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Determination of Arsenic Species by High-Performance Liquid Chromatography–Hydride Generation–(Ultrasonic Nebulizer)–Atomic Fluorescence Spectrometry
MICROCHEMICAL JOURNAL ARTICLE NO.
54, 184–194 (1996)
0094
Determination of Arsenic Species by High-Performance Liquid Chromatography–Hydride Generation–(Ultrasonic Nebulizer)– Atomic Fluorescence Spectrometry ´ GNES WOLLER, ZOLTA´N MESTER, A
AND
PE´TER FODOR1
Department of Chemistry and Biochemistry, University of Horticulture and Food Industry, 29-35 Villa´nyi, H-1114 Budapest, Hungary A technique to determine four species of arsenic (AsIII, AsV, dimethylarsinic (DMAs), and monomethylarsonic (MMAs) acids) where high-performance liquid chromatography was coupled to an atomic fluorescence spectrometer using an ultrasonic nebulizer as an interface has been further improved by the use of hydride generation. The effect of hydride conditions on signal intensities has been investigated. The detection limits for AsIII, AsV, DMAs, and MMAs were 2.5, 6, 3.2, and 2 ng, respectively (at 250 mm3 volume injected). The linearity for all four arsenic species were in the range 25–1000 ng. q 1996 Academic Press, Inc.
INTRODUCTION
Speciation studies has become more and more popular in the past decades for a large scientific community. Because these techniques address only a fraction of the total metal present in the sample therefore requires ultra-sensitive methods (ng/liter range in water and ng/g in soil matrices). This problem has been solved most frequently by coupling a powerful separation technique (usually chromatography) and a sensitive, selective detection system, e.g., atomic absorption spectrometry (AAS), inductively coupled plasma–mass spectrometry (ICP-MS), inductively coupled plasma–atomic emission spectrometry (ICP-AES), etc. These techniques are usually called ‘‘hyphenated’’ techniques. A very important field of speciation studies today is arsenic speciation. A possible solution for the speciation of four toxicologically important arsenic species (AsIII, AsV, dimethylarsinic (DMAs), and monomethylarsonic (MMAs) acids) has been published in our previous paper (1), where a C18 Rutin column coupled to a high-performance liquid chromatography pump has been used for the separation; for detection a hydrogen diffusion-flame-based atomic fluorescence spectrometer (AFS) has been coupled to the chromatographic system. Desolvation and nebulization needed for the AFS technique has been carried out by an ultrasonic nebulizer (USN). The mentioned hyphenated technique has shown relatively good separation for the investigated species, although the limit of detection (LOD) was too high for the analyses of biological and environmental samples. The analytical potential of hydride generation (HG) was first reported by Holak (2) in 1969; since then the technique has become a widely accepted method for determining elements which form volatile hydrides. These elements are 1
To whom correspondence should be addressed. 184
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antimony, arsenic, bismuth, germanium, lead, selenium, tin, and tellurium. It is well documented in the literature (3, 14) that HG has been used to improve sensitivity and detection limits for spectrometric determinations successfully in many cases. It may be used in combination with many different detection systems, including flame- and graphite-furnace atomic absorption spectrometry, atomic emission spectrometry, and colorimetry. The hydride generation technique is commonly used for the determination of trace amounts of arsenic. Sodium tetrahydroborate, when added to an acidic solution, produces nascent hydrogen, and this was reported as an efficient method for producing arsines for use in spectrometric analysis (4). First a sodium borohydride pellet method was used for hydride generation where the acidified sample was added to pellets of the reductant. Later solutions have become more popular. The advantages of using solution rather than pellets of sodium borohydride was reported by McDaniel et al. (5); sodium borohydride (NaBH4 ) solution with hydrochloric acid (HCl) matrix (6) is now the most common reagent for the generation of arsines for spectroscopic analysis. A great advantage of hydride generation techniques is the separation of the above mentioned hydride forming elements from the matrix media which can result in a relatively interference free background spectrum (from the spectroscopical point of view). Another benefit is the increased sensitivity caused by more efficient introduction of hydrides into the flame compared to any pneumatic nebulizer and the good dissociation conditions of hydrides even in cool flames. A potential drawback of the method is that it requires the element to be in a particular oxidation state before the hydride may be formed. Although sodium borohydride is able to reduce As(V) in acidic solution to As(III) and then to the hydride (AsH3 ) the resultant signal is generally depressed by 20–30% relative to that for As(III) (7). Another disadvantage of HG is the numerous sources of interferences during hydride generation (8, 9). The task of the present paper was to find possible ways to increase sensitivity and detection limits of an already working system that has been developed for the speciation of four arsenic (1). The LOD of the referred system for As(III), As(V), DMAs, and MMAs were 35, 50, 20, and 20 ng, respectively. Hydride generation as a promising derivatization technique has given us the hope to improve this LOD by at least an order of magnitude (10, 11, 14). EXPERIMENTAL
Instrumentation A Shimadzu Model LC-7A HPLC pump was attached to a sample injection valve (sixport rheodyne system, LMIM, Hungary). A 250-mm3 sample loop was used for sample introduction. The analytical column was a Bio Separation Technologies (BST) C18 Rutin column (25 1 4.6 mm i.d., 10-mm particle size). A four-channel peristaltic pump (Ismatec MS-CA 4, Switzerland), a 100-cm long mixing coil, and the aerosol chamber of a Cetac Model 5000 Ultrasonic Nebulizer (CETAC, Omaha, NE) functioned as a continuous hydride generator. The piezoelectronic transducer of the USN was not in use during
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FIG. 1. Schematic diagram of the HPLC–HG–(USN)–AFS system used for arsenic speciation.
