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Determination of arsenic species in environmental samples: use of the alga Chlorella vulgaris for arsenic(III) retention
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Determination of arsenic species in environmental samples: use of the alga Chlorella vulgaris for arsenic( III ) retention A. Taboada-de la Calzada, M.C. Villa-Lojo, E. Beceiro-Gonzaè lez*, E. Alonso-Rodr|èguez, D. Prada-Rodr|èguez
Analytical Chemistry Department, University of A Corunìa, Campus da Zapateira s / n, 15071 A Corunì a, Spain An overview is given of current analytical methodologies for arsenic speciation in environmental samples. Most of these are conventional instrumental methods ^ mainly chromatographic techniques ( HPLC, GC, etc. ) coupled with a variety of detectors. However, methods using micro-organisms are increasingly being applied for the removal of metal ions and other metal species from aqueous solutions. The use of the alga Chlorella vulgaris is proposed for the separation of arsenic( III ) from the other arsenic species. The arsenic concentration is measured by hydride generation atomic absorption spectrometry. z1998 Elsevier Science B.V. Keywords: Arsenic speciation; Speciation of arsenic in alga; Chlorella vulgaris alga, arsenic speciation
*Corresponding author. Abbreviations: AAS, atomic absorption spectrometry ; ABAs, p-aminobenzene arsonate; AFS, atomic fluorescence spectrometry; AsB, arsenobetaine; AsC, arsenocholine; CE, capillary electrophoresis; CZE, capillary zone electrophoresis; DDAB, didodecyldimethylammonium bromide; DIN, direct injector nebulizer; DMA, dimethyl arsinate; ETAAAS, electrothermal atomic absorption spectrometry; FID, flame ionization detector; GC, gas chromatography; HGAAS, hydride generation atomic absorption spectrometry; HPLC, high-performance liquid chromatography ; ICPAES, inductively coupled plasma atomic emission spectrometry; ICP-MS, inductively coupled plasma mass spectrometry ; IEC, ion-exclusion chromatography ; MMA, monomethyl arsonate; MES, microwave emission spectrometric system; MIP, microwave-induced plasma; p-APA, paminophenylarsonate; PhAs, phenyl arsonate; Ph3 As, triphenyl arsonate; PID, photoionization detector; PMB, phenylmagnesium bromide; PTFE, polytetrafluorethylene; SDDC, silver diethyldithiocarbamate; SFC, supercritical fluid chromatography; SIC, suppressed ion chromatography; STAT, slotted tube atom trap; TBA, tetrabutylammonium ; TMA, trimethylarsenic ; TMAs, tetramethylarsonium; USN, ultrasonic nebulization; UV, ultraviolet
1. Introduction Agricultural herbicides, pesticides, insecticides, fossil fuels and metal smelting are common anthropogenic sources of arsenic. Among the common arsenic compounds in the environment, of particular interest is arsenite, which is 10 times more toxic than arsenate and 70 times more toxic than the methylated species, MMA and DMA [ 1 ]. Arsenobetaine and arsenocholine are considered to be non-toxic [ 2 ]. Thess facts indicate why it would be of priority interest to develop methods for the selective determination of As( III ). Numerous instrumental methods for As speciation are reported in the literature. Most of them are based on chromatographic separation techniques coupled with selective and sensitive detectors such as AAS, HGAAS, ICP-MS, and ICP-AES ( see Table 1 ). Although these techniques show great sensitivity and selectivity they usually require very expensive equipment, and other alternative speciation methods that use inexpensive materials could be of technical and commercial interest. It is known that a number of micro-organisms such as yeast, bacteria and algae adsorb ions from their surroundings and lead to their biotransformation into other species [ 3^5 ]. Although most of these microbiological methods have been used for the removal or preconcentration pf metal ions, the possibility is now being explored of employing them for analytical purposes and to selectively separate different species of an element depending on their toxicity. Thus a simple alternative speciation method can use living micro-organisms. For this reason we have initiated the study of the biosorption properties of C. vulgaris with respect to arsenite, arsenate, MMA and DMA and for performing the separation of arsenic species.
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Table 1 Instrumental techniques for arsenic speciation Methods
The best As species determined
LOD (Wg l31 )
Reference
Spectrophotometric
As( III ), As(V ) As( III ), As(V ) As( III ), As(V ) As( III ), As(V ), MMA, DMA As( III ), As(V ), MMA, DMA As( III ), As(V ), MMA, DMA, AsB, AsC, TMAs As( III ), As(V ), MMA, DMA As( III ), As(V ),MMA, DMA, AsB, AsC As( III ), As(V ), MMA, DMA As( III ), As(V ), MMA, DMA, p-APA As( III ) As(V ) As( III ), As(V ) As( III ), As(V ), MMA, DMA As( III ), As(V ), MMA,DMA, AsB, AsC As( III ), As(V ), MMA, DMA,TMAs As( III ), As(V ), MMA, DMA As( III ), AsB As( III ), As(V ), MMA, DMA As( III ), As(V ), MMA, DMA, AsB, AsC As( III ), As(V ), MMA, DMA As( III ), As(V ), DMA
100, 100 0.03, 1 ^ ^ ^ 1100, 1400, 1400, 700, 300, 500, 400 1, 1.6, 1.2, 4.7 5, 8, 6, 6, 4, 4 0.5, 0.5, 0.5, 0.5 4, 20, 3.2, 6.5, 9.3 114 120 s 3000 3.5, 9.2, 3.8, 21.3 2.6, 9.6, 13, 9.8, 7.9, 6.1 ^ 1, 5, 1.2, 6 1, 5 4.9, 6, 3.6, 1.2 0.5, 0.3, 1, 1, 0.5, 0.5 0.14, 0.2, 0.08, 0.08 90, 60, 120 76, 40, 70 0.1, 0.02, ^, ^ 10, 10 0.0018 0.0008 0.05, ^ 87.5 5 0.13
[6] [7] [8] [9]
FIA^HG-AAS FIA^HG-ICP-AES HPLC^AAS HPLC^HG-AAS HPLC^UV^HG-AAS IC^HG-AAS SIC^Electrochemical SIC^Conductimetric IEC^PID HPLC^HG-ICP-AES HPLC^UV^HG-ICP-AES HPLC^ETAAS HPLC^HG-MIP-AES HPLC^ICP-MS HPLC^USN-AFS CZE^UV CZE^Conductivity CE^ICP-MS GC^FID HG^GC^PID GC^MES SFC^FID Voltammetric ICP-AES Voltammetric ICP-MS
As( III ), As(V ), MMA, DMA As( III ), As(V ) As( III ), As(V ) As( III ), As(V ) As( III ), As(V ) As( III ) As( III )
2. Arsenic speciation using instrumental techniques 2.1. Spectrophotometric methods
Originally, the pH solution control was used in the determination of arsenite and arsenate, so Howard and Arbab-Zavar [ 6 ] modi¢ed the SDDC spectrophotometric procedure for the determination of arsenite and arsenate. This procedure has interferences from trace metals and methylated arsenic species, but these can be prevented using chelation solvent extraction or ion exchange. Palanivelu et al. [ 7 ] describe a highly sensitive spectrophotometric method for the determination of arsenite and arsenate. Absorbances are measured with a Carl Zeiss PMQII spectrophotometer with 10 mm quartz cells. The determination is made as arsenic triiodide, after an extractive separation into benzene. As(V ) is determined by measuring total arsenic after
