VJ
Time (mIn)
%i^w 9
10
11
Time (mIn)
Figure 4:
Chromatograms obtained by Labs. 1,2 and 3 using (A) LC-HG-QFAAS, (B) LC-ICPMS and (C) LC-UV-HG-ICP/AES techniques (chromatographic conditions described in the text)
301
Arsenic spedation in environmental analysis
[Ch.l2
Table 3:
Detection limits (in ng As) of the four detection techniques considered in this study (ICP/AES, UV-HG-ICP/AES, HG-QFAAS and ICPMS)
Compound
LC-ICP/AES (Lab.l)
LC-UV-HGICP/AES (Lab.3)
LC-HG QFAAS (Lab.l)
LC-ICPMS (Lab.2)
Asail)
120 130 100 130 110 120
0.26 0.96 1.3 0.98 0.79 0.61
0.15 0.84 0.33 0.43 — —
0.03 0.02 0.02 0.01 0.01
1
As(V) MMA DMA Asbet Aschol
Table 4:
0.01
1
Coefficients of variation expressed in % of the four detection techniques considered
Compound
LC-ICP/AES (Lab.l)^
LC-UV-HGICP/AES (Lab.3)'
LC-HG QFAAS (Lab.l)^
LC-ICPMS (Lab.2)'
As(III) As(V) MMA DMA Asbet Aschol
2.42 3.90 4.71 2.76 3.55 2.37
5.5 5.2 6.3 6.8 5.0 4.6
3.12 3.67 3.46 2.95 — -—
2.2
1
1.6 4.1 2.5 2.5
2.4
I
short term reproducibility considered for eight consecutive injections of a solution 10 />tg.mr^(in As). long term reproducibility determined by injecting ten times in three non consecutive days a solution approximatively five times the LOD. short term reproducibility determined by injecting eight consecutive times a solution 50 ng.mr^(in As) of each compound . short term reproducibility determined by injecting six consecutive times a solution 0.1 mg.ml'^of each compound . 12.4
Conclusions
Several coupling techniques using liquid chromatography as the separation method and ICP/AES, HG-QFAAS, UV-HG-ICP/AES or ICP-MS as detectors are now available for arsenic speciation in environmental matrices. Nevertheless, some inter-comparisons within the BCR programme on arsenic speciation in marine organisms and sediments have shown discrepancies between the results of the different laboratories involved. Further efforts are
302
[Ch.l2
Arsenic speciation in environmental analysis
necessary to improve the quality of the measurements, particularly at sample preparation step as well as on the quantification procedure. Concerning the development of new methods for arsenic speciation, capillary zone electrophoresis (CZE) appears a priori to be suitable since it is best dedicated to ion separation. As(III), As(V), DMA and MMA have already been separated at low concentrations using CZE [59] and the technique has been evaluated in a BCR intercomparison study (Figure 5).However, when applied to extracts of environmental samples the detection limits (UV detector, 190 nm) are too high for the arsenic species. CZE technique can be coupled with mass spectrometry but the low masses of the compounds do not allow good detection limits. Improvements are also needed in the clean-up procedure of the extracts. Additionally the extraction leads to a significant dilution and therefore requires very low detection limits (5 to 10 ng.ml"^) which are best fitted by ICP-MS or HG-GFAAS. Another development would be the obtention of specific extractants designed for anion extraction. This would possibly lead to pre-concentration using either liquid-liquid extraction or substituted resins. ARSENITE TARGET VALUE
T 5
cone, ( m g / k g ) LC-ICP/MS LC-HAAS LC-HICP
CZE LC-ETAAS mean of ali individual values mean of mean values
DMA TARGET VALUE
T 4
5
6
LC-ICP/MS •
LC-HAAS
LC-racp
-*^-^
GC-HAAS
LC-ETAAS mean of all individual values mean of means values
Figure 5:
Results of a BCR intercomparison study for As speciation in a mixture of standard solutions containing 5 mg.kg"^ of the different species. Example of As(III) and DMA.