analysis. A Cetac Model 2050 ‘‘Heated and Refrigerated Constant Temperature Bath’’ provided the required conditions for desolvation. Removal of the drain from the nebulizer was carried out with a peristaltic pump (Raimin Instruments Co., Wolburn, MA). The nebulizer was further connected to an AFS (PSA Excalibur, PS Analytical, Sevenoaks, Kent, UK) that utilized an arsenic boosted-discharge hollow cathode lamp (Superlamp, Photron, Victoria, Australia) as an excitation source. Measurements were carried out around the resonance wavelength of arsenic (193.7 nm) using a multi-reflectance filter having a spectral bandpass between 20 and 40 nm. Argon functioned as a carrier gas in the USN, and mixed with the hydrogen, supported the diffusion flame. The constant gas flows were maintained by Cole–Palmer rotameters (Niles, IL). Data collection and evaluation was totally automated by Borwin Chromatographic software (JMBS, Grenoble, France). All peaks were evaluated by their peak height. A schematic diagram of the system is shown in Fig. 1. Reagents Arsenite stock solution was prepared by dissolving 1.320 g of As2O3 in 25 cm of 0.5 mol dm03 NaOH solution and then diluting the solution to 1 dm3 with 0.6 mol dm03 HCl. AsV stock solution was obtained from Merck. MMAs stock solution was prepared from strychrotonin solution (Chinoin, Budapest), and the DMAs was obtained from Fluka. The 1000 mg dm03 stock solutions of MMAs, DMAs, AsIII, and AsV were further diluted in deionized water daily. The DDAB solution (0.01 mol dm03) was prepared by adding 0.5% (v/v) methanol and 0.1% (v/v) of 0.01 mol dm03 DDAB solution to the Na2HPO4 buffer solution (20 mmol dm03). The pH was set to 6.0 by the addition of NaH2PO4 solution containing the same amount of phosphate, methanol, and DDAB as the eluent. Chemicals for hydride generation. The (2% (m/v)) sodium tetrahydroborate solution was prepared daily by dissolving NaBH4 powder (Aldrich) in 0.5% (m/v) NaOH solution, and the HCl was obtained from Reanal (Hungary). Reanal (Hungary) potas-
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sium iodide and sodium thiosulfate were used for preparing the 0.1, 1, and 10% solutions for reducing As(V) to As(III). Procedures Column modification. Column modification was carried out according to procedure described elsewhere (12). Arsenic speciation. 250 mm3 of the working standard solutions of a mixture of arsenic species is injected directly onto the HPLC column through the injection port (Fig. 1). After the separation part of the system, first HCl and then NaBH4 solutions are introduced into the stream of the column effluent. Hydride generation takes place in a mixing coil, and the generated gas phase is separated from the liquid phase continuously in the aerosol chamber of the USN. The transducer of the ultrasonic nebulizer was not in use during our measurements; the nebulizer in this case functioned only as a gas–liquid separator and desolvation system. A continuous stream of argon transfers the generated hydrides and hydrogen from the nebulizer to the AFS. The hydrogen–argon diffusion flame is maintained by externally introduced hydrogen where the hydrogen gas enters the system via a Y-shape connection tube about 25
TABLE 1 Operating Conditions for the HPLC–HG–(USN)–AFS System
Column Column temperature Sample loop size Mobile phase Pump flow rate
Chromatography BST C18 Rutin column (25 1 4.6 mm i.d., 10-mm particle size) 247C 250 mm3 20 mmol dm03 Na2HPO4 –NaH2PO4 buffer (pH 6.0) / 0.1% (v/v) of 1002 mol dm03 DDAB / 0.5 % (v/v) methanol 1 ml min01 Hydride generation conditions 2% (m/v) NaBH4 solution dissolved in 0.5% (m/v) NaOH (1.7 ml min01) 1.5 mol dm03 HCl (1.7 ml min01) Ultrasonic nebulizer
Heating temperature Cooling temperature
1407C 57C Atomic fluorescence detector
Primary current Boost current
27.5 mA 35 mA Gas flow rates
Argon Hydrogen
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cm from the flame. (Tube lengths were reduced as much as possible to minimize dead volumes.) All chromatographic peaks were evaluated by their peak heights. Experimental conditions for hydride generation were optimized, and the parameters can be seen in Table 1. RESULTS AND DISCUSSION
As described in the Experimental section, in the present system we utilized only the desolvation power of the USN. The piezoelectronic transducer was turned off during analysis because the nebulization process was not essential; the HG technique converts the analyte into vapor phase. The small and cold hydrogen diffusion-flame in the AFS apparatus demands the analyte to be in a gaseous form since the flame cannot handle a large solvent load. The HG beside that which forms gaseous analyte has another benefit; it produces enough hydrogen in the chemical reaction to maintain the flame (13). To reduce the instability of the system we used externally introduced hydrogen (gas flow rates in Table 1 correspond only to the externally introduced gas, and do not include the amount that has been formed as a result of chemical reaction). The aerosol chamber of the nebulizer functioned as a gas–liquid separator, and the nebulization section of the U-500 USN was used without any change in the original design. The performance of gas–liquid separation was fairly good: no memory effect has been found. As the analyte