[ 10 ] [ 11 ] [ 12 ] [ 13 ] [ 14 ] [ 15 ] [ 16 ] [ 17 ] [ 18 ] [ 19 ] [ 20 ] [ 21 ] [ 22 ] [ 23 ] [ 24 ] [ 25 ] [ 26 ] [ 27 ] [ 28 ] [ 29 ] [ 30 ] [ 31 ] [ 32 ]
the reduction of arsenate with potassium iodide. The results are more sensitive than those based on reaction with SDDC, and superior to the £uorescence method based on the use of rhodamine B. This method has the advantage of lack of interferences and permits precise determination of trace amounts of arsenic in natural and synthetic samples. Tamari et al. [ 8 ] presented a new co-precipitation method for the spectrophotometric determination of arsenite and arsenate in groundwater. Both species are co-precipitated with thorium(V ) hydroxide at pH 9; after centrifugation and dilution with hydrochloric acid the absorbance was measured on a spectrophotometer by the usual SDDS method. Continuous hydride generation, using NaBH4 as reductant, in conjunction with AAS and ICP-AES, was used by Anderson et al. [ 9 ] to form arsine selectively from arsenate, arsenite, MMA and DMA. It was shown that pH is not the only factor to affect the reduction of individual arsenic species, but that other factors
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such as kinetic control and complexation are also involved. 2.2. High-performance liquid chromatography separation coupled with different detectors
Nowadays, the speciation methods commonly used involve an initial separation of the different analyte species in the sample matrix by chromatographic techniques. The method most employed for the separation of arsenic species is HPLC in its various forms. The molecular forms of As which are subjected to speciation analysis are the anions, As( III ), As(V ), MMA and DMA, or the cations AsB, AsC or TMA. The separation techniques used are anion-exchange HPLC with either isocratic or gradient-step elution, or cation exchange with isocratic elution. Ion-pair HPLC has also been used. In order to separate arsenic anions and cations in a single run, a column-switching system involving a combination of anion exchange and reversed-phase separation has been developed. After separation by HPLC the As species are detected on-line by several detection systems such as UV, AAS, HG-AAS, ICP-AES, ICP-MS, or off-line by ETA-AAS. The combination of HPLC with AAS provides a tool that allows the separation and identi¢cation of known arsenic species as well as detecting and giving retention times for unknown arsenic compounds, but it is only suitable for samples containing more than 100 Wg / ml of arsenic. Hansen et al. [ 10 ] applied this online system for the speciation of arsenic species. As( III ), As(V ), MMA and DMA were separated from each other and from the co-injected cationic arsenic compounds on an organic polymeric anionexchange column with 0.1 M carbonate at pH = 10.3 as the mobile phase. AsB, AsC and TMA were separated from each other and from the co-injected anionic species on a silica-based cation-exchange column, with pyridine at pH 2.65 as the mobile phase. For signal enhancement a STAT was installed. The atom trap was made from a quartz tube and was attached by screws to the burner head. The interfacing was established by a vented PTFE capillary tube connecting the HPLC column to the nebulizer of the atomic absorption spectrometer and using a H2 ^Ar £ame. The detection limits are suf¢ciently low for the selected applications, although these detection limits are 150^470 times worse than those from the ICP-MS. The methods that employ hydride generation between the HPLC and AAS present better sensitivity.
Their disadvantage is that only molecules that produce volatile arsines can be detected. Hakala and Pyy [ 11 ] studied arsenic exposure and its monitoring in urine by using a system with on-line HPLC^HG-AAS. The separation of As( III ), As(V ), MMA and DMA is carried out on a C18 reversed-phase column with 10 mM TBA^20 mM phosphate at pH 6.0 as the mobile phase. Because AsB and AsC do not generate hydrides, a preoxidation to an inorganic arsenical is necessary, as by the on-line combination of K2 S2 O8 with UV light. Loèpez et al. [ 12 ] have incorporated the on-line thermo-oxidation to the speciation of As( III ), As(V ), MMA, DMA, AsB and AsC. They used an anion column with 17 mM phosphate at pH 6.0 as the mobile phase. The ef£uent of the HPLC was merged with a persulfate stream before entering the thermo-reactor consisting of a loop of PTFE tubing dipped in a powdered-graphite oven heated to 140³C. After cooling in an ice-bath, HCl and NaBH4 were added on-line to generate the arsine. A similar HPLC system, with the same chromatographic column and with a post-column reaction to achieve complete formation of volatile arsines from the methylated species and As(V ), was developed for the speciation of As( III ), As(V ), MMA and DMA [ 13 ]. Ricci et al. [ 14 ] presented a technique that separates the species using ion-exchange chromatography followed by continuous hydride generation and atomic absorption detection. The separation of the arsenic species, As( III ), As(V ), MMA, DMA and p-APA, is achieved by using a standard Dionex 3U500 mm anion-separator column. A mixture of NaHCO3 , Na2 CO3 and Na2 B4 O7 is used as the mobile phase for the separation of As(V ), MMA and p-APA, and Na2 B4 O7 is used in the case of As( III ) and DMA. The ef£uent exiting the column led via microbore tubing directly into a hydride generator^AAS detection system. McGeehan and Naylor [ 15 ] described a direct simultaneous measurement of arsenic and selenium inorganic species using a suppressed-ion chromatography system with a Dionex 4000i. A Na2 CO3 ^ NaHCO3 solution is used as the mobile phase and an electrochemical and a conductimetric detector were used in series with a dual-channel integrator to quantify As( III ) and As(V ). Although SIC achieves lower detection limits than IC, neither has suf¢cient sensitivity for the analysis of many environmental samples. The major advantage of SIC is its ability to separate and quantify the ionic species of arsenic and selenium without oxidation / reduction pretreatments.
