Simultaneous Arsenic and Chromium Speciation by HPLC/ICP-MS in Environmental Waters

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1 Simultaneous Arsenic and Chromium Speciation by HPLC/ICP-MS in Environmental Waters Introduction Many elements can exist in a variety of oxidation states which have differing impacts on health and the environment. As a result, it is necessary to quantify the individual oxidation states of these elements within a sample rather than simply the total element content for an accurate assessment of their impact. Two elements in this category are chromium and arsenic. Trivalent chromium (Cr III) is an essential nutrient, while hexavalent chromium (Cr VI) is toxic, does not occur naturally, and results only from anthropogenic activities. Arsenic exists in a variety of forms with the trivalent form (As III) being the most toxic, followed by the pentavalent form (As V). Other common forms of arsenic include organically bound monomethyl arsenic (MMA), dimethyl arsenic (DMA) and arsenobetaine (AsB). The speciation and quantification of both chromium and arsenic are best accomplished by using HPLC to separate the species and ICP-MS to detect them. However, HPLC separations can take ten minutes per element and require different mobile phases to separate different elements. Under these constraints, each sample would have to be analyzed twice to determine both the chromium and arsenic species present. A significant problem encountered when attempting to measure low levels of chromium and arsenic in environmental waters is matrix interference. Carbon, chloride and calcium are common elements typically found in these types of samples. Carbon (ArC + ) interferes with the chromium isotopes, while chloride (ArCl +, CaCl + ) interferes with the only arsenic isotope. Consequently, elimination of these interfering matrix components is required to enable low-level detection of chromium and arsenic. The goal of this work was to develop a method for simultaneous chromium and arsenic speciation in environmental water samples in less than five minutes. With this approach, each sample would only have to be analyzed once. ICP-MS with a Dynamic Reaction Cell (DRC ) was chosen as the detector because of its ability to provide the lowest possible detection limits by eliminating the effects of common interferences. It should be noted that a current concern regarding speciation is the pre-analysis preservation of species in a sample. It is known Authors Kenneth R. Neubauer, Wilhad M. Reuter, Pamela A. Perrone, Zoe A. Grosser PerkinElmer Life and Analytical Sciences 710 Bridgeport Avenue Shelton, CT USA HPLC / ICP-MS A P P L I C A T I O N N O T E

2 that several species readily interconvert between oxidation states so that the concentration of species analyzed is not necessarily representative of that which exists in the environment. This is a separate area of research and debate and beyond the scope of this work. Experimental HPLC Conditions Separation was accomplished using the PerkinElmer Series 200 Binary HPLC Pump, Autosampler and Vacuum Degasser. A 3 cm Pecosphere column with 3 µm C8 packing (Part No , PerkinElmer Life and Analytical Sciences, Shelton, CT USA) was used for the separation. Other details of the HPLC conditions are given in Table 1. The mobile phase consisted of 1mM tetrabutylammonium hydroxide (TBAH) and 0.5 mm ethylenediaminetetraaceticacid dipotassium salt dihydrate (EDTA) (Aldrich Chemical Company, Milwaukee, WI USA), and 5% methanol (Fisher Scientific, Pittsburgh, PA USA). The ph was adjusted to 7.2 using dilute nitric acid and ammonium hydroxide (Fisher Scientific). All solutions were made up in 18 MΩ distilled deionized water. These conditions were chosen because they provided the best compromise between peak shape and retention time for all of the chromium and arsenic species investigated. Prior to analysis, the column was conditioned for 30 minutes with the mobile phase flowing at 1.5 ml/min; this was required to properly equilibrate the column. Upon completion of analyses at the end of each day, the column was washed for 15 minutes with a 5/95 mixture of methanol/water to remove the buffer/ salts from the column, followed by a 15 minute wash with a 70/30 methanol/water mixture to prevent the column from drying out. For overnight storage, the column remained connected to the HPLC system; the ends of the column were capped for long-term storage. ICP-MS Conditions Detection of chromium and arsenic species was accomplished with an ELAN DRC II (PerkinElmer SCIEX, Concord, ON Canada); detailed instrumental conditions are given in Table 2. Oxygen was chosen as the reaction gas because it reduces the ArC + interference on Cr + at m/z 52 and reacts readily with As + to form AsO + (m/z 91), a new species which does not suffer from ArCl + and CaCl + interferences at m/z 75. Table 1. HPLC Conditions HPLC System Column Mobile Phase Total chromium and arsenic concentrations were also measured without speciation, using conventional nebulization into the ICP-MS. For these analyses, all instrumental parameters were the same as for the speciation analysis except for the reaction-cell conditions, which are shown in Table 3. The reaction-cell conditions differ between analyses because of the different matrices involved: for speciation, the matrix is the mobile phase; for total analyses, the matrix is the water samples. Standard and Sample Preparation All standards and samples were prepared in a solvent mixture similar to the mobile phase PerkinElmer Series 200 Binary Pump, Autosampler and Vacuum Degasser Pecosphere C8; 3 µm particles; 3 cm 1 mm TBAH mm EDTA (potassium salt) + 5% methanol ph 7.2 ph Adjustment Dilute HNO 3, NH 4 OH Injection Volume 50 µl Flow Rate Auto Sampler Flush Solvent Table 2. ICP-MS Conditions Instrument Nebulizer Spray Chamber RF Power 1.5 ml/min 5% methanol ELAN DRC II (PerkinElmer SCIEX) Quartz Concentric Quartz Cyclonic 1500 W Analytes Cr + (m/z 52); AsO + (m/z 91) Reaction Gas RPq 0.55 Dwell Time Analysis Time O 0.6 ml/min 500 milliseconds (per analyte) 150 seconds Table 3. Reaction Cell Conditions for Total As and Cr Determination Analyte m/z Reaction Gas Flow (ml/min) RPq Cr 52 NH AsO 91 O