[Ch.l2
Arsenic spedation in environmental analysis
303
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304
[38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59]
Arsenic spedation in environmental analysis
[Ch.l2
R.A. Stockton and KJ. Irgolic,/. Environ, Anal Chem,, 6, 313 (1979) E.A. Woolson and N.J. Aharonson, /. Assoc, Off, Anal Chem,, 63, 523 (1980) T.M. Vickery, H.E. Howell and M.T. Paradise, Anal Chem,, 51, 1880 (1979) L. Ebdon, S. Hill and R.W. Ward, Analyst, 112, 1 (1987) G.K. Low, G.E. BaUey and S.J. Buchanan, Anal Chim, Acta, 197, 327 (1987) J.P. McCarthy, J.A. Caruso and F.L.Fricke, J. Chromatogr. Sci.,21, 389 (1983) G. Rauret, R. Rubio, I. Peralta and J. Alberti, /. Liquid Chromatogr,, 16,3531 (1993) W. Nisamaneepong, M. Ibrahim and T.W. Gilbert, /. Chromatogr, Scl, 22,473 (1984) W.R. Cullen and M. Dodd, Appl Organomet, Chem,, 2, 1 (1988) C.I. Brockbank, G.E. Batley and C,Y.,C,IJO^, Environ, Tech, Letters, 9, 1361 (1988) R.H. Atallah and D.A. Kalman, Talanta, 38, 167 (1991) R. Rauret, J. Alberti and G. Rauret, Intern, J, Environ, Anal Chem,, 52, 203 (1993) E.H. Larsen, G. Pritzl and S.H. Hansen, /. Anal At, Spectrom., in press C. Demesmay, M. Olle and M. Porthault, Fresenius J. Anal Chem., 348, 205 (1994) J.R. Dean, S. Munro and L. Ebdon, /. Anal Atom, Spectrom., 2, 607 (1987) J.J. Thompson and R.S. Houk, Anal Chem., 58, 2541 (1986) D. Beauchemin, M.E. Bednas, S.S.Berman and J.W. McLaren, Anal Chem., 60,2209 (1988) E.H. Evans and L. Ebdon, /. Anal Atom, Spectrom,, 4, 299 (1988) J.M. Riviello, A. Sirirako, R.M. Manabe, R. Roehl and M. Alforque, 4(12), 25 (1991) Y. Shibata and M. Morita, Anal Chem,, 61, 2116 (1989) C. Demesmay, M. Olle and M. Porthault, unpublished results P. Morin, M.B. Amran, S. Favier, R. Heimburger and M. Leroy, Fresenius J, Anal Chem,, 342, 357 (1992)
285
12. Arsenic speciation in environmental matrices B. AmianS F. Lagarde\ MJ.F.Leroy\ A. Lamotte^ C. Demesmay^, M. 011e\ M. Albeif, G. Rauref and J.F. Lopez-Sdnchez' 1. 2. 3.
Laboiatoiie de Chiime Min^fale et Analytique, URA 405 da CNRS, E.H.I.C.S.,1 me Blaise Pascal F-67008 Strasbourg C6dex, France S^vice Ceatral d'Analyse, VSR 59 da CNRS, Echangeur de Solaize, BP 22 F-69390 Vemaison, France Universitat de Barcelona, Dqiartament de Quimica Analitica, Avda Diagonal 647 0828 Barcelona, Spain
Arsenic is widely distributed in the environment because of its natural origin and its industrial production. Arsenic compounds are mainly used in agriculture to prepare insecticides, herbicides and fungicides. They are also used as cotton dessicants or wood preservatives and in medicine as bactericides or parasiticides. The natural presence of arsenic is essentially due to the emergence of groundwaters containing high concentrations of that element and to volcanism. Terrestrial crust contains about 3 mg.kg"^As. In sea water as well as in freshwater, arsenic is present at the /ig.kg*^ level. In soils, contents are in the range of 0.05 to 0.2 mg.kg"\ Marine organisms contain very high arsenic moieties (1-100 mg.kg^). Arsenic occurs in various organic and inorganic species with several oxidation states (-3, 0, +3 and +5). The compounds most commonly found are arsenite and arsenate ions (As(III) and As(V)), monomethylarsonic and dimethylarsinic acids (MMA and DMA), arsine, di- and trimethylarsine as well as other organoarsenical compounds such as arsenobetaine (Asbet), arsenocholine (Aschol), arsenolipids and arsenosugars. Corresponding formula are presented in Table 1. The toxicity of arsenic depends on its chemical form. Contrary to lead or mercury, inorganic species of arsenic are more toxic than organic compounds as shown in Table 2. It can be seen that the toxicity of As(III) and As(V) is to be compared to that of strychnine, which is known to be a violent poison. On the contrary, DMA and MMA are as toxic as aspirin. Asbet and Aschol are roughly not toxic.
286
Arsenic spedation in environmental analysis
Table 1:
Chemical formula of some arsenic compounds
[Ch.l2
OH
I
O = As - OH Arsenious acid (As(III))
CH3
O = As - OH
I
I
O = As - OH
OH Arsenic acid (As(V))
CH3
CH3
CH3
I
0 = As-CH3
I OH Monomethyiarsonic acid (MMA)
I
I
CH3 - As+ - CH2 - COO- CH3 - As"*" - CH2 - CH2 - OH, X"
I
CH3
CH3
OH Dimethylarsinic acid (DMA)
Arsenobetaine
Arsenocholine
OH
I O = As - C6H5
I OH Phenylarsonic acid
Table 2:
Lethal Dose 50 of some arsenic compounds (LD50: dose which is fatal to onehalf a population of experimental animals) Compounds Arsine Potassium arsenite Arsenic trioxide Calcium arsenate Phenylarsonic acid Monomethyiarsonic acid (MMA) Dimethylarsinic acid (DMA) Strychnine Aspirin Arsenobetaine (Asbet) Arsenocholine (Aschol)
LD<5o (mg.kg'l weight of rat) 3 14 20 20 50 700-1800 700 - 2600 16 1000-1600 > 10000 > 10000
1
Arsenic speciation in environmental analysis
[Ch.l2
287
In the environment, arsenic and its compounds may be submitted to physical, chemical and biochemical transformations with or without modification of their oxidation states, and/or mineralization, adsorption and precipitation processes. Figure 1 describes all the complex phenomena which may occur in the different compartments.