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Hemmings and Jones [ 16 ] presented an IEC method to measure As( III ) and As(V ). Any iron is an important interference which is removed by a cation-exchange procedure before the separation of the arsenic species by using a Dionex 2010i ion chromatograph. The arsenic speciation is carried out by using an HPICE-AS1 ion-exclusion column with a photometric detector. The main problem is the lack of sensitivity of this detector for the determination of arsenic species at trace levels. Another technique described in the literature for As speciation is HPLC^HG-ICP-AES. In this case, only a few authors use hydride generation to detect arsines in the gas phase. A gas^liquid separator was used by Rauret et al. [ 17 ] to minimize the volume of solution reaching the plasma torch and to improve the separation of volatile hydrides. These authors used a silicabased anion-exchange column to separate the arsenic species As( III ), As(V ), MMA and DMA with a phosphate buffer, pH = 6.75 as the mobile phase. Rubio et al. [ 18 ] developed the separation of As( III ), As(V ), MMA, DMA, AsB and AsC on an anion-exchange column using a phosphate buffer as the mobile phase. On-line UV-photo-oxidation was used by these authors at the exit of the chromatographic column for the decomposition of AsB and AsC to As(V ). ETA-AAS can also be used as a detector after separation by HPLC. Although the detection of arsenic species using on-line techniques is the ideal, off-line detection by ETA-AAS is still advantageous owing to the good sensitivity, but the fairly long time required for the analysis of a series of collected fractions may be inconvenient for practical analytical work. Another problem is the loss of analyte at the pre-atomization step. In this way, Larsen [ 19 ] studied conventional and fast-furnace programs, with and without palladium^magnesium nitrate as a chemical modi¢er. The on-line coupling of vesicle-mediated HPLC to low-power argon MIP detection was applied to the arsenic speciation of As( III ), As(V ), MMA and DMA in natural waters and in human urine by Costa-Fernaèndez et al. [ 20 ]. They used a C18 -bonded silica stationary phase with a mobile phase prepared with NaH2 PO4 ( 5 mM ) in MeOH ( 1%) and 0.01 mM vesicles of DDAB at pH=5.75. The presence of DDAB vesicles in the mobile phase improves the detection limits. The volatile species formed are separated from the liquid and directed to the low-pressure MIP system after passing through a desiccator ( H3 PO4 ). The low cost of the detection / excitation source and its maintenance and the low detection limits are com-
parable to those obtained with other more powerful sources such as ICP. Several workers have reported on arsenic speciation by HPLC^ICP-MS. The bene¢ts of coupling plasma mass-spectrometric detection with chromatographic separation include speci¢city, the ability to separate interferences from peaks of interest, multi-element capability, and low levels of detection. Beauchemin et al. [ 21 ] coupled various forms of HPLC with ICPMS for arsenic speciation. They studied ion-pairing and ion-exchange HPLC to separate As( III ), As(V ), MMA, DMA and AsB. They found that anion exchange has resolution inferior to ion-pairing chromatography, but it appears to be less susceptible to matrix interferences. Sheppard et al. [ 22 ] used a column Wescan Anion / R-IC to separate As( III ), As(V ), MMA and DMA from each other and from 40 Ar35 Cl , which is formed with the plasma gas and the chloride present in samples. The eluents used were 2% propan-1-ol and carbonate buffer 50 mM at pH 7.5. The column was directly connected to the nebulizer of the ICP by polyplex tubing. The sensitivity of this method is limited by the sample dilution that is necessary to diminish the high chloride concentration. Demesmay et al. [ 23 ] used an ICP-MS detector coupled to an HPLC system to determine As( III ), As(V ), MMA, DMA, AsB and AsC. The interface was established through PTFE capillary tubing. They used an anion-exchange column with a mobile phase of phosphate buffer with 2% acetonitrile. To reach an optimum separation of the six arsenic species an ionic-strength step gradient was necessary. Most hydride-forming elements can be detected by using AFS in the UV region below 250 nm. AFS is not being widely used, one possible reason being that, until recently, there was no reliable high-intensity excitation source. The appearance of the boosted-discharge hollow cathode lamp provided a very good excitation source for AFS detection. The main problem with this technique lies in the interface between the HPLC and AFS. Woller et al. [ 24 ] used the coupling HPLC^USN-AFS to determine As( III ), As(V ), MMA and DMA. They used a USN different from previous designs and found better detection limits than other authors. 2.3. Capillary electrophoresis
Schlegel et al. [ 25 ] presented a modi¢cation of the high-performance capillary electrophoresis ^ capillary zone electrophoresis ^ which uses two different
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detection systems, photometric and conductimetric. The CZE shows excellent suitability for arsenic speciation, and the separation of different species is possible. With the photometric detector, As( III ), As(V ) and DMA are determined, and with the conductimetric detector, As(V ), DMA, ABAs and PhAs. Another CE technique for determining As( III ), As(V ), MMA and DMA is presented by Lin et al. [ 26 ]. They introduced a different detection system ( ICP-MS ) and developed a novel interface which is based on a DIN. This interface can be used under different CE conditions and it allows for the DIN to be optimized independently from the CE system to give the most ef¢cient sample introduction into the ICPMS. This method therefore combines the high separation ef¢ciency of the CE with the high elemental sensitivity and selectivity of the ICP-MS. 2.4. Gas chromatographic separation coupled with different detectors
Beckermann [ 27 ] presented an elegant method for the determination of MMA and DMA in biological samples. Most of the methods proposed for this speciation are based on the conversion by sodium borohydride of the arsenic into the corresponding methylarsine compounds, but their volatilities make it necessary to convert MMA and DMA into stable derivatives. With this method, the methylarsenic acids are derivatized with thioglycolic acid methyl ester, to yield lipophilic species which can be determined by GC using a FID. Another technique, presented by two different groups of authors [ 28,29 ], is based on selective hydride generation, liquid-nitrogen-cooled trapping, and gas chromatography with helium-dischargetype photo-ionization detection. It is applied to the simultaneous determination of As( III ) and Sb ( III ) under weak-acid conditions and to As( III ), As(V ) and Sb( III ), Sb(V ) under strong-acid conditions. Talmi et al. [ 30 ] presented a method consisting of gas chromatography coupled with a MES detector for arsenic and antimony determination in environmental samples. The analytical procedure is based on the following steps: sample pretreatment, co-crystallization of As( III ) and Sb( III ) with thionalide, and reaction of the dry precipitate with PMB. When the phenylation had been completed, the excess of PMB was decomposed to prevent oxidation of Ph3 As, then a portion of the organic layer was injected into the column with a gradient program, and determined by MES.
2.5. Supercritical £uid chromatography
Laintz et al. [ 31 ] described a novel technique based on a simultaneous separation and quanti¢cation of As( III ) and As(V ) ( after reduction with potassium iodide and sodium thiosulfate ), achieved by extraction with lithium bis( tri£uoroethyl )dithiocarbamate followed by supercritical £uid chromatography with FID. The extraction technique reduces organic matrix interferences and preconcentrates the metal species by conversion into chelates suitable for SFC analysis. 2.6. Voltammetric methods
Pretty et al. [ 32 ] presented an on-line, anodic stripping-voltammetry £ow cell with detection by ICPAES and ICP-MS, for As( III ) determination. With this method, polyatomic interferences which arise from chloride in sample matrices are eliminated. The detection by ICP-AES has insuf¢cient sensitivity in many cases, but lower detection limits can be obtained with a hydride generator coupled to the ICP. The detection by ICP-MS enhances the signals.
3. Arsenic speciation using micro-organisms Many micro-organisms, such as algae, fungi, yeast and bacteria, have been used for biosorption of toxic elements by binding to the cell surface and intracellularly. This property has been applied largely to the preconcentration of metal ions, mainly in industry, the environment, etc. [ 33 ], but its application to metal speciation has recently been proposed. The interest in the use of biological substrates can be explained because cells have a small and uniform size, present many active binding sites, and have a low production cost. Because metabolic activity is not essential for metal uptake, this may be carried out by living or dead micro-organisms. The biosorption process may be performed in two ways: in batches without immobilized substrates and using micro-organisms immobilized on a suitable support [ 34,35 ]. The capacity of some algae to accumulate arsenic has been known for several years [ 36 ], and has been used to eliminate this element from industrial ef£uents [ 33 ]. Yamaoka et al. [ 37^39 ] studied the accumulation of arsenic by Duniella sp. and the effect of various elements on this accumulation. The capacity of the alga Chlorella vulgaris to transform inorganic arsenic compounds [ 40^42 ] and to reduce As( III ) to As(V )
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[ 43,44 ] has been evaluated. Goessler et al. [ 45 ] studied the uptake of arsenic compounds by three Chlorella species. Bioaccumulation and biomethylation of inorganic arsenic by the marine alga Polyphysa peniculus [ 46 ] and the blue-green alga Nostoc sp. [ 47 ] were also investigated. Five bacteria (Proteus sp., Escherichia coli, Flavobacterium sp., Corynebacterium sp. and Pseudomonas sp. ) were used to determine arsenic uptake and distribution into the cells [ 48 ]. According to the work mentioned above the relationship between different micro-organisms and arsenic is widely demonstrated, but this fact has not yet been exploited as a practical way for arsenic spe-
ciation. In our laboratory we have studied the retention capacity of the alga Chlorella vulgaris with respect to As( III ), As(V ), MMA and DMA, as a possible method of separation of As( III ), the most toxic species, from the others.
4. Case study: use of the alga for the As( III ) retention Our study demonstrates that Chlorella vulgaris can be used to selectively separate about 50% of the most toxic arsenic species, As( III ), from the other species, As(V ), MMA and DMA. It has been proved that
Fig. 1. Analytical procedure for the As( III ) retention by Chlorella vulgaris.