enters into the face-plate of the transducer through a glass sample tube, separation takes place in the nebulization chamber according to the rules of gravitation. The gaseous hydrides and hydrogen had already been formed in the mixing coil and as they entered into the chamber the carrier gas (Ar) transported them toward the desolvation section of the system. The liquid drain is carried away from the bottom of the vessel by a peristaltic pump. The construction of the system provides a water trap layer as well. Several home-made design gas–liquid separators have been tried but they all suffered from strong sources of noise because of pulsation caused by the inadequate overflow. Effect of Hydride Generation Conditions on Net Signal The optimal working conditions for the HPLC–USN–AFS system have already been determined (1) and these parameters served as starting points for our further investigation. The efficiency of HG depends primarily on the concentrations of HCl and NaBH4 . In the present system the flow rates of these components were kept constant at 1.7 ml/min. According to our previous investigations and the literature, the following conditions have been selected for the determination of AsIII, AsV, DMAs, and MMAs by HG: NaBH4 (mol dm03) 03
HCl (mol dm )
0.25
0.5
1.5
0.75
1.5
3.0
Results of the signal optimization of four arsenic species at different HG parameters can be seen in Figs. 2A, 2B, and 2C. It can be concluded from the figure that the efficiency of HG in the cases of As(III) and MMAs is virtually independent from the
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FIG. 2. Influence of hydrochloric acid (mol dm03) and sodium borohydrate (A, 0.5; B, 1.0; C, 2.0% (m/v)) concentrations on AsIII (1), DMAs (2), MMAs (3), and AsV (4) signal intensities at 1 mg dm03 injected concentrations.
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MESTER, WOLLER, AND FODOR TABLE 2 Comparison of LODs of Different Systems for Arsenic Speciation
Species
LC– ICP/AES
LC–HG– ICP/AES
LC–ICP/MS
LC–USN–AFS
LC–HG– (USN)–AFS
As (III) As (V) MMAs DMAs
120 130 100 130
0.26 0.96 1.3 0.98
0.01 0.03 0.02 0.02
35 50 20 20
2.5 6 2 3.2
Note. Numbers represent ng of arsenic14.
concentrations of the reductant and the acid. The increasing NaBH4 concentration hardly increased the signal intensities, and with increasing acid concentration, no significant change was measured during the experiment. Regarding the other two compounds (As(V) and DMAs), the concentration of the acid and reductant had a strong effect on the net signal. A direct proportion has been observed between the concentration of NaBH4 solution and the efficiency of HG in the case of these components. Arsenic (V) responded with a higher signal to the higher HCl concentrations, whereas the signal of DMAs decreased in response to higher HCl concentrations. As a result of the HG reagent optimization we can conclude that in the case of the determination of all four arsenic species only a compromised optimum can be achieved. At the determination of either methylized or inorganic compounds in separate systems real optimum exists regarding the HG conditions. As a result of our experiments for running the system we have selected the concentrations of 1.5 mol dm03 for HCl (1.7 ml min01) and 2% (m/v) for NaBH4 (1.7 ml min01). The effect of hydrochloride acid on the appearance of the chromatograms is presented in Fig. 3. From the top the concentrations of HCl are 0.75, 1.5, and 3.0 mol dm03. No change in the intensity of As(III) and MMAs was noticed as the HCl concentration was increased; the intensity of the DMAs signal eluting second decreased and the intensity of the As(V) signal increased. It is important to note that the reproducibility of retention times of the species is extremely good in spite of the manual mode of injection. According to the literature (7, 9), the reduction of arsenic (V) to arsine is slower than that of As(III). In batch systems this results in peak shift and in 20–30% lower peak absorbance. In continuous systems Welz (9) has found sensitivity for As(V) an order of magnitude lower because of the kinetically slower reaction. A lot of studies have discussed the possible ways of eliminating this type of interference, however the cold flow-injection systems were not often investigated before. The majority of pre-reduction techniques applied for As(V) reduction were heated systems. In our experiments we investigated the effect of added potassium iodide (KI) (0.1, 1, and 10%) and sodium thiosulfate (0.1, 1, and 10%) on the signal of As(V) in a cold flow-injection system. The sodium thiosulfate solution was introduced directly into the eluent stream in two ways, before and after the introduction of the HCl solution. The KI solution was introduced together with the acid. Our conclusion was that in the given system, the above mentioned pre-reduction
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FIG. 3. The effect of different concentrations of hydrochloride acid solutions on the appearance of the chromatograms at 2% (m/v) NaBH4 concentration. From the top toward the bottom the HCl concentrations are 0.75, 1.5, and 3.0 mol dm03. The four arsenic species (identified by their retention times (RT)) are the following: RT Å 3.4 As(III), RT Å 4.3 DMAs, RT Å 5.9 MMAs, and RT Å 12.4 As(V).