NATURAL AND ARTIFICIAL SOURCES : ATMOSPHERE, ADSORPTION ON ATMOSPHERIC PARTICULES
As (inorganic) oxidation 4
BIOSPHERE
(CH3)2AsH, (CH3)3As •
-(CH3)3As0
(CH3)3As'^CH2C00-
(FISHES, MAMMAL. ,
(BACTERIA, FUNGI)
(CH3)2AsOCHjCOOH
t
•
(CH-;)2AsO(OH) -*-arsenosonbosides*-(CH3)2AsOCH2CH20H (ALGAE) I (CH3)AsO(OH)2 f (CH3)3As^CH2CH20H,XAs(ni) • MICROORGANISMS MICROORGANISMS A As(V) As(iin -
HYDROSPHERE
^
r X | # . Fe(llI).As(V) COMinJIXES | - ^ ^ Fe(III) Asdll)
-
(A.B,C,D) LITOSPHERE
- - ik- •
aerobic medium
anaerobic medium
- | " B ~ | - Fe(Un Aadll)
aerobic medium
"^ FeAsS. AS2S3 -tc ! • — ^
Figure 1:
Bio-geochemical cycle of arsenic A: deposition and co-precipitation, B: dissolution, C: diffusion and reaction with sulfides, D: adsorption
288
Arsenic spedation in environmental ana^is
[Ch.l2
Speciation of arsenic has already been studied but has generally been performed on calibrant solutions. A Measurements and Testing Programme (BCR/EC) project concerning arsenic speciation in marine organisms is in progress. The aim is to prepare certified materials (mussels and tuna fish tissues) for sixarsenic species: As(III), As(V), MMA, DMA, Asbet and Aschol. Different interlaboratory studies have been conducted and have shown the necessity of validating an analytical methodology. 12.1
Critical review of existing methods
The first methodology developed for arsenic speciation was based on the hydride generation technique [1]. Since then, other methods have been developed and detection limits have considerably decreased. Nowadays, it is possible to analyse samples containing only some tens ng.g'^As of each individual species. Most of the techniques described in the literature are coupling methods using the hydride generation technique or liquid chromatography (LC) as separation methods and atomic absorption spectrometry (AAS), or inductively coupled plasma emission spectroscopy (ICP/AES) as detection technique. Direct current plasma emission spectroscopy (DCP/AES), or mass spectrometry have also been used. In this part, we present the most commonly used techniques, their advantages and their drawbacks. 12.1.1 Hydride generation method 12.1,1.1 Hydride generation and separation This derivatization method was originally developed for selenium speciation [2] but has been successfully applied to the determination of arsenite and arsenate ions, monomethylarsonic and dimethylarsinic acids and trimethylarsenoxide. In an acidic medium, some arsenic compounds may be reduced to volatile arsines and carried by an inert gas flow to a specific detector. Using suitable conditions, it is possible to generate arsines quantitatively and/or selectively. Main parameters are pH, as well as the nature and concentration of the reducing agent and the acid. In order to avoid problems due to the hydride generation kinetics, a liquid nitrogen trap is placed after the reactor. After reduction of the arsenic compounds, hydrides are carried to the trap where they are condensed. After complete reaction, the trap is progressively heated and hydrides are volatilized according to their respective boiling points. This technique, also used for tin speciation, is known as the "cold trap method". Arsines formed are further separated by a gas chromatograph or analysed directly. Many authors have studied the selectivity of hydride generation as a function of reactional parameters in the case of arsenic [3-9]. Anderson and coworkers (1986) have shown that it was possible to selectively generate hydrides from As(III), As(V), MMA and DMA. Conditions described allow the rapid determination of As(III), DMA, As(III) + As(V) and total arsenic concentrations [8].
[Ch.l2
Arsenic spedation in environmental ana^is
12.1.1.2
289
Detection
Whatever the detection technique used, the main advantages of the hydride generation technique are: analyte/matrix separation improvement of the introduction system: the quantity introduced to the detector is 50 to 100 higher than with pneumatic nebulizers analyte preconcentration large choice of detection techniques easy on-line and automatized use This method has, nevertheless, several drawbacks: quantification is possible only for compounds forming volatile hydrides (arsenobetaine and arsenocholine, for example, can not be converted into arsines) the presence of some elements (Ni, Co, Cu, ...)decreases the efficiency of hydride generation one must strictly control the reaction medium to obtain good results. The hydride generation technique was first coupled to flame atomic absorption spectrometry (FAAS) [10]. The analyte is removed from the matrix so that classical interferences observed in A AS are reduced or even eliminated. Nevertheless, FAAS is not sensitive enough to allow the determination of arsenic traces in environmental samples and flames have been progressively replaced by quartz furnaces [8,11] which offer better control of atomization and lead to more suitable detection limits. Using this technique, inorganic species (arsenite and arsenate), monomethylarsonic and dimethylarsinic acids have been quantified in waters, sediments and biological tissue extracts [3,8,9,12-14]with detection limits in the /xg.l'^ range. Atomic emission spectrometry is not often used because of its higher cost. Hydride generation has been only coupled with a direct current plasma system for the determination of arsenite, arsenate and total arsenic in waters and fishes [15]. The most sophisticated system has been developed by Kaise et al [16]. Hydrides are generated, condensed, volatilized, separated by gas chromatography and then analysed in a mass spectrometer. The most intense peaks correspond to m/z = 76 and 78 for ASH3, 90 for CH3ASH2 and (CH3)2AsH, 103 and 120 for (CH3)3As. 12.1.2 Liquid Chromatography coupled with specific detectors Liquid chromatography is particularly suitable for the determination of arsenic compounds because of the hydrophilic and ionic or ionizable character of these species. This technique presents several advantages when compared to the previous one. Indeed, no derivatization is needed before separation, several types of chromatography may be used and a large choice of stationary phase/mobile phase couples are available. Nevertheless, classical detectors such as UV/Vis or electrochemical detectors are not sensitive enough and a specific detector (absorption or emission spectrometry) has to be used.