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As( III ) retention by the alga occurs in a strongly basic medium at room temperature and As(V ), MMA and DMA retention is not signi¢cant ( see Fig. 1 ). These conditions are easily obtained in a laboratory. The alga was harvested in two different macronutrient media, one with limited phosphate and nitrate concentrations ( A ), and the other with a suf¢cient amount of these essential nutrients ( B ). It can be seen that the alga ( A ), with limited concentrations of these nutrients, appears to have a higher retention ( 52%) than the other ( 40%). Since AsO33 3 has chem3 and NO , it is possible that ical similarity to PO33 4 3 those nutrients could be replaced by AsO33 3 . The Chlorella vulgaris alga needs no particular pretreatment, so with ageing alga there were no signi¢cant changes in As( III ) retention, and it has been observed that the use of dry alga gave similar results. However, the best retention was obtained when the living alga was used with limited essential nutrients, ( A ). When the alga A was used the retention increased with the alga volume, and the best retention was obtained with 100 ml. This situation probably results from different accumulation mechanisms. In one of these, the binding of the arsenic species to the cell surface occurs by adsorption, and the other mechanism consists of transport of As( III ) through the cell membrane: this latter one needs biological activity. The method does not require too much time, 10 min of contact time between the alga and the solution containing As( III ) is enough to give optimum retention.
5. Conclusion HG-AAS is the classical method most used for arsenic determination, but this technique does not permit speciation without a previous separation of the species. If the separation is performed by co-precipitation, pH, etc., interferences may occur and a previous extraction or ion exchange would be necessary to avoid this. Thus, a liquid chromatography separation coupled to different detectors is mainly employed; this also allows the speciation of inorganic and organic arsenic species. HPLC^AAS does not show detection limits low enough for most of the environmental applications, but the methods that employ hydride generation between HPLC separation and AAS detection ( HPLC^HG-AAS ) improve the sensitivity. The trouble is that a diminution of the sensitivity is observed when thermo-oxidation, to release AsB and AsC as As(V ), is used.
Speciation methods using HPLC^HG-ICP-AES do not have enough sensitivity in many cases and give higher detection limits than with an AAS detector. When thermo-oxidation is employed to convert AsB and AsC, as in the above-mentioned case, the detection limits become worse relative to HPLC^UV-HGAAS. When off-line detection by ETA-AAS is coupled with HPLC the main advantage is an increase in the sensitivity, but it is a tedious method and has disadvantages for practical analytical work. Another problem is the loss of analyte at the pre-atomization step. Arsenic speciation by HPLC^ICP-MS or CE^ICPMS combines the bene¢ts of coupling plasma mass spectrometric detection with chromatographic separation. These techniques show good speci¢city and the ability to separate interferences from peaks of interest, along with multi-element capability and low levels of detection, but they have the inconvenience of high instrumental and maintenance cost. Similar advantages and disadvantages are shown when HPLC^ MIP is used. HPLC^USN-AFS is not used very frequently for arsenic speciation, probably because it is dif¢cult to obtain a suitable interface, although this technique shows excellent detection limits. Other liquid chromatography methods, such as SIC and IEC, using photometric, conductimetric or electrochemical detectors present low sensitivity, and only As( III ) and As(V ) can be determined. The same situation is observed when CE is used with photometric or conductimetric detectors, but in this case DMA can also be determined. With GC, the main problem at the moment is that the technique is limited to the determination of As( III ) and As(V ); its principal advantage is that it presents excellent sensitivity. Supercritical £uid chromatography is a novel technique that does not have great application for arsenic speciation at the moment, because it also is limited to the determination of As( III ) and As(V ). However, in contrast to GC it presents low sensitivity. When voltammetric methods are coupled with ICP^ AES, they do not show enough sensitivity, and when they are coupled with ICP^MS they have the same disadvantages as HPLC^ICP-MS. The high sophistication required of the coupled systems makes their implementation dif¢cult for routine analysis. Recently, interest has grown in areas where analytical chemistry and biology interplay. There is a need for simple analytical methods to separate species of several analytes characterized by different toxicity.
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One simple alternative is to use biological substrates to distinguish between toxic and non-toxic species. The use of biological micro-organisms has been known for a long time, but its application to metal speciation is now being initiated. There are no references in the literature about arsenic speciation with such materials. However, the use of living micro-organisms for As( III ) speciation could give a new perspective for speciation of this element. The aim of this paper is to extend the applicability of Chlorella vulgaris to the selective determination of arsenic species. We propose the use of the green alga Chlorella vulgaris to selectively separate As( III ) from the other common arsenic species, As(V ), MMA and DMA. Although total As( III ) separation is not reached, one advantage of the use of this alga for As( III ) separation is that it provides a simple and economic procedure suitable for routine analysis.
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[ 18 ] R. Rubio, A. Pradoè, J. Albert|è, G. Rauret, Anal. Chim. Acta 283 ( 1993 ) 160. [ 19 ] E.H. Larsen, J. Anal. Atom. Spectrom. 6 ( 1991 ) 375. [ 20 ] J.M. Costa-Fernaèndez, F. Lunzer, R. Pereiro-Garc|èa, A. Sanz Medel, N. Bordel-Garc|èa, J. Anal. Atom. Spectrom. 10 ( 1995 ) 1019. [ 21 ] D. Beauchemin, K.W.M. Siu, J.W. McLaren, S.S. Berman, J. Anal. Atom. Spectrom. 4 ( 1989 ) 285. [ 22 ] B.S. Sheppard, J.A. Caruso, D.T. Heitkemper, K.A. Wolnik, Analyst ( Lond. ) 117 ( 1992 ) 971. [ 23 ] C. Demesmay, M. Olle, M. Porthault, Fresenius J. Anal. Chem. 348 ( 1994 ) 205. [ 24 ] A. Woller, Z. Mester, P. Fodor, J. Anal. Atom. Spectrom. 10 ( 1995 ) 609. [ 25 ] D. Schlegel, J. Mattusch, R. Wennrich, Fresenius J. Anal. Chem. 354 ( 1996 ) 535. [ 26 ] Y. Lin, V. Lopez-Avila, J.J. Zhu, D.R. Wiederin, W.F. Beckert, Anal. Chem. 67 ( 1995 ) 2020. [ 27 ] B. Beckerman, Anal. Chim. Acta 135 ( 1982 ) 77. [ 28 ] M. Yamamoto, S. Tanaka, Y. Hashimoto, Appl. Organomet. Chem. 6 ( 1992 ) 351. [ 29 ] L.S. Cutter, G.A. Cutter, M.L.C. San Diego-McGlone, Anal. Chem. 63 ( 1991 ) 1138. [ 30 ] Y. Talmi, V.E. Norvell, Anal. Chem. 47 ( 1975 ) 1510. [ 31 ] K.E. Laintz, G.M. Shieh, C.M. Wai, J. Chromatogr. Sci. 30 ( 1992 ) 120. [ 32 ] J.R. Pretty, E.A. Blubaugh, J.A. Caruso, Anal. Chem. 65 ( 1993 ) 3396. [ 33 ] S. Maeda, T. Kumamoto, M. Yomemoto, S. Nakajima, T. Takeshita, S. Higashi, K. Ueno, Sep. Sci. Technol. 18 ( 1983 ) 375. [ 34 ] T. Peèrez-Corona, Y. Madrid, C. Caèmara, Anal. Chim. Acta 345 ( 1997 ) 249. [ 35 ] Y. Madrid, C. Cabrera, T. Peèrez-Corona, C. Caèmara, Anal. Chem. 67 ( 1995 ) 750. [ 36 ] G. Lunde, Environ. Health Perspect. 19 ( 1977 ) 47. [ 37 ] Y. Yamaoka, O. Takimura, Agric. Biol. Chem. 50 ( 1986 ) 185. [ 38 ] Y. Yamaoka, O. Takimura, H. Fuse, Appl. Organomet. Chem. 2 ( 1988 ) 359. [ 39 ] Y. Yamaoka, O. Takimura, H. Fuse, K. Kamimura, Appl. Organomet. Chem. 4 ( 1990 ) 261. [ 40 ] S. Maeda, R. Inoue, T. Kozono, T. Tokuda, A. Ohki, T. Takeshita, Chemosphere 20 ( 1990 ) 101. [ 41 ] S. Maeda, A. Ohki, T. Tokuda, M. Ohmine, Appl. Organomet. Chem. 4 ( 1990 ) 251. [ 42 ] S. Maeda, A. Ohki, K. Kusadome, T. Kuroiwa, I. Yoshifuku, K. Naka, Appl. Organomet. Chem. 6 ( 1992 ) 213. [ 43 ] R. Jeanjean, F. Blasco, C. Gaudin, C.R. Hebd. Seances Acad. Sci. Ser. D 272 ( 1971 ) 64. [ 44 ] F. Blasco, C. Gaudin, R. Jeanjean, C.R. Hebd. Seances Acad. Sci. Ser. D 273 ( 1971 ) 812. [ 45 ] W. Goessler, J. Lintschinger, J. Szaèkovaè, P. Mader, J. Kopecky, J. Doucha, K.J. Irgolic, Appl. Organomet. Chem. 11 ( 1997 ) 57. [ 46 ] W.R. Cullen, H. Li, S.A. Pergantis, G.K. Eigendorf, L.G. Harrison, Chemosphere 28 ( 1994 ) 5. [ 47 ] S. Maeda, K. Mawatari, A. Ohki, K. Naka, Appl. Organomet. Chem. 7 ( 1993 ) 467. [ 48 ] M. Shariatpanahi, A.C. Anderson, A.A. Abdelghani, Trace Subst. Environ. Health 16 ( 1982 ) 170.