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FIG. 4. HPLC–HG–(USN)–AFS chromatogram of the four investigated arsenic species under optimal working conditions. Peak identification: RT Å 3.88 As(III), RT Å 4.89 DMAs, RT Å 6.6 MMAs and RT Å 10.91 As(V). The concentration of injected arsenic species were 1 mg dm03.
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steps weren’t successful, the KI had no effect, and, instead of increasing the signal of As(V), the sodium thiosulfate has decreased it. We are planning to apply these pre-reduction steps in a heated flow-injection system in the future. Analytical Conditions The LOD (last column of Table 2) was calculated on the basis of 3 S criterion using the results of from nine replicated measurements. Linearity of the system for the four investigated arsenic species was in the range 25–1000 ng. The chromatogram of the four arsenic species can be seen in Fig. 4. The flow rate was kept at 0.8 ml/min during the elution of the first three peaks and after them it was adjusted to 2 ml/min to a give better appearance to the chromatogram. The resolution of the first three components could be further improved by changing the eluent conditions although in the case of peak height evaluation this resolution power proved to be satisfactory. The lower sensitivity of As(V) compared to As(III), MMAs, and DMAs could be explained by the lower efficiency of hydride generation. CONCLUSIONS
Comparison of LODs of different systems for arsenic speciation are shown in Table 2. The data are in good accordance with the literature that presents some orders of magnitude enhancement in many cases when using HG. As we expected, the LODs of the present system improved an order of magnitude compared to results measured in a system without HG (last two columns of Table 2). The HPLC–HG–(USN)– AFS system is a fairly robust and stable system with a very simple mode of detection. The cost of the system is much less than the cost of the commonly used sophisticated methods (ICP, ICP-MS detectors). Routine analyses of environmental and biological samples are possible on the system because the LODs are in the required range in most cases. Unfortunately, with the introduction of HG the possible sources of interferences increased. The origin of these interferences might be the liquid phase (hydride generation kinetics, generation efficiency) or the gaseous phase (transport kinetics, transport efficiency); gaseous phase interference can take place during the atomization process as well. The investigation of this phenomenon and to find solutions to minimize the interferences are the next steps in the development of the HPLC–HG–(USN)–AFS system. ACKNOWLEDGMENT This research was partly supported by the Hungarian Scientific Research Foundation (OTKA) Grant T014329.
REFERENCES 1. 2. 3. 4. 5. 6.
´ .; Mester, Z.; Fodor, P. J. Anal. At. Spectrom. 1995, 10, 629. Woller, A Holak, W. Anal. Chem. 1969, 41, 1712. Robbins, W. B.; Caruso, J. A. Anal. Chem. A. 1979, 51, 889. Braman, R. S.; Justen, L. L.; Foreback, C. C. Anal. Chem. 1972, 44, 2195–2199. McDaniel, M.; Shendrikar, A. D.; Reiszner, K. D.; West, P. W. Anal. Chem. 1976, 48, 2240–2243. Thompson, M.; Pahlavanpour, B.; Walton, S. J.; Kirkbright, G. F. Analyst 1978, 103, 568–579.
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7. Francesconi, K. A.; Edmonds, J. S.; Morita, M. In Arsenic in the Environment, Part I: Cycling and Characterization, ed. (J. O. Nriagu, Ed.), Vol. 9, pp. 189–219. Wiley, New York, 1994. 8. Smith, A. E. Analyst 1975, 100, 300–306. 9. Welz, B.; Stauss, P. Spectrochim. Acta B. 1993, 48, 951–976. 10. Nakahara, T. Prog. Anal. Atom. Spectrosc. 1983, 6, 163. 11. Huang, B.; Zhang, Z.; Zeng, X. Specrochim. Acta B. 1987, 42, 129–137. 12. Liu, Y. M.; Ferna´ndez Sa´nchez, M. L.; Gonza´lez, E. B.; Salz-Mendel, A. J. Anal. At. Spectrom. 1993, 8, 816. 13. Corns, W. T.; Stockwell, P. B.; Ebdon, L.; Hill, S. J. J. Anal. At. Spectrom. 1993, 8, 71. 14. Quevauviller, Ph.; Maier, E. A.; Griepink, B. (Eds.) Quality Assurance for Environmental Analysis, pp. 288–303. Elsevier, Amsterdam, 1995.