290
Arsenic spedation in environmental analysis
[Ch.l2
12.1.2.1 Separation by ion-pair chromatography Separations have been performed using apolar stationary phases constituted of C18 silica [17-20] or polymeric resin [21-23]. The ion-pairing agent is generally tetrabutylammonium phosphate [17,18] or hydroxide [20-23]. Brickman et al [24] have achieved the separation in the presence of tetraheptylammonium nitrate whereas Larsen has investigated Cg-Ciz alkylammoniums as well as butanesulfonate [19]. Elution is achieved using an isocratic regime or a gradient step program. Analysis time is in the range of 10 to 40 minutes. 12.1.2.2 Separation by ion-exchange chromatography Silica based [18,20,25-27] or polymeric [19,29-35] stationary phases grafted with strong anion-exchange groups (quaternary ammoniums) as well as weak anion-exchange columns [28] have been investigated. Compounds are eluted using a phosphate, carbonate or acetate buffer as mobile phase. pH is in the 6-7 range. The addition of an organic modifier has been found to decrease hydrophobic interactions [23-25]. Analysis times are also in the range of 10 to 40 minutes. These separations have been performed on calibrant solutions and on natural samples: pesticides and herbicides residues [29],urine [28,30,36],biological samples [25,26,32] and waters [31]. Some authors have mentioned the degradation of silica ionexchange columns after few weeks of extensive use [27,30,33]. 12.1.2.3 Detection by atomic absorption spectrometry Coupling HPLC with FAAS does not present any major technological problems. Nevertheless, the low sensitivity of the system (detection limit for arsenic : 1 mg.l"^) due to the difficult introduction of the sample to the flame and to the attenuation of the arsenic radiation intensity by radicals formed in the flame, has considerably limited its application in the arsenic speciation field. Several studies using electrothermal atomic absorption spectrometry (ETAAS) as the detector have shown that sensitivity can be improved by a factor of 10 to 100 when compared to FAAS. Nevertheless, coupling a continuous separation technique (HPLC) to a sequential detector is not easy. Two types of interface have been used, leading to off-line and on-line coupling methods: in the first [37], a graphite furnace sampler is used as a fraction collector . Better resolution is obtained but analysis time is considerably increased, in the on-line method [38-40], effluent fractions are collected and periodically analysed. This technique requires large chromatographic peaks because 30 to 60 s are needed for each determination. HPLC-ETAAS has been developed for arsenic speciation using anion-exchange [38,39]and reverse phase liquid chromatography [24]. Mobile phases, generally more complex, lead to an important increase of the background noise and require the use of a powerful apparatus equipped with a Zeeman background correction system [41]. It has been used for arsenic speciation in waters [24], pesticides [29,39] and soils [24]. Detection limits are in the nanogramme range. However, elution of significant quantities of organic materials together with arsenic compounds disturbs the detection. Moreover, some species are able to be volatilized without atomization if large amounts of salts or carbonaceous compounds are present in the sample.
[Ch.l2
Arsenic spedation in environmental analysis
291
12.1.2.4 Detection by inductively coupled plasma atomic emission spectrometry The compatibility of flow rates used in HPLC and ICP/AES (generally 1 ml.min^) allows a very easy coupling between these two techniques. A PTFE tube of adequate internal diameter is connected from the exit of the column to the nebulizer of the apparatus. This coupling method has been developed for arsenic speciation with ion-exchange [18,26, 32,42-44] and ion-pair liquid chromatography [17,21,45].Detection limits are some tens of nanogrammes for ion-exchange chromatography or some hundreds of nanogrammes for ionpair liquid chromatography. Some attempts to decrease the detection limits by improving the introduction system have been realized but no conclusive results have been obtained. The detection limits obtained allow the determination of the most abundant arsenic compound found in marine food, ie, arsenobetaine, but are generally not low enough to reach the other concentrations. 12.1.2.5 LC procedures involving hydride generation Matrix interferences observed in LC-AAS may be suppressed and detection limits improved by a post-column derivatization: for example the reduction of arsenic species into volatile arsine increases the quantity of analyte introduced in the atomization cell. Hydrides are carried by an inert gas current into a heated quartz cell where they are atomized and detected. Detection limits are in the range of few hundreds of picogrammes As, which are particularly suited for arsenic determination in environmental samples. ICP-AES detection may also be used after hydride generation in a simple coupling configuration: a PTFE tube is connected from the column to the entrance of the reduction coil, the nebulizer of the ICP/AES is removed and the exit of the gas-liquid separator is directly connected to the plasma. This modification may be realized by the user himself but some hydride generation kits are commercially available. The use of hydride generation for arsenic speciation increases sensitivity (some nanogrammes) but restricts the application of the technique to species which are able to form hydrides ie. As(III) , As(V), MMA and DMA. Some authors have proposed a photolitic reaction to convert Asbet, MMA and DMA in As(V) [46,47]. Nevertheless, the long period of time (hours) required for quantitative photolysis of Asbet makes it unsuitable for "on-line" measurements. The UV irradiation with addition of peroxodisulfate has been used for Asbet, DMA, MMA and As(V) determination in a LC-UV-HG-ICP system [48]. Recently, Rauret et al have studied the photo-oxidation of Asbet and Aschol by UV irradiation in persulfate media in order to convert these inert compounds in simple molecules able to produce volatile hydrides which could be determined by LC-UV-HGICP/AES [49]. The experimental conditions such as the photoreactor design, use of persulfate in different media, irradiation time and the effect of the power lamp have been optimized and it was shown that all the arsenic species of environmental interest are able to be converted into As(V) and consequentiy to be quantified by the LC-UV-HG-ICP/AES technique. Detection limits are very close to those obtained with HG-ICP/AES detection.