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A. Taboada-de la Calzada and M.C. Villa-Lojo are graduates of the University of Santiago de Compostela and are graduate students at the University of A Corunìa. E. Beceiro-Gonzaèlez and E. Alonso-Rodr|èguez are Doctors in Chemistry, with PhD degrees from the University of Santiago de Compostela and are lecturers in analytical chemistry at the University of A Corunìa.
D. Prada-Rodr|èguez is a Doctor in Chemistry, with a PhD degree from the University of Santiago de Compostela, and is Professor of Analytical Chemistry and Head of the Analytical Chemistry Department, University of A Corunìa, Campus da Zapateira s / n, 15071 A Corunìa, Spain.
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Determination of arsenic species in environmental samples: use of the alga Chlorella vulgaris for arsenic( III ) retention A. Taboada-de la Calzada, M.C. Villa-Lojo, E. Beceiro-Gonzaè lez*, E. Alonso-Rodr|èguez, D. Prada-Rodr|èguez
Analytical Chemistry Department, University of A Corunìa, Campus da Zapateira s / n, 15071 A Corunì a, Spain An overview is given of current analytical methodologies for arsenic speciation in environmental samples. Most of these are conventional instrumental methods ^ mainly chromatographic techniques ( HPLC, GC, etc. ) coupled with a variety of detectors. However, methods using micro-organisms are increasingly being applied for the removal of metal ions and other metal species from aqueous solutions. The use of the alga Chlorella vulgaris is proposed for the separation of arsenic( III ) from the other arsenic species. The arsenic concentration is measured by hydride generation atomic absorption spectrometry. z1998 Elsevier Science B.V. Keywords: Arsenic speciation; Speciation of arsenic in alga; Chlorella vulgaris alga, arsenic speciation
*Corresponding author. Abbreviations: AAS, atomic absorption spectrometry ; ABAs, p-aminobenzene arsonate; AFS, atomic fluorescence spectrometry; AsB, arsenobetaine; AsC, arsenocholine; CE, capillary electrophoresis; CZE, capillary zone electrophoresis; DDAB, didodecyldimethylammonium bromide; DIN, direct injector nebulizer; DMA, dimethyl arsinate; ETAAAS, electrothermal atomic absorption spectrometry; FID, flame ionization detector; GC, gas chromatography; HGAAS, hydride generation atomic absorption spectrometry; HPLC, high-performance liquid chromatography ; ICPAES, inductively coupled plasma atomic emission spectrometry; ICP-MS, inductively coupled plasma mass spectrometry ; IEC, ion-exclusion chromatography ; MMA, monomethyl arsonate; MES, microwave emission spectrometric system; MIP, microwave-induced plasma; p-APA, paminophenylarsonate; PhAs, phenyl arsonate; Ph3 As, triphenyl arsonate; PID, photoionization detector; PMB, phenylmagnesium bromide; PTFE, polytetrafluorethylene; SDDC, silver diethyldithiocarbamate; SFC, supercritical fluid chromatography; SIC, suppressed ion chromatography; STAT, slotted tube atom trap; TBA, tetrabutylammonium ; TMA, trimethylarsenic ; TMAs, tetramethylarsonium; USN, ultrasonic nebulization; UV, ultraviolet
1. Introduction Agricultural herbicides, pesticides, insecticides, fossil fuels and metal smelting are common anthropogenic sources of arsenic. Among the common arsenic compounds in the environment, of particular interest is arsenite, which is 10 times more toxic than arsenate and 70 times more toxic than the methylated species, MMA and DMA [ 1 ]. Arsenobetaine and arsenocholine are considered to be non-toxic [ 2 ]. Thess facts indicate why it would be of priority interest to develop methods for the selective determination of As( III ). Numerous instrumental methods for As speciation are reported in the literature. Most of them are based on chromatographic separation techniques coupled with selective and sensitive detectors such as AAS, HGAAS, ICP-MS, and ICP-AES ( see Table 1 ). Although these techniques show great sensitivity and selectivity they usually require very expensive equipment, and other alternative speciation methods that use inexpensive materials could be of technical and commercial interest. It is known that a number of micro-organisms such as yeast, bacteria and algae adsorb ions from their surroundings and lead to their biotransformation into other species [ 3^5 ]. Although most of these microbiological methods have been used for the removal or preconcentration pf metal ions, the possibility is now being explored of employing them for analytical purposes and to selectively separate different species of an element depending on their toxicity. Thus a simple alternative speciation method can use living micro-organisms. For this reason we have initiated the study of the biosorption properties of C. vulgaris with respect to arsenite, arsenate, MMA and DMA and for performing the separation of arsenic species.