3 (1 mm TBAH mm EDTA; ph = 7.2) and allowed to sit for at least 30 minutes prior to analysis. Samples were diluted by at least a factor of two. Higher dilution factors were used for certain samples containing high analyte levels, so that the final concentration was within the range of the calibration curve. It was necessary for the diluted samples to sit for at least 30 minutes in mobile phase so that all species were equilibrated with the mobile phase. All standards were made by dilution of the following 1000 mg/l stock solutions: chromium (III) and arsenic (V) (PE Pure, PerkinElmer Life and Analytical Sciences) and chromium (VI) and arsenic (III) (SpexCertiprep, Metuchen, NJ USA). Samples were obtained from various sources (see Tables 4 and 5) and collected in plastic bottles (polyethylene or polypropylene), without acid preservation. Lake and river waters were filtered by gravity through Whatman 40 filter paper prior to analysis to remove particulate material. Bottled waters were purchased in a local grocery store and allowed to degas at room temperature. Residential waters were collected directly from faucets of private homes. Figure 1. Chromatogram of four chromium and arsenic species separated and quantitated in a single analysis. Each species is present at 1 µg/l. Results and Discussions Figure 1 shows a chromatogram of 1 µg/l chromium and arsenic standards. These selected ion chromatograms were acquired simultaneously by monitoring two different masses on the ICP-MS: m/z 52 for Cr + and m/z 91 for AsO +. This figure clearly shows that all four species can be detected simultaneously within a single analysis in less than three minutes. For these studies, oxygen was chosen as the reaction gas for the removal of ArC +. Although oxygen reacts readily with ArC +, it is known that ammonia is more efficient at removing this interference. However, for this study, ammonia was not used since it also reacts strongly with As +, thus removing it from the ion stream. Therefore, the elevated chromium baseline that was observed in Figure 1 resulted from the incomplete removal of ArC + as well as chromium contamination in the mobile phase. Although the elevated baseline may appear to be problematic, it did not affect the signal-to-noise ratio (S/N) for the chromatographic peaks. Subsequent data show that 100 ng/l Cr could still be detected. Figure 2. Chromatogram of Glendale, CA municipal water sample. The concentrations shown are those read by the instrument; they are not corrected for dilution. Calibration curves were established with 0.5, 1.0 and 5.0 µg/l standards; each species yielded an R 2 > 0.999, thus demonstrating the linearity of the technique. Peak areas were used for all quantitative measurements. Sample chromatograms appear in Figures 2-4 which are representative of the samples analyzed. Figure 2 shows Figure 3. Chromatogram of a residential well-water sample. The concentration shown is that read by the instrument; it is not corrected for dilution.