292
Arsenic spedation in environmental anal^is
[Ch.l2
12.1.2.6 HPLC coupled to ICP-MS This coupling method has been used for the speciation of arsenic with ion exchange, ionpair, or gel permeation chromatography [22,28,33,34,50-57]. Detection limits are very low (20-200 pg As) and the detector is linear over a wide dynamic range. This technique has been successfully applied to the determination of arsenic compounds in seafood products and sediments [50, 52]. 12.2
Means of validation
Some reference materials certified for total arsenic concentration are commercially available. In the environmental field and more particularly concerning the biological tissues and sediments CRM's, the Measurements and Testing Programme (BCR) of the Commission of the European Communities provides 7 materials: -
CRM CRM CRM CRM CRM CRM CRM
185 (bovine liver) : 186 (pig kidney) : 278 (mussel tissue) : 422 (cod muscle) : 277 (estuarine sediment): 280 (lake sediment) : 320 (river sediment) :
2 4 + 3 fig.g^ 63 ± 9 /xg.g' 5.9 ± 0.2/xg.g' 21.1 ± 0.5iLtg.g' 47.3 ± 1.6/xg.g'^ 51.0 ± 2.4^g.g' 76.7 ± 3A fig.g'
The National Research Council Canada (NRCC) has also prepared two fish tissues certified for their total arsenic content: - DORM-1 (dogfish muscle) : - DOLT-1 (dogfish liver) :
17.7 ± 2.1 fig.g' 10.1 ± lAfxg.g'
Finally, the National Institute of Standards and Technology (NIST) produced two sediments: - SRM 1646 (estuarine sediment) : - SRM 2704 (buffalo river sediment) :
11.6 ± 1.3/xg.g"^ 23.4 ± 0.8/ig.g"'
Speciation studies have been conducted on DORM-1 CRM [50,54,57]and have shown that 91 to 96 % of the arsenic extracted in the aqueous phase is present as Asbet. Nevertheless, no reference material certified for arsenic species is available. A BCR project which aims to elaborate reference materials of this type is now in progress. Pilot laboratories are the Laboratoire de Chimie Minerale et Analytique (Strasbourg, F) and the Service Central d'Analyse (CNRS, Vemaison, F). At the beginning, four materials (soil, sediment, fish and mussel tissues) and six arsenic species (As(III), As(V), DMA, MMA, Asbet and Aschol) were proposed. Nevertheless, the idea of certifying soils and sediments was abandonned because of the difficulty of collecting the appropriate materials.
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293
Moreover, the easy interconversion between As(III) and As(V) has led the organizers to restrict the quantification to As(III)+As(V), Asbet, Aschol, DMA and MMA. Intercomparison studies involving 10 to 20 laboratories have been organized using the classical step by step approach: 123456-
Preparation of the calibrants Study of pure calibrants or mixtures of pure calibrant solutions Study of mixtures of calibrants containing interfering compounds Study of clean extracts Study of raw extracts Study of real samples
Each step involves one or two intercomparison exercises and stability studies of the solutions distributed to the participants. With respect to calibrants, As(III), As(V), DMA and MMA are commercially available and can be used to prepare calibrant solutions. On the contrary, Asbet and Aschol have to be synthesized. The preparation has been achieved at the Laboratoire des Materiaux Organiques (Solaize, France) and characterized by elemental analysis, mass spectrometry and inductively coupled plasma atomic emission spectrometry. Calibrant solutions have been prepared in freshly boiled deionized water and their stability studied at 4 °C,25 °Cand 40 °C.It has been proved that solutions are stable at least for six months if kept in the dark at 4 °C. If storage conditions are not respected, oxidation of As(III) into As(V) and degradation of methylated species may occur. Stability studies performed for steps 3, 4 and 5 have shown that solutions which do not contain inorganic arsenic are stable at -40 °C and 4 °C in the dark. In the presence of mineral arsenic, interconversion between As(III) and As(V) is observed, this phenomenon depending on the temperature and on the sample composition. The different intercomparison studies have helped the participants to improve their methods of determination and have allowed the identification of several sources of error (problem of calibration, column washing and pre-conditioning, sample treatment, presence of chlorides for ICP-MS detection etc.) which have been solved. The 6th step has been started in the second half of 1994 and the certification campaign of mussels and fish tissues has been organized at the end of 1994. 12.3
Description of a validated technique
Sample treatment and arsenic speciation procedures described in this paragraph are the methods used in laboratories 1 (Laboratoire de Chimie Minerale et Analytique, Strasbourg), 2 (Service Central d'Analyse, Vemaison) and 3 (Universitat de Barcelona, E). These procedures have been established for the six more commonly found arsenic species in sediments and seafood products: As(III), As(V), Asbet, Aschol, DMA and MMA.