0165-9936/98/$19.00 PII S 0 1 6 5 - 9 9 3 6 ( 9 8 ) 0 0 0 0 2 - 8
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Table 1 Instrumental techniques for arsenic speciation Methods
The best As species determined
LOD (Wg l31 )
Reference
Spectrophotometric
As( III ), As(V ) As( III ), As(V ) As( III ), As(V ) As( III ), As(V ), MMA, DMA As( III ), As(V ), MMA, DMA As( III ), As(V ), MMA, DMA, AsB, AsC, TMAs As( III ), As(V ), MMA, DMA As( III ), As(V ),MMA, DMA, AsB, AsC As( III ), As(V ), MMA, DMA As( III ), As(V ), MMA, DMA, p-APA As( III ) As(V ) As( III ), As(V ) As( III ), As(V ), MMA, DMA As( III ), As(V ), MMA,DMA, AsB, AsC As( III ), As(V ), MMA, DMA,TMAs As( III ), As(V ), MMA, DMA As( III ), AsB As( III ), As(V ), MMA, DMA As( III ), As(V ), MMA, DMA, AsB, AsC As( III ), As(V ), MMA, DMA As( III ), As(V ), DMA
100, 100 0.03, 1 ^ ^ ^ 1100, 1400, 1400, 700, 300, 500, 400 1, 1.6, 1.2, 4.7 5, 8, 6, 6, 4, 4 0.5, 0.5, 0.5, 0.5 4, 20, 3.2, 6.5, 9.3 114 120 s 3000 3.5, 9.2, 3.8, 21.3 2.6, 9.6, 13, 9.8, 7.9, 6.1 ^ 1, 5, 1.2, 6 1, 5 4.9, 6, 3.6, 1.2 0.5, 0.3, 1, 1, 0.5, 0.5 0.14, 0.2, 0.08, 0.08 90, 60, 120 76, 40, 70 0.1, 0.02, ^, ^ 10, 10 0.0018 0.0008 0.05, ^ 87.5 5 0.13
[6] [7] [8] [9]
FIA^HG-AAS FIA^HG-ICP-AES HPLC^AAS HPLC^HG-AAS HPLC^UV^HG-AAS IC^HG-AAS SIC^Electrochemical SIC^Conductimetric IEC^PID HPLC^HG-ICP-AES HPLC^UV^HG-ICP-AES HPLC^ETAAS HPLC^HG-MIP-AES HPLC^ICP-MS HPLC^USN-AFS CZE^UV CZE^Conductivity CE^ICP-MS GC^FID HG^GC^PID GC^MES SFC^FID Voltammetric ICP-AES Voltammetric ICP-MS
As( III ), As(V ), MMA, DMA As( III ), As(V ) As( III ), As(V ) As( III ), As(V ) As( III ), As(V ) As( III ) As( III )
2. Arsenic speciation using instrumental techniques 2.1. Spectrophotometric methods
Originally, the pH solution control was used in the determination of arsenite and arsenate, so Howard and Arbab-Zavar [ 6 ] modi¢ed the SDDC spectrophotometric procedure for the determination of arsenite and arsenate. This procedure has interferences from trace metals and methylated arsenic species, but these can be prevented using chelation solvent extraction or ion exchange. Palanivelu et al. [ 7 ] describe a highly sensitive spectrophotometric method for the determination of arsenite and arsenate. Absorbances are measured with a Carl Zeiss PMQII spectrophotometer with 10 mm quartz cells. The determination is made as arsenic triiodide, after an extractive separation into benzene. As(V ) is determined by measuring total arsenic after
[ 10 ] [ 11 ] [ 12 ] [ 13 ] [ 14 ] [ 15 ] [ 16 ] [ 17 ] [ 18 ] [ 19 ] [ 20 ] [ 21 ] [ 22 ] [ 23 ] [ 24 ] [ 25 ] [ 26 ] [ 27 ] [ 28 ] [ 29 ] [ 30 ] [ 31 ] [ 32 ]
the reduction of arsenate with potassium iodide. The results are more sensitive than those based on reaction with SDDC, and superior to the £uorescence method based on the use of rhodamine B. This method has the advantage of lack of interferences and permits precise determination of trace amounts of arsenic in natural and synthetic samples. Tamari et al. [ 8 ] presented a new co-precipitation method for the spectrophotometric determination of arsenite and arsenate in groundwater. Both species are co-precipitated with thorium(V ) hydroxide at pH 9; after centrifugation and dilution with hydrochloric acid the absorbance was measured on a spectrophotometer by the usual SDDS method. Continuous hydride generation, using NaBH4 as reductant, in conjunction with AAS and ICP-AES, was used by Anderson et al. [ 9 ] to form arsine selectively from arsenate, arsenite, MMA and DMA. It was shown that pH is not the only factor to affect the reduction of individual arsenic species, but that other factors
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such as kinetic control and complexation are also involved. 2.2. High-performance liquid chromatography separation coupled with different detectors
Nowadays, the speciation methods commonly used involve an initial separation of the different analyte species in the sample matrix by chromatographic techniques. The method most employed for the separation of arsenic species is HPLC in its various forms. The molecular forms of As which are subjected to speciation analysis are the anions, As( III ), As(V ), MMA and DMA, or the cations AsB, AsC or TMA. The separation techniques used are anion-exchange HPLC with either isocratic or gradient-step elution, or cation exchange with isocratic elution. Ion-pair HPLC has also been used. In order to separate arsenic anions and cations in a single run, a column-switching system involving a combination of anion exchange and reversed-phase separation has been developed. After separation by HPLC the As species are detected on-line by several detection systems such as UV, AAS, HG-AAS, ICP-AES, ICP-MS, or off-line by ETA-AAS. The combination of HPLC with AAS provides a tool that allows the separation and identi¢cation of known arsenic species as well as detecting and giving retention times for unknown arsenic compounds, but it is only suitable for samples containing more than 100 Wg / ml of arsenic. Hansen et al. [ 10 ] applied this online system for the speciation of arsenic species. As( III ), As(V ), MMA and DMA were separated from each other and from the co-injected cationic arsenic compounds on an organic polymeric anionexchange column with 0.1 M carbonate at pH = 10.3 as the mobile phase. AsB, AsC and TMA were separated from each other and from the co-injected anionic species on a silica-based cation-exchange column, with pyridine at pH 2.65 as the mobile phase. For signal enhancement a STAT was installed. The atom trap was made from a quartz tube and was attached by screws to the burner head. The interfacing was established by a vented PTFE capillary tube connecting the HPLC column to the nebulizer of the atomic absorption spectrometer and using a H2 ^Ar £ame. The detection limits are suf¢ciently low for the selected applications, although these detection limits are 150^470 times worse than those from the ICP-MS. The methods that employ hydride generation between the HPLC and AAS present better sensitivity.
Their disadvantage is that only molecules that produce volatile arsines can be detected. Hakala and Pyy [ 11 ] studied arsenic exposure and its monitoring in urine by using a system with on-line HPLC^HG-AAS. The separation of As( III ), As(V ), MMA and DMA is carried out on a C18 reversed-phase column with 10 mM TBA^20 mM phosphate at pH 6.0 as the mobile phase. Because AsB and AsC do not generate hydrides, a preoxidation to an inorganic arsenical is necessary, as by the on-line combination of K2 S2 O8 with UV light. Loèpez et al. [ 12 ] have incorporated the on-line thermo-oxidation to the speciation of As( III ), As(V ), MMA, DMA, AsB and AsC. They used an anion column with 17 mM phosphate at pH 6.0 as the mobile phase. The ef£uent of the HPLC was merged with a persulfate stream before entering the thermo-reactor consisting of a loop of PTFE tubing dipped in a powdered-graphite oven heated to 140³C. After cooling in an ice-bath, HCl and NaBH4 were added on-line to generate the arsine. A similar HPLC system, with the same chromatographic column and with a post-column reaction to achieve complete formation of volatile arsines from the methylated species and As(V ), was developed for the speciation of As( III ), As(V ), MMA and DMA [ 13 ]. Ricci et al. [ 14 ] presented a technique that separates the species using ion-exchange chromatography followed by continuous hydride generation and atomic absorption detection. The separation of the arsenic species, As( III ), As(V ), MMA, DMA and p-APA, is achieved by using a standard Dionex 3U500 mm anion-separator column. A mixture of NaHCO3 , Na2 CO3 and Na2 B4 O7 is used as the mobile phase for the separation of As(V ), MMA and p-APA, and Na2 B4 O7 is used in the case of As( III ) and DMA. The ef£uent exiting the column led via microbore tubing directly into a hydride generator^AAS detection system. McGeehan and Naylor [ 15 ] described a direct simultaneous measurement of arsenic and selenium inorganic species using a suppressed-ion chromatography system with a Dionex 4000i. A Na2 CO3 ^ NaHCO3 solution is used as the mobile phase and an electrochemical and a conductimetric detector were used in series with a dual-channel integrator to quantify As( III ) and As(V ). Although SIC achieves lower detection limits than IC, neither has suf¢cient sensitivity for the analysis of many environmental samples. The major advantage of SIC is its ability to separate and quantify the ionic species of arsenic and selenium without oxidation / reduction pretreatments.