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54, 184–194 (1996)
0094
Determination of Arsenic Species by High-Performance Liquid Chromatography–Hydride Generation–(Ultrasonic Nebulizer)– Atomic Fluorescence Spectrometry ´ GNES WOLLER, ZOLTA´N MESTER, A
AND
PE´TER FODOR1
Department of Chemistry and Biochemistry, University of Horticulture and Food Industry, 29-35 Villa´nyi, H-1114 Budapest, Hungary A technique to determine four species of arsenic (AsIII, AsV, dimethylarsinic (DMAs), and monomethylarsonic (MMAs) acids) where high-performance liquid chromatography was coupled to an atomic fluorescence spectrometer using an ultrasonic nebulizer as an interface has been further improved by the use of hydride generation. The effect of hydride conditions on signal intensities has been investigated. The detection limits for AsIII, AsV, DMAs, and MMAs were 2.5, 6, 3.2, and 2 ng, respectively (at 250 mm3 volume injected). The linearity for all four arsenic species were in the range 25–1000 ng. q 1996 Academic Press, Inc.
INTRODUCTION
Speciation studies has become more and more popular in the past decades for a large scientific community. Because these techniques address only a fraction of the total metal present in the sample therefore requires ultra-sensitive methods (ng/liter range in water and ng/g in soil matrices). This problem has been solved most frequently by coupling a powerful separation technique (usually chromatography) and a sensitive, selective detection system, e.g., atomic absorption spectrometry (AAS), inductively coupled plasma–mass spectrometry (ICP-MS), inductively coupled plasma–atomic emission spectrometry (ICP-AES), etc. These techniques are usually called ‘‘hyphenated’’ techniques. A very important field of speciation studies today is arsenic speciation. A possible solution for the speciation of four toxicologically important arsenic species (AsIII, AsV, dimethylarsinic (DMAs), and monomethylarsonic (MMAs) acids) has been published in our previous paper (1), where a C18 Rutin column coupled to a high-performance liquid chromatography pump has been used for the separation; for detection a hydrogen diffusion-flame-based atomic fluorescence spectrometer (AFS) has been coupled to the chromatographic system. Desolvation and nebulization needed for the AFS technique has been carried out by an ultrasonic nebulizer (USN). The mentioned hyphenated technique has shown relatively good separation for the investigated species, although the limit of detection (LOD) was too high for the analyses of biological and environmental samples. The analytical potential of hydride generation (HG) was first reported by Holak (2) in 1969; since then the technique has become a widely accepted method for determining elements which form volatile hydrides. These elements are 1
To whom correspondence should be addressed. 184
0026-265X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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antimony, arsenic, bismuth, germanium, lead, selenium, tin, and tellurium. It is well documented in the literature (3, 14) that HG has been used to improve sensitivity and detection limits for spectrometric determinations successfully in many cases. It may be used in combination with many different detection systems, including flame- and graphite-furnace atomic absorption spectrometry, atomic emission spectrometry, and colorimetry. The hydride generation technique is commonly used for the determination of trace amounts of arsenic. Sodium tetrahydroborate, when added to an acidic solution, produces nascent hydrogen, and this was reported as an efficient method for producing arsines for use in spectrometric analysis (4). First a sodium borohydride pellet method was used for hydride generation where the acidified sample was added to pellets of the reductant. Later solutions have become more popular. The advantages of using solution rather than pellets of sodium borohydride was reported by McDaniel et al. (5); sodium borohydride (NaBH4 ) solution with hydrochloric acid (HCl) matrix (6) is now the most common reagent for the generation of arsines for spectroscopic analysis. A great advantage of hydride generation techniques is the separation of the above mentioned hydride forming elements from the matrix media which can result in a relatively interference free background spectrum (from the spectroscopical point of view). Another benefit is the increased sensitivity caused by more efficient introduction of hydrides into the flame compared to any pneumatic nebulizer and the good dissociation conditions of hydrides even in cool flames. A potential drawback of the method is that it requires the element to be in a particular oxidation state before the hydride may be formed. Although sodium borohydride is able to reduce As(V) in acidic solution to As(III) and then to the hydride (AsH3 ) the resultant signal is generally depressed by 20–30% relative to that for As(III) (7). Another disadvantage of HG is the numerous sources of interferences during hydride generation (8, 9). The task of the present paper was to find possible ways to increase sensitivity and detection limits of an already working system that has been developed for the speciation of four arsenic (1). The LOD of the referred system for As(III), As(V), DMAs, and MMAs were 35, 50, 20, and 20 ng, respectively. Hydride generation as a promising derivatization technique has given us the hope to improve this LOD by at least an order of magnitude (10, 11, 14). EXPERIMENTAL
Instrumentation A Shimadzu Model LC-7A HPLC pump was attached to a sample injection valve (sixport rheodyne system, LMIM, Hungary). A 250-mm3 sample loop was used for sample introduction. The analytical column was a Bio Separation Technologies (BST) C18 Rutin column (25 1 4.6 mm i.d., 10-mm particle size). A four-channel peristaltic pump (Ismatec MS-CA 4, Switzerland), a 100-cm long mixing coil, and the aerosol chamber of a Cetac Model 5000 Ultrasonic Nebulizer (CETAC, Omaha, NE) functioned as a continuous hydride generator. The piezoelectronic transducer of the USN was not in use during
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FIG. 1. Schematic diagram of the HPLC–HG–(USN)–AFS system used for arsenic speciation.