294
Arsenic spedation in environmental analysis
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12J.1 Sample treatment The sample preparation is one of the most difficult steps for trace element speciation in natural samples. The entire procedure has to be achieved in such a way that no loss or contamination or change in the speciation happens. 12.3.1.1 Analysis of seafood materials Fresh material is collected, washed with sea water and stored at O^'C. In the case of shellfish, shells are opened by freezing at -25 °C. After that, products are lyophilized so that the moisture content does not exceed 8-9 %, The powder obtained is homogeneized, pulverized and filtered on a 125 ^m sieve. Further treatment applied by Lab.l to this type of sample is based on a water/methanol extraction followed by a clean-up with ether as shown in Figure 2. Methanol/water evaporation should not be taken to dryness because of the possibility of volatilization and/or degradation. In the extraction step, the final volume indicated corresponds to the minimal volume required to take the sample out from the flask . It may be reduced if possible but practically can not be lower than 5 ml. Indeed, the solution becomes too viscous and further purification can not be achieved. Filtration may be replaced by centrifugation in the extraction step. Use of an ultrasonic bath has been prefered to mechanical stirring because sample powder is more easily dispersed that way. In order to check that the procedure does not induce any transformation of the species of interest and leads to 100 % recovery, a codfish powder was: first spiked with calibrant solutions of the six species of interest and then extracted and purified (I) secondly extracted, purified and spiked (II). Speciation was then performed using LC-ICP/AES (for Asbet and Aschol) and LC-HGQFAAS coupling techniques. The same results were obtained by procedures I and II. Moreover, total arsenic concentration independently determined by EDXRF (Energy Dispersive X-Ray Fluorescence) on the starting powder was found equal to the sum of Asbet, DMA and As(V) concentrations, the only As species detected in the sample. Finally, concentrations found for each compound in procedures I and II corresponded to the sum: concentration in the sample + concentration of the spike. 12.3.1.2 Sediment analysis In sediments, the most commonly found arsenic species are As(III), As(V), DMA and MMA. Extraction may be performed by classical acid attack in beaker or tubes or by microwave digestion after homogenization, pulverization and filtration on a 125 fxra sieve. The advantages of microwave digestion are both the reduction of analysis times and the possibility of automatization.
Arsenic spedation in environmental analysis
[Ch.l2
SAMPLE POWDER
295
1000 mg.
METHANOL / WATER (1: 1) - (5 x 10 mL)
;r
ULTRASONIC BATH
RE-EXTRACTION
-N SOLID V^
FILTRATION
f LIQUID 1
EVAPORATION
WATER r EXTRACT
^ E T H E R (5 X 10 mU
fsEPARATION
ORGANIC PHASE
J
\ '
i
- ( RE-EXTRACTION J
AQUEOUS PHASE
PURIFIED EXTRACT
Figure 2: Extraction/Clean-up procedure for arsenic speciation in seafood products
296
Arsenic spedation in environmental analysis
[Ch.l2
Two procedures have been developed by laboratory 2 for sediment analysis [58]: Digestion with a mixture of hydrochloric and nitric acids: 7 ml HCl and 3 ml HNO3 are added to 200 mg powder sample and introduced into a microwave digestion apparatus (Microdigest A 300 type, Prolabo, power: 0-200 W) using 20 % of maximum power for 5 minutes and then 25 % maximal power for 10 minutes. The same quantity of HCI/HNO3 mixture, 1 ml H2O2 and 5 ml water are successively added to the solution and heated at 30 % for 10 minutes, 20 % for 5 minutes and 25 % for 5 minutes respectively. It was observed by HPLC-ICPMS that, under those conditions, As(III) is totally converted into As(V) but DMA and MMA are preserved. This procedure allows the determination of mineral arsenic, DMA and MMA. Digestion with orthophosphoric acid: The best extraction yields are obtained using orthophosphoric acid 0.3 mol.l'^ at pH 1.3 stirring the solution (100 mg powder and 15 ml acid) for 10 minutes in the same microwave system as previously described. In those conditions, As(III) oxidation is slight. DMA and MMA can quantified. 12,3.2 Arsenic spedation The separation technique used by laboratories 1, 2 and 3 is liquid chromatography. Lab. 1 performed the separation using an ion-pair reverse system followed by ICP/AES and HGQFAAS techniques whereas labs. 2 and 3 used ion-exchange chromatography with ICP-MS and UV-HG-ICP/AES detections respectively. Arsenic was measured at a wavelength of 193.7 nm. 12.3.2,1 Procedures used: Laboratory 1 Determination of arsenobetaine by ICP/AES The first approach is based on the direct connection between the chromatographic system and the atomic emission spectrophotometer. The interface between both is made of a PTFE tube (250 jLcm, length: as short as possible) which is connected from the exit of the column to the nebulizer of the apparatus. Taking into account the poor sensitivity of the technique (see paragraph 12.1), only arsenobetaine can be determined this way in seafood products. 200 yil of the purified extract is injected onto a Hamilton PRP-1 ion-pair column using a solution of tetrabutylammonium phosphate (TBAP, 0.5mmol.r\pH 9.5) as a mobile phase. The eluate is directly introduced into the ICP/AES nebulizer and analysed. Determination of As(III), As(V), DMA and MMA by LC-HG-QFAAS Chromatographic conditions were modified to separate the four arsenic species forming hydrides in one run. This time, 200 yl of the sample was injected onto the same column but with a mixture of TBAP (10 mM) and TBAOH (Tetrabutylammoniumhydroxide, lOmmol.l"') at pH 6.15 as a mobile phase. The pH of the mobile phase as well as buffer concentration have been optimized. In the derivatization phase, the type of acid, its concentration and borohydride concentration have been considered. H2SO4 (0.5mol.r\l ml.min"^) and NaBH4