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Hemmings and Jones [ 16 ] presented an IEC method to measure As( III ) and As(V ). Any iron is an important interference which is removed by a cation-exchange procedure before the separation of the arsenic species by using a Dionex 2010i ion chromatograph. The arsenic speciation is carried out by using an HPICE-AS1 ion-exclusion column with a photometric detector. The main problem is the lack of sensitivity of this detector for the determination of arsenic species at trace levels. Another technique described in the literature for As speciation is HPLC^HG-ICP-AES. In this case, only a few authors use hydride generation to detect arsines in the gas phase. A gas^liquid separator was used by Rauret et al. [ 17 ] to minimize the volume of solution reaching the plasma torch and to improve the separation of volatile hydrides. These authors used a silicabased anion-exchange column to separate the arsenic species As( III ), As(V ), MMA and DMA with a phosphate buffer, pH = 6.75 as the mobile phase. Rubio et al. [ 18 ] developed the separation of As( III ), As(V ), MMA, DMA, AsB and AsC on an anion-exchange column using a phosphate buffer as the mobile phase. On-line UV-photo-oxidation was used by these authors at the exit of the chromatographic column for the decomposition of AsB and AsC to As(V ). ETA-AAS can also be used as a detector after separation by HPLC. Although the detection of arsenic species using on-line techniques is the ideal, off-line detection by ETA-AAS is still advantageous owing to the good sensitivity, but the fairly long time required for the analysis of a series of collected fractions may be inconvenient for practical analytical work. Another problem is the loss of analyte at the pre-atomization step. In this way, Larsen [ 19 ] studied conventional and fast-furnace programs, with and without palladium^magnesium nitrate as a chemical modi¢er. The on-line coupling of vesicle-mediated HPLC to low-power argon MIP detection was applied to the arsenic speciation of As( III ), As(V ), MMA and DMA in natural waters and in human urine by Costa-Fernaèndez et al. [ 20 ]. They used a C18 -bonded silica stationary phase with a mobile phase prepared with NaH2 PO4 ( 5 mM ) in MeOH ( 1%) and 0.01 mM vesicles of DDAB at pH=5.75. The presence of DDAB vesicles in the mobile phase improves the detection limits. The volatile species formed are separated from the liquid and directed to the low-pressure MIP system after passing through a desiccator ( H3 PO4 ). The low cost of the detection / excitation source and its maintenance and the low detection limits are com-
parable to those obtained with other more powerful sources such as ICP. Several workers have reported on arsenic speciation by HPLC^ICP-MS. The bene¢ts of coupling plasma mass-spectrometric detection with chromatographic separation include speci¢city, the ability to separate interferences from peaks of interest, multi-element capability, and low levels of detection. Beauchemin et al. [ 21 ] coupled various forms of HPLC with ICPMS for arsenic speciation. They studied ion-pairing and ion-exchange HPLC to separate As( III ), As(V ), MMA, DMA and AsB. They found that anion exchange has resolution inferior to ion-pairing chromatography, but it appears to be less susceptible to matrix interferences. Sheppard et al. [ 22 ] used a column Wescan Anion / R-IC to separate As( III ), As(V ), MMA and DMA from each other and from 40 Ar35 Cl , which is formed with the plasma gas and the chloride present in samples. The eluents used were 2% propan-1-ol and carbonate buffer 50 mM at pH 7.5. The column was directly connected to the nebulizer of the ICP by polyplex tubing. The sensitivity of this method is limited by the sample dilution that is necessary to diminish the high chloride concentration. Demesmay et al. [ 23 ] used an ICP-MS detector coupled to an HPLC system to determine As( III ), As(V ), MMA, DMA, AsB and AsC. The interface was established through PTFE capillary tubing. They used an anion-exchange column with a mobile phase of phosphate buffer with 2% acetonitrile. To reach an optimum separation of the six arsenic species an ionic-strength step gradient was necessary. Most hydride-forming elements can be detected by using AFS in the UV region below 250 nm. AFS is not being widely used, one possible reason being that, until recently, there was no reliable high-intensity excitation source. The appearance of the boosted-discharge hollow cathode lamp provided a very good excitation source for AFS detection. The main problem with this technique lies in the interface between the HPLC and AFS. Woller et al. [ 24 ] used the coupling HPLC^USN-AFS to determine As( III ), As(V ), MMA and DMA. They used a USN different from previous designs and found better detection limits than other authors. 2.3. Capillary electrophoresis
Schlegel et al. [ 25 ] presented a modi¢cation of the high-performance capillary electrophoresis ^ capillary zone electrophoresis ^ which uses two different
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detection systems, photometric and conductimetric. The CZE shows excellent suitability for arsenic speciation, and the separation of different species is possible. With the photometric detector, As( III ), As(V ) and DMA are determined, and with the conductimetric detector, As(V ), DMA, ABAs and PhAs. Another CE technique for determining As( III ), As(V ), MMA and DMA is presented by Lin et al. [ 26 ]. They introduced a different detection system ( ICP-MS ) and developed a novel interface which is based on a DIN. This interface can be used under different CE conditions and it allows for the DIN to be optimized independently from the CE system to give the most ef¢cient sample introduction into the ICPMS. This method therefore combines the high separation ef¢ciency of the CE with the high elemental sensitivity and selectivity of the ICP-MS. 2.4. Gas chromatographic separation coupled with different detectors
Beckermann [ 27 ] presented an elegant method for the determination of MMA and DMA in biological samples. Most of the methods proposed for this speciation are based on the conversion by sodium borohydride of the arsenic into the corresponding methylarsine compounds, but their volatilities make it necessary to convert MMA and DMA into stable derivatives. With this method, the methylarsenic acids are derivatized with thioglycolic acid methyl ester, to yield lipophilic species which can be determined by GC using a FID. Another technique, presented by two different groups of authors [ 28,29 ], is based on selective hydride generation, liquid-nitrogen-cooled trapping, and gas chromatography with helium-dischargetype photo-ionization detection. It is applied to the simultaneous determination of As( III ) and Sb ( III ) under weak-acid conditions and to As( III ), As(V ) and Sb( III ), Sb(V ) under strong-acid conditions. Talmi et al. [ 30 ] presented a method consisting of gas chromatography coupled with a MES detector for arsenic and antimony determination in environmental samples. The analytical procedure is based on the following steps: sample pretreatment, co-crystallization of As( III ) and Sb( III ) with thionalide, and reaction of the dry precipitate with PMB. When the phenylation had been completed, the excess of PMB was decomposed to prevent oxidation of Ph3 As, then a portion of the organic layer was injected into the column with a gradient program, and determined by MES.
2.5. Supercritical £uid chromatography
Laintz et al. [ 31 ] described a novel technique based on a simultaneous separation and quanti¢cation of As( III ) and As(V ) ( after reduction with potassium iodide and sodium thiosulfate ), achieved by extraction with lithium bis( tri£uoroethyl )dithiocarbamate followed by supercritical £uid chromatography with FID. The extraction technique reduces organic matrix interferences and preconcentrates the metal species by conversion into chelates suitable for SFC analysis. 2.6. Voltammetric methods
Pretty et al. [ 32 ] presented an on-line, anodic stripping-voltammetry £ow cell with detection by ICPAES and ICP-MS, for As( III ) determination. With this method, polyatomic interferences which arise from chloride in sample matrices are eliminated. The detection by ICP-AES has insuf¢cient sensitivity in many cases, but lower detection limits can be obtained with a hydride generator coupled to the ICP. The detection by ICP-MS enhances the signals.