analysis. A Cetac Model 2050 ‘‘Heated and Refrigerated Constant Temperature Bath’’ provided the required conditions for desolvation. Removal of the drain from the nebulizer was carried out with a peristaltic pump (Raimin Instruments Co., Wolburn, MA). The nebulizer was further connected to an AFS (PSA Excalibur, PS Analytical, Sevenoaks, Kent, UK) that utilized an arsenic boosted-discharge hollow cathode lamp (Superlamp, Photron, Victoria, Australia) as an excitation source. Measurements were carried out around the resonance wavelength of arsenic (193.7 nm) using a multi-reflectance filter having a spectral bandpass between 20 and 40 nm. Argon functioned as a carrier gas in the USN, and mixed with the hydrogen, supported the diffusion flame. The constant gas flows were maintained by Cole–Palmer rotameters (Niles, IL). Data collection and evaluation was totally automated by Borwin Chromatographic software (JMBS, Grenoble, France). All peaks were evaluated by their peak height. A schematic diagram of the system is shown in Fig. 1. Reagents Arsenite stock solution was prepared by dissolving 1.320 g of As2O3 in 25 cm of 0.5 mol dm03 NaOH solution and then diluting the solution to 1 dm3 with 0.6 mol dm03 HCl. AsV stock solution was obtained from Merck. MMAs stock solution was prepared from strychrotonin solution (Chinoin, Budapest), and the DMAs was obtained from Fluka. The 1000 mg dm03 stock solutions of MMAs, DMAs, AsIII, and AsV were further diluted in deionized water daily. The DDAB solution (0.01 mol dm03) was prepared by adding 0.5% (v/v) methanol and 0.1% (v/v) of 0.01 mol dm03 DDAB solution to the Na2HPO4 buffer solution (20 mmol dm03). The pH was set to 6.0 by the addition of NaH2PO4 solution containing the same amount of phosphate, methanol, and DDAB as the eluent. Chemicals for hydride generation. The (2% (m/v)) sodium tetrahydroborate solution was prepared daily by dissolving NaBH4 powder (Aldrich) in 0.5% (m/v) NaOH solution, and the HCl was obtained from Reanal (Hungary). Reanal (Hungary) potas-
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sium iodide and sodium thiosulfate were used for preparing the 0.1, 1, and 10% solutions for reducing As(V) to As(III). Procedures Column modification. Column modification was carried out according to procedure described elsewhere (12). Arsenic speciation. 250 mm3 of the working standard solutions of a mixture of arsenic species is injected directly onto the HPLC column through the injection port (Fig. 1). After the separation part of the system, first HCl and then NaBH4 solutions are introduced into the stream of the column effluent. Hydride generation takes place in a mixing coil, and the generated gas phase is separated from the liquid phase continuously in the aerosol chamber of the USN. The transducer of the ultrasonic nebulizer was not in use during our measurements; the nebulizer in this case functioned only as a gas–liquid separator and desolvation system. A continuous stream of argon transfers the generated hydrides and hydrogen from the nebulizer to the AFS. The hydrogen–argon diffusion flame is maintained by externally introduced hydrogen where the hydrogen gas enters the system via a Y-shape connection tube about 25
TABLE 1 Operating Conditions for the HPLC–HG–(USN)–AFS System
Column Column temperature Sample loop size Mobile phase Pump flow rate
Chromatography BST C18 Rutin column (25 1 4.6 mm i.d., 10-mm particle size) 247C 250 mm3 20 mmol dm03 Na2HPO4 –NaH2PO4 buffer (pH 6.0) / 0.1% (v/v) of 1002 mol dm03 DDAB / 0.5 % (v/v) methanol 1 ml min01 Hydride generation conditions 2% (m/v) NaBH4 solution dissolved in 0.5% (m/v) NaOH (1.7 ml min01) 1.5 mol dm03 HCl (1.7 ml min01) Ultrasonic nebulizer
Heating temperature Cooling temperature
1407C 57C Atomic fluorescence detector
Primary current Boost current
27.5 mA 35 mA Gas flow rates
Argon Hydrogen
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cm from the flame. (Tube lengths were reduced as much as possible to minimize dead volumes.) All chromatographic peaks were evaluated by their peak heights. Experimental conditions for hydride generation were optimized, and the parameters can be seen in Table 1. RESULTS AND DISCUSSION
As described in the Experimental section, in the present system we utilized only the desolvation power of the USN. The piezoelectronic transducer was turned off during analysis because the nebulization process was not essential; the HG technique converts the analyte into vapor phase. The small and cold hydrogen diffusion-flame in the AFS apparatus demands the analyte to be in a gaseous form since the flame cannot handle a large solvent load. The HG beside that which forms gaseous analyte has another benefit; it produces enough hydrogen in the chemical reaction to maintain the flame (13). To reduce the instability of the system we used externally introduced hydrogen (gas flow rates in Table 1 correspond only to the externally introduced gas, and do not include the amount that has been formed as a result of chemical reaction). The aerosol chamber of the nebulizer functioned as a gas–liquid separator, and the nebulization section of the U-500 USN was used without any change in the original design. The performance of gas–liquid separation was fairly good: no memory effect has been found. As the analyte