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297
(1 % in NaOH 0.1 %, 1 ml.min*^) were added to the eluate and the solution was introduced into the gas-liquid separator of the hydride generation system (Perkin Elmer FIAS 400, shown in Figure 3a). The separator includes a membrane which prevents the liquid entering the quartz atomization cell. Nevertheless, the mobile phase used becomes very effervescent when acid and borohydride are added and membrane is often wet if no precaution is taken; one solution consists of inserting a spring into the separator. The argon flow rate was set at a value of about 75 ml.minVhich leads to the best compromise between reproducibility and sensitivity. Cell temperature was about 900 ^'Cand the slit width was set at 0.7 nm. An EDL lamp (20 mA) was used. 123,2,2 Procedures used: Laboratory 2 Determination of As(III), As(V), DMA and MMA by LC-HG-QFAAS The same method as Lab. 1 has been used. Only the LC conditions and hydride generation system (FIAS 200, Perkin Elmer) were different. 100 jLil of sample solution was injected onto a Hamilton PRP X-100 anion exchange column and a gradient elution was performed using a flow rate of 1 ml.min'^ As mobile phases solution A (NH4)2P04/(NH4)2HP04, 10 moLl\ pH 6.2) and solution B ((NH4)2HP04, 100 mol.r\pH 8) were used. The following gradient program was performed : 100 % A for 3.4 min, decreasing to 50 % A in 0.1 min and maintained for 3 min. 100 % A was reached again in 0.1 min and maintained for 7 min. This mobile phase did not become effervescent when acid and reductant were added. H2SO4 1 mol.r^(1.3ml.min'^) and NaBH4 (1 % in NaOH 0.1 %, 1.3 ml.min"^) were used as reactants for hydride generation. Reaction takes place in a PTFE tube (25 cm length, 0.5 mm i.d.).Detection limits were similar to those obtained by Lab.l. Determination of all the species by HPLC-ICP-MS [51] LC conditions were adapted to the specific detector used. Separation was performed using the mobile phases described previously but adding 2 % CH3CN to enhance the sensitivity of the method. NH4* counter ions are prefered to Na* ions because signals observed in ICPMS are greatly affected by the presence of alkaline ions. Parameters of the ICP-MS apparatus (VG Plasma Quad 2) were optimized each day with a solution of 20 ng.ml'^As in buffer A in order to reach the highest possible signal. Coupling between LC and ICPMS system was achieved with a simple PTFE connection as previously described. As was determined at m/z 75. The signal was recorded on a Shimadzu CR3 A integrator connected to the analog output of the electron multiplier (pulse counting mode). A resistance-capacity filter was used in order to lower the background noise. High concentrations of easily ionizable cations such as Na* or K^ could interfere with Aschol determination because of their similar retention times. Chloride ions, which may combine with argon to produce an interfering peak at m/z 75 in ICP-MS, cannot disturb arsenic speciation in this system since they elute later than the arsenic compounds of interest.
298
Arsenic speciation in environmental analysis
quartz
[Ch.l2
fiiniacc
M intcrfecc
acid borohydride peristaltic pump
peristaltic pump gas-liquid separator
HonochroiiMtor
Potassium Persulphate Plasma Torch
^/W-^
/////////// LC Pump
Figure 3:
UV Photoreactor
Schematic of the (A) LC-HG-QFAAS and (B) LC-UV-HG-ICP/AES systems used for arsenic speciation
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Arsenic spedation in environmental analysis
299
123.2,3 Procedures used: Laboratory 3 Determination by HPLC-UV-HG-ICP/AES As already said, ICP/AES allows the detection of Asbet if its concentration is high enough (for example, in seafood products) and HG-ICP/AES is limited to the quantification of species which are able to form hydrides. In order to determine all the species on line and in one experiment. Lab.3 has developed a method in which they are all converted into As(V) in a photo-reactor located just after the LC chromatograph. The As(V) obtained is introduced into an hydride generation system and determined by ICP/AES. Working conditions have been optimized in order to obtain the best detection limits. The signal is filtered by using a Fourier Transform in order to better evaluate the peak height. A photoreactor was developed to be easily coupled on line between the exit of the HPLC column and the entrance to the reduction chamber. The length and the internal diameter of the PTFE coil and flow rate, were considered in order to minimize the dispersion of the chromatographic peaks, since this depends on L, r and, according to the equation: Tcr^L ay^ = k
(j) 24 Dj^
r : internal radius of the capillary (|): liquid flow rate Djn : molecular diffusion coefficient for analyte in solvent k : proportionality factor which depends on the flow profile in the capillary The variance can be minimized when the length of the tube is increased and its internal diameter is reduced. Thus, 0.35 mm, the narrowest easily available PTFE tube was chosen. The optimal length of the tube was determined by the time necessary to complete the photooxidation reaction. This photo-oxidation permits "on line" coupling between chromatographic separation and hydride generation. The eluate is introduced in the photo-reactor connected to the outlet of the column with addition of 3 % K2S208in3 % NaOH (0.2 ml.min"^) as photo-oxidation reagent. The vapour emerging from the photo-reactor is introduced into the hydride generation system. As hydride generation system, HCl 8 mol.l' (1 ml.min^) and NaBH4 1 % in NaOH 0.5 % (1 ml.min"^) are used. The resulting solution reaches the gas-liquid separator and then the plasma torch (see Figure 3b). Figure 4 presents some chromatograms obtained by the arsenic speciation methods developed in the three laboratories. Detection limits and reproducibility of the techniques are shown in Tables 3 and 4.