4 a municipal water supply sample that contains a high level of chromium and low levels of arsenic; Figure 3 is a residential well-water sample containing elevated arsenic and low-level chromium; Figure 4 is another municipal water supply sample containing low levels of both chromium and arsenic. Tables 4 and 5 summarize the results from the various water samples studied which included: two environmental sources, two municipal water supplies, a residential well and three different commercially available bottled waters. These data show good agreement between the speciated results (as determined by HPLC/ICP-MS) and total elemental analyses (measured by ICP-MS without the HPLC) even at low levels, thus demonstrating the overall effectiveness of the method. Figures 5 and 6 show chromium and arsenic chromatograms obtained from 3 residential well waters in the same city (Danbury, CT USA). Figure 5 shows that the amount and species of chromium vary among the residences, while Figure 6 demonstrates that each water supply contained only As (V), all at about the same quantity. These results show how the levels and species of chromium and arsenic can vary within a community. An interesting aspect of Figure 6 is that the As (V) peaks do not occur at the same retention time, a reproducible phenomenon. When a total-metal analysis was performed on these samples, it was noticed that they contained differing levels of minerals (i.e., Na, Mg, K, Ca, Fe) with Water 1 having the highest mineral content and Water 3 containing the lowest level. A subsequent study was performed to explore the effect of salt concentration on the retention times of the chromium and arsenic species. Chromium and arsenic standards (at 1µg/L) were made in 25, 100 and 500 mg/l sodium chloride solutions and analyzed. The results appear in Figures 7 and 8 and show that the salt concentration of a sample affects the retention times of Cr (III) and As (V). As the salt concentration increases, the retention times of Cr (III) and As (V) increase, while the retention times of Cr (VI) and As (III) are unaffected. This trend explains the observation in Figure 6. The effect of salt concentration can probably be mitigated by increasing the ionic strength (i.e., adding a buffer) to the mobile phase. This will be the focus of a future study. Two other common arsenic species usually studied along with As (III) and As (V) are monomethyl arsenic (MMA) and dimethyl arsenic (DMA). Using a chromatographic method which separates these four arsenic species, it was found that MMA and DMA were not present in any of the water samples analyzed. Therefore, the focus of this work included only the arsenic species which were observed: As (III) and As (V). Figure 4. Chromatogram of Shelton, CT municipal water sample. The concentrations shown are those read by the instrument; they are not corrected for dilution. Table 4. Chromium Results Sample Cr (III) Cr (VI) Total Cr (µg/l) (µg/l) (µg/l) Connecticut River 0.07 Lake Mohegan Shelton, CT Water Glendale, CA Water Oxford, CT Water Bottled Water A Bottled Water B Bottled Water C = None detected Table 5. Arsenic Results Sample As (III) As (V) Total As (µg/l) (µg/l) (µg/l) Connecticut River Lake Mohegan Shelton, CT Water Glendale, CA Water Oxford, CT Water Bottled Water A Bottled Water B Bottled Water C = None detected 4

5 Conclusions This study demonstrated the feasibility of rapid, simultaneous chromium and arsenic speciation in environmental waters using HPLC/ICP-MS. By careful selection of chromatographic conditions, Cr (III), Cr (VI), As (III) and As (V) can be separated in a single run in under three minutes. Low-level detection is accomplished using DRC ICP-MS to eliminate the effects of interferences on chromium and arsenic. This method was then applied to a variety of environmental water samples from rivers, lakes, municipal water supplies and residential well waters, as well as from bottled waters. Chromium and arsenic levels below 100 ng/l were measured, as shown in the chromatograms of water samples. Judging from the signal-tonoise ratios observed in these chromatograms, lower levels can probably be detected. The results from this work demonstrated that this method can serve as a rapid screening tool to quantitatively determine these species in a number of water sources. Figure 5. Chromium chromatograms from three residential well waters in Danbury, CT. Figure 6. Arsenic chromatograms from three residential well waters in Danbury, CT. Figure 7. Effect of salt concentration on chromium speciation. Each chromium standard is present at 1 µg/l. Figure 8. Effect of salt concentration on arsenic speciation. Each arsenic standard is present at 1 µg/l. 5

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