enters into the face-plate of the transducer through a glass sample tube, separation takes place in the nebulization chamber according to the rules of gravitation. The gaseous hydrides and hydrogen had already been formed in the mixing coil and as they entered into the chamber the carrier gas (Ar) transported them toward the desolvation section of the system. The liquid drain is carried away from the bottom of the vessel by a peristaltic pump. The construction of the system provides a water trap layer as well. Several home-made design gas–liquid separators have been tried but they all suffered from strong sources of noise because of pulsation caused by the inadequate overflow. Effect of Hydride Generation Conditions on Net Signal The optimal working conditions for the HPLC–USN–AFS system have already been determined (1) and these parameters served as starting points for our further investigation. The efficiency of HG depends primarily on the concentrations of HCl and NaBH4 . In the present system the flow rates of these components were kept constant at 1.7 ml/min. According to our previous investigations and the literature, the following conditions have been selected for the determination of AsIII, AsV, DMAs, and MMAs by HG: NaBH4 (mol dm03) 03
HCl (mol dm )
0.25
0.5
1.5
0.75
1.5
3.0
Results of the signal optimization of four arsenic species at different HG parameters can be seen in Figs. 2A, 2B, and 2C. It can be concluded from the figure that the efficiency of HG in the cases of As(III) and MMAs is virtually independent from the
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FIG. 2. Influence of hydrochloric acid (mol dm03) and sodium borohydrate (A, 0.5; B, 1.0; C, 2.0% (m/v)) concentrations on AsIII (1), DMAs (2), MMAs (3), and AsV (4) signal intensities at 1 mg dm03 injected concentrations.
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MESTER, WOLLER, AND FODOR TABLE 2 Comparison of LODs of Different Systems for Arsenic Speciation
Species
LC– ICP/AES
LC–HG– ICP/AES
LC–ICP/MS
LC–USN–AFS
LC–HG– (USN)–AFS
As (III) As (V) MMAs DMAs
120 130 100 130
0.26 0.96 1.3 0.98
0.01 0.03 0.02 0.02
35 50 20 20
2.5 6 2 3.2
Note. Numbers represent ng of arsenic14.
concentrations of the reductant and the acid. The increasing NaBH4 concentration hardly increased the signal intensities, and with increasing acid concentration, no significant change was measured during the experiment. Regarding the other two compounds (As(V) and DMAs), the concentration of the acid and reductant had a strong effect on the net signal. A direct proportion has been observed between the concentration of NaBH4 solution and the efficiency of HG in the case of these components. Arsenic (V) responded with a higher signal to the higher HCl concentrations, whereas the signal of DMAs decreased in response to higher HCl concentrations. As a result of the HG reagent optimization we can conclude that in the case of the determination of all four arsenic species only a compromised optimum can be achieved. At the determination of either methylized or inorganic compounds in separate systems real optimum exists regarding the HG conditions. As a result of our experiments for running the system we have selected the concentrations of 1.5 mol dm03 for HCl (1.7 ml min01) and 2% (m/v) for NaBH4 (1.7 ml min01). The effect of hydrochloride acid on the appearance of the chromatograms is presented in Fig. 3. From the top the concentrations of HCl are 0.75, 1.5, and 3.0 mol dm03. No change in the intensity of As(III) and MMAs was noticed as the HCl concentration was increased; the intensity of the DMAs signal eluting second decreased and the intensity of the As(V) signal increased. It is important to note that the reproducibility of retention times of the species is extremely good in spite of the manual mode of injection. According to the literature (7, 9), the reduction of arsenic (V) to arsine is slower than that of As(III). In batch systems this results in peak shift and in 20–30% lower peak absorbance. In continuous systems Welz (9) has found sensitivity for As(V) an order of magnitude lower because of the kinetically slower reaction. A lot of studies have discussed the possible ways of eliminating this type of interference, however the cold flow-injection systems were not often investigated before. The majority of pre-reduction techniques applied for As(V) reduction were heated systems. In our experiments we investigated the effect of added potassium iodide (KI) (0.1, 1, and 10%) and sodium thiosulfate (0.1, 1, and 10%) on the signal of As(V) in a cold flow-injection system. The sodium thiosulfate solution was introduced directly into the eluent stream in two ways, before and after the introduction of the HCl solution. The KI solution was introduced together with the acid. Our conclusion was that in the given system, the above mentioned pre-reduction
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FIG. 3. The effect of different concentrations of hydrochloride acid solutions on the appearance of the chromatograms at 2% (m/v) NaBH4 concentration. From the top toward the bottom the HCl concentrations are 0.75, 1.5, and 3.0 mol dm03. The four arsenic species (identified by their retention times (RT)) are the following: RT Å 3.4 As(III), RT Å 4.3 DMAs, RT Å 5.9 MMAs, and RT Å 12.4 As(V).