300
Arsenic spedation in environmental analysis
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AS(tll)
AS (III) AsBat
AsChol 10 15 Time (min)
A»(V)
DMA MMA
A8(V)
J IJ VJ
Time (mIn)
%i^w 9
10
11
Time (mIn)
Figure 4:
Chromatograms obtained by Labs. 1,2 and 3 using (A) LC-HG-QFAAS, (B) LC-ICPMS and (C) LC-UV-HG-ICP/AES techniques (chromatographic conditions described in the text)
301
Arsenic spedation in environmental analysis
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Table 3:
Detection limits (in ng As) of the four detection techniques considered in this study (ICP/AES, UV-HG-ICP/AES, HG-QFAAS and ICPMS)
Compound
LC-ICP/AES (Lab.l)
LC-UV-HGICP/AES (Lab.3)
LC-HG QFAAS (Lab.l)
LC-ICPMS (Lab.2)
Asail)
120 130 100 130 110 120
0.26 0.96 1.3 0.98 0.79 0.61
0.15 0.84 0.33 0.43 — —
0.03 0.02 0.02 0.01 0.01
1
As(V) MMA DMA Asbet Aschol
Table 4:
0.01
1
Coefficients of variation expressed in % of the four detection techniques considered
Compound
LC-ICP/AES (Lab.l)^
LC-UV-HGICP/AES (Lab.3)'
LC-HG QFAAS (Lab.l)^
LC-ICPMS (Lab.2)'
As(III) As(V) MMA DMA Asbet Aschol
2.42 3.90 4.71 2.76 3.55 2.37
5.5 5.2 6.3 6.8 5.0 4.6
3.12 3.67 3.46 2.95 — -—
2.2
1
1.6 4.1 2.5 2.5
2.4
I
short term reproducibility considered for eight consecutive injections of a solution 10 />tg.mr^(in As). long term reproducibility determined by injecting ten times in three non consecutive days a solution approximatively five times the LOD. short term reproducibility determined by injecting eight consecutive times a solution 50 ng.mr^(in As) of each compound . short term reproducibility determined by injecting six consecutive times a solution 0.1 mg.ml'^of each compound . 12.4
Conclusions
Several coupling techniques using liquid chromatography as the separation method and ICP/AES, HG-QFAAS, UV-HG-ICP/AES or ICP-MS as detectors are now available for arsenic speciation in environmental matrices. Nevertheless, some inter-comparisons within the BCR programme on arsenic speciation in marine organisms and sediments have shown discrepancies between the results of the different laboratories involved. Further efforts are
302
[Ch.l2
Arsenic speciation in environmental analysis
necessary to improve the quality of the measurements, particularly at sample preparation step as well as on the quantification procedure. Concerning the development of new methods for arsenic speciation, capillary zone electrophoresis (CZE) appears a priori to be suitable since it is best dedicated to ion separation. As(III), As(V), DMA and MMA have already been separated at low concentrations using CZE [59] and the technique has been evaluated in a BCR intercomparison study (Figure 5).However, when applied to extracts of environmental samples the detection limits (UV detector, 190 nm) are too high for the arsenic species. CZE technique can be coupled with mass spectrometry but the low masses of the compounds do not allow good detection limits. Improvements are also needed in the clean-up procedure of the extracts. Additionally the extraction leads to a significant dilution and therefore requires very low detection limits (5 to 10 ng.ml"^) which are best fitted by ICP-MS or HG-GFAAS. Another development would be the obtention of specific extractants designed for anion extraction. This would possibly lead to pre-concentration using either liquid-liquid extraction or substituted resins. ARSENITE TARGET VALUE
T 5
cone, ( m g / k g ) LC-ICP/MS LC-HAAS LC-HICP
CZE LC-ETAAS mean of ali individual values mean of mean values
DMA TARGET VALUE
T 4
5
6
LC-ICP/MS •
LC-HAAS
LC-racp
-*^-^
GC-HAAS
LC-ETAAS mean of all individual values mean of means values
Figure 5:
Results of a BCR intercomparison study for As speciation in a mixture of standard solutions containing 5 mg.kg"^ of the different species. Example of As(III) and DMA.
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Arsenic spedation in environmental analysis
303
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