3. Arsenic speciation using micro-organisms Many micro-organisms, such as algae, fungi, yeast and bacteria, have been used for biosorption of toxic elements by binding to the cell surface and intracellularly. This property has been applied largely to the preconcentration of metal ions, mainly in industry, the environment, etc. [ 33 ], but its application to metal speciation has recently been proposed. The interest in the use of biological substrates can be explained because cells have a small and uniform size, present many active binding sites, and have a low production cost. Because metabolic activity is not essential for metal uptake, this may be carried out by living or dead micro-organisms. The biosorption process may be performed in two ways: in batches without immobilized substrates and using micro-organisms immobilized on a suitable support [ 34,35 ]. The capacity of some algae to accumulate arsenic has been known for several years [ 36 ], and has been used to eliminate this element from industrial ef£uents [ 33 ]. Yamaoka et al. [ 37^39 ] studied the accumulation of arsenic by Duniella sp. and the effect of various elements on this accumulation. The capacity of the alga Chlorella vulgaris to transform inorganic arsenic compounds [ 40^42 ] and to reduce As( III ) to As(V )
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[ 43,44 ] has been evaluated. Goessler et al. [ 45 ] studied the uptake of arsenic compounds by three Chlorella species. Bioaccumulation and biomethylation of inorganic arsenic by the marine alga Polyphysa peniculus [ 46 ] and the blue-green alga Nostoc sp. [ 47 ] were also investigated. Five bacteria (Proteus sp., Escherichia coli, Flavobacterium sp., Corynebacterium sp. and Pseudomonas sp. ) were used to determine arsenic uptake and distribution into the cells [ 48 ]. According to the work mentioned above the relationship between different micro-organisms and arsenic is widely demonstrated, but this fact has not yet been exploited as a practical way for arsenic spe-
ciation. In our laboratory we have studied the retention capacity of the alga Chlorella vulgaris with respect to As( III ), As(V ), MMA and DMA, as a possible method of separation of As( III ), the most toxic species, from the others.
4. Case study: use of the alga for the As( III ) retention Our study demonstrates that Chlorella vulgaris can be used to selectively separate about 50% of the most toxic arsenic species, As( III ), from the other species, As(V ), MMA and DMA. It has been proved that
Fig. 1. Analytical procedure for the As( III ) retention by Chlorella vulgaris.
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As( III ) retention by the alga occurs in a strongly basic medium at room temperature and As(V ), MMA and DMA retention is not signi¢cant ( see Fig. 1 ). These conditions are easily obtained in a laboratory. The alga was harvested in two different macronutrient media, one with limited phosphate and nitrate concentrations ( A ), and the other with a suf¢cient amount of these essential nutrients ( B ). It can be seen that the alga ( A ), with limited concentrations of these nutrients, appears to have a higher retention ( 52%) than the other ( 40%). Since AsO33 3 has chem3 and NO , it is possible that ical similarity to PO33 4 3 those nutrients could be replaced by AsO33 3 . The Chlorella vulgaris alga needs no particular pretreatment, so with ageing alga there were no signi¢cant changes in As( III ) retention, and it has been observed that the use of dry alga gave similar results. However, the best retention was obtained when the living alga was used with limited essential nutrients, ( A ). When the alga A was used the retention increased with the alga volume, and the best retention was obtained with 100 ml. This situation probably results from different accumulation mechanisms. In one of these, the binding of the arsenic species to the cell surface occurs by adsorption, and the other mechanism consists of transport of As( III ) through the cell membrane: this latter one needs biological activity. The method does not require too much time, 10 min of contact time between the alga and the solution containing As( III ) is enough to give optimum retention.
5. Conclusion HG-AAS is the classical method most used for arsenic determination, but this technique does not permit speciation without a previous separation of the species. If the separation is performed by co-precipitation, pH, etc., interferences may occur and a previous extraction or ion exchange would be necessary to avoid this. Thus, a liquid chromatography separation coupled to different detectors is mainly employed; this also allows the speciation of inorganic and organic arsenic species. HPLC^AAS does not show detection limits low enough for most of the environmental applications, but the methods that employ hydride generation between HPLC separation and AAS detection ( HPLC^HG-AAS ) improve the sensitivity. The trouble is that a diminution of the sensitivity is observed when thermo-oxidation, to release AsB and AsC as As(V ), is used.
Speciation methods using HPLC^HG-ICP-AES do not have enough sensitivity in many cases and give higher detection limits than with an AAS detector. When thermo-oxidation is employed to convert AsB and AsC, as in the above-mentioned case, the detection limits become worse relative to HPLC^UV-HGAAS. When off-line detection by ETA-AAS is coupled with HPLC the main advantage is an increase in the sensitivity, but it is a tedious method and has disadvantages for practical analytical work. Another problem is the loss of analyte at the pre-atomization step. Arsenic speciation by HPLC^ICP-MS or CE^ICPMS combines the bene¢ts of coupling plasma mass spectrometric detection with chromatographic separation. These techniques show good speci¢city and the ability to separate interferences from peaks of interest, along with multi-element capability and low levels of detection, but they have the inconvenience of high instrumental and maintenance cost. Similar advantages and disadvantages are shown when HPLC^ MIP is used. HPLC^USN-AFS is not used very frequently for arsenic speciation, probably because it is dif¢cult to obtain a suitable interface, although this technique shows excellent detection limits. Other liquid chromatography methods, such as SIC and IEC, using photometric, conductimetric or electrochemical detectors present low sensitivity, and only As( III ) and As(V ) can be determined. The same situation is observed when CE is used with photometric or conductimetric detectors, but in this case DMA can also be determined. With GC, the main problem at the moment is that the technique is limited to the determination of As( III ) and As(V ); its principal advantage is that it presents excellent sensitivity. Supercritical £uid chromatography is a novel technique that does not have great application for arsenic speciation at the moment, because it also is limited to the determination of As( III ) and As(V ). However, in contrast to GC it presents low sensitivity. When voltammetric methods are coupled with ICP^ AES, they do not show enough sensitivity, and when they are coupled with ICP^MS they have the same disadvantages as HPLC^ICP-MS. The high sophistication required of the coupled systems makes their implementation dif¢cult for routine analysis. Recently, interest has grown in areas where analytical chemistry and biology interplay. There is a need for simple analytical methods to separate species of several analytes characterized by different toxicity.
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One simple alternative is to use biological substrates to distinguish between toxic and non-toxic species. The use of biological micro-organisms has been known for a long time, but its application to metal speciation is now being initiated. There are no references in the literature about arsenic speciation with such materials. However, the use of living micro-organisms for As( III ) speciation could give a new perspective for speciation of this element. The aim of this paper is to extend the applicability of Chlorella vulgaris to the selective determination of arsenic species. We propose the use of the green alga Chlorella vulgaris to selectively separate As( III ) from the other common arsenic species, As(V ), MMA and DMA. Although total As( III ) separation is not reached, one advantage of the use of this alga for As( III ) separation is that it provides a simple and economic procedure suitable for routine analysis.
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A. Taboada-de la Calzada and M.C. Villa-Lojo are graduates of the University of Santiago de Compostela and are graduate students at the University of A Corunìa. E. Beceiro-Gonzaèlez and E. Alonso-Rodr|èguez are Doctors in Chemistry, with PhD degrees from the University of Santiago de Compostela and are lecturers in analytical chemistry at the University of A Corunìa.
D. Prada-Rodr|èguez is a Doctor in Chemistry, with a PhD degree from the University of Santiago de Compostela, and is Professor of Analytical Chemistry and Head of the Analytical Chemistry Department, University of A Corunìa, Campus da Zapateira s / n, 15071 A Corunìa, Spain.
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