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FIG. 4. HPLC–HG–(USN)–AFS chromatogram of the four investigated arsenic species under optimal working conditions. Peak identification: RT Å 3.88 As(III), RT Å 4.89 DMAs, RT Å 6.6 MMAs and RT Å 10.91 As(V). The concentration of injected arsenic species were 1 mg dm03.
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steps weren’t successful, the KI had no effect, and, instead of increasing the signal of As(V), the sodium thiosulfate has decreased it. We are planning to apply these pre-reduction steps in a heated flow-injection system in the future. Analytical Conditions The LOD (last column of Table 2) was calculated on the basis of 3 S criterion using the results of from nine replicated measurements. Linearity of the system for the four investigated arsenic species was in the range 25–1000 ng. The chromatogram of the four arsenic species can be seen in Fig. 4. The flow rate was kept at 0.8 ml/min during the elution of the first three peaks and after them it was adjusted to 2 ml/min to a give better appearance to the chromatogram. The resolution of the first three components could be further improved by changing the eluent conditions although in the case of peak height evaluation this resolution power proved to be satisfactory. The lower sensitivity of As(V) compared to As(III), MMAs, and DMAs could be explained by the lower efficiency of hydride generation. CONCLUSIONS
Comparison of LODs of different systems for arsenic speciation are shown in Table 2. The data are in good accordance with the literature that presents some orders of magnitude enhancement in many cases when using HG. As we expected, the LODs of the present system improved an order of magnitude compared to results measured in a system without HG (last two columns of Table 2). The HPLC–HG–(USN)– AFS system is a fairly robust and stable system with a very simple mode of detection. The cost of the system is much less than the cost of the commonly used sophisticated methods (ICP, ICP-MS detectors). Routine analyses of environmental and biological samples are possible on the system because the LODs are in the required range in most cases. Unfortunately, with the introduction of HG the possible sources of interferences increased. The origin of these interferences might be the liquid phase (hydride generation kinetics, generation efficiency) or the gaseous phase (transport kinetics, transport efficiency); gaseous phase interference can take place during the atomization process as well. The investigation of this phenomenon and to find solutions to minimize the interferences are the next steps in the development of the HPLC–HG–(USN)–AFS system. ACKNOWLEDGMENT This research was partly supported by the Hungarian Scientific Research Foundation (OTKA) Grant T014329.
REFERENCES 1. 2. 3. 4. 5. 6.
´ .; Mester, Z.; Fodor, P. J. Anal. At. Spectrom. 1995, 10, 629. Woller, A Holak, W. Anal. Chem. 1969, 41, 1712. Robbins, W. B.; Caruso, J. A. Anal. Chem. A. 1979, 51, 889. Braman, R. S.; Justen, L. L.; Foreback, C. C. Anal. Chem. 1972, 44, 2195–2199. McDaniel, M.; Shendrikar, A. D.; Reiszner, K. D.; West, P. W. Anal. Chem. 1976, 48, 2240–2243. Thompson, M.; Pahlavanpour, B.; Walton, S. J.; Kirkbright, G. F. Analyst 1978, 103, 568–579.
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7. Francesconi, K. A.; Edmonds, J. S.; Morita, M. In Arsenic in the Environment, Part I: Cycling and Characterization, ed. (J. O. Nriagu, Ed.), Vol. 9, pp. 189–219. Wiley, New York, 1994. 8. Smith, A. E. Analyst 1975, 100, 300–306. 9. Welz, B.; Stauss, P. Spectrochim. Acta B. 1993, 48, 951–976. 10. Nakahara, T. Prog. Anal. Atom. Spectrosc. 1983, 6, 163. 11. Huang, B.; Zhang, Z.; Zeng, X. Specrochim. Acta B. 1987, 42, 129–137. 12. Liu, Y. M.; Ferna´ndez Sa´nchez, M. L.; Gonza´lez, E. B.; Salz-Mendel, A. J. Anal. At. Spectrom. 1993, 8, 816. 13. Corns, W. T.; Stockwell, P. B.; Ebdon, L.; Hill, S. J. J. Anal. At. Spectrom. 1993, 8, 71. 14. Quevauviller, Ph.; Maier, E. A.; Griepink, B. (Eds.) Quality Assurance for Environmental Analysis, pp. 288–303. Elsevier, Amsterdam, 1995.
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