Determination of Arsenic and Bismuth in Biological Materials by Total Reflection X-ray Fluorescence After Separation and Collection of Their Hydrides JU� RGEN MESSERSCHMIDT, ALEX VON BOHLEN, FRIEDRICH ALT AND REINHOLD KLOCKENKA� MPER* Institut fu� r Spektrochemie und Angewandte Spektroskopie (ISAS), Bunsen-KirchhoV-Str. 11, D-44139 Dortmund, Germany A combined procedure was developed for the determination of EXPERIMENTAL As and Bi in biological materials by total reflection X-ray Accessories fluorescence (TXRF).The materials were first digested by an open wet decomposition method. The separation of both Equipment elements from the sample matrix was then achieved by For sample decomposition an open digestion device was used, generation of their volatile hydrides and subsequent trapping in consisting of an aluminium heating block with drill-holes for a collection solution. The quantitative determination of As and the insertion of 10 ml quartz glass tubes.The separation and Bi was performed simultaneously by TXRF using Y as collection of the element hydrides was carried out in a laborainternal standard. The procedure was applied to several tory-built hydride generation and collection system (Fig. 1), diVerent biological certified reference materials. The recovery consisting of a peristaltic pump (LKB 2132, Pharmacia, rate determined for mass fractions between 40 ng g-1 and Freiburg, Germany), a 10 ml quartz glass tube, a system for 10 mg g-1 was 90–100% after correction with aqueous the supply of NaBH4 solution and carrier gas (N2), glass standard solutions.The RSD for the whole procedure ranged capillary tubes (Blaubrand intra end 2 ml and intra mark 50 ml; from 8 to 14% and the detection limits were about 10 ng g-1 Brand, Wertheim, Germany), an angular tube, a collection for As and 20 ng g-1 for Bi. vessel (1.5 ml Eppendorf-type vessel ) and a heating device (Reacti-Therm 18800, Pierce, Rockford, IL, USA).Keywords: Arsenic; bismuth; hydride generation; total reflection X-ray fluorescence; biological materials An EXTRA II TXRF spectrometer, equipped with Mo and W tubes for X-ray excitation (Rich. Seifert & Co., Ahrensburg, Germany) and a QX 2000 Si(Li) detector and analyzer (Link; Oxford Instruments, High Wycombe, Buckinghamshire, UK) Sample solutions to be prepared for analysis by total reflection including a software package were used to record and process X-ray fluorescence (TXRF) have to fulfil certain conditions in the spectra.order to make use of the high sensitivity of this technique. The solutions after sample decomposition should contain few matrix residues and the sample solvent (e.g., water, nitric acid) Reagents should have high volatility. About 10 ml of the final solution All reagents used were of analytical-reagent or high-purity have to be pipetted onto clean quartz glass carriers and dried grade (Merck, Darmstadt, Germany).Water was de-ionized by evaporation. A dry residue of only a few micrograms or by a Milli-Q water purification system (Millipore, Bedford, less deposited as a thin layer several micrometres thick and MA, USA). High-purity grade nitric acid was prepared by several millimetres in diameter has to be applied for TXRF measurements. The detection limits of the method can deteriorate by up to three orders of magnitude if the matrix content is too high.1 However, such interference can be reduced by using suitable techniques of analyte/matrix separation.Apart from extraction or chromatographic separation, which often only reduces the problem, volatilization of the analyte and its collection in a suitable solvent can be a successful approach. In this paper, a hydride separation/collection technique and its adaptation to TXRF is described. It is well known that a number of elements (e.g., As, Se, Sb, Bi) can be reduced to their corresponding hydrides.These can be transferred via a carrier gas stream (nitrogen, argon) into a heated quartz tube, positioned in an atomic absorption spectrometer. Another possibility is the coupling of a hydride generation system to an inductively coupled plasma spectrometer. These techniques have been well known for many years2–5 and used successfully in routine analysis.6,7 A combination of hydride generation, collection of the volatile hydrides in a suitable solvent and Fig. 1 Hydride generation and collection system: (a) 10 ml quartz multi-element determination by TXRF was first proposed by glass tube (with digested sample material) and introduction system for HaVer et al.,8 but the method was only applied to aqueous NaBH4 solution and carrier gas, (b) angular tube, (c) glass capillary standard solutions.A similar technique is described in this (Blaubrand intra mark, 50 ml ), (d) glass capillary (Blaubrand intra paper and was applied to biological certified reference end, 2 ml ), (e) collection vessel (1.5 ml Eppendorf-type vessel ), (f ) heating device (105–110 °C) and (g) silicone rubber tubing.materials. Journal of Analytical Atomic Spectrometry, November 1997, Vol. 12 (1251–1254) 1251sub-boiling distillation (H. Ku� rner, Rosenheim, Germany) of was placed in a collection vessel. The vessel was placed in the heating device of the hydride generation/collection apparatus analytical-reagent grade acid, and then stored in quartz glass bottles.shown in Fig. 1 and pre-heated for 30 s (block temperature 105–110 °C). The quartz glass tube with the digested sample For the hydride generation, a NaBH4 solution was prepared by dissolving 3 g of NaBH4 (Merck) in 100 ml of 0.01 M NaOH. solution was connected to the collection vessel by capillaries. By means of a peristaltic pump, the NaBH4 solution was This solution was prepared just before use and was stored in a polyethylene bottle. pumped into the quartz glass tube (60 ml h-1, 45 s).The volatile hydrides were transported to the collection vessel by Stock standard solutions (1000 mg l-1) of As, Se, Sb and Bi were prepared from Merck Titrisol solutions; a stock standard a stream of nitrogen which acted as a carrier gas (20 ml min-1). The nitrogen was allowed to flow through the apparatus for a solution of Y (1000 mg l-1) was purchased from Aldrich (Milwaukee, WI, USA). Working standard solutions of each further 90 s in order to complete the hydride collection.The collection vessel was then removed from the heating device element were prepared daily from the stock solutions. and closed. Samples TXRF measurements and quantitative determinations First, the procedure (hydride generation, collection and TXRF Aliquots of 30, 50 or 100 ml of the collection solution were measurement) was tested using aqueous standard solutions dried on quartz glass carriers by means of IR radiation at a prepared by dilution of stock standard solutions.Subsequently, temperature of about 80 °C. TXRF spectra were recorded in a standard or certified reference materials (SRMs or CRMs) preset live time of 300–1000 s depending on the analyte concen- were subjected to the whole procedure including their tration in the sample. Yttrium was used as an internal standard decomposition by an open digestion method. The following element for quantification. The mass of the analyte element materials were chosen: Bovine Liver (NIST SRM 1577a), present in the collection solution was determined from the Orchard Leaves (NIST SRM 1571), Tomato Leaves (NIST following equation: SRM 1573), Plankton (BCR CRM 414) and Tea Leaves (NIES No. 7). All the materials were supplied by Promochem (Wesel, Germany). mx= Nx/Sx NY/SY ·mY (1) where m indicates the mass, N the measured net intensity and Procedure S the relative sensitivity of either the analyte, x, or the internal The whole procedure consists of the following steps: (i) open standard, Y.9 The sensitivity values were known from previous wet decomposition of the samples, (ii ) generation of the investigations on aqueous standard solutions;10 the mass mY hydrides AsH3, SeH2, SbH3 and BiH3 and their collection in was chosen to be 4 ng.The mass fraction cx of the analyte in a small volume of a collection solution and (iii ) measurement the chosen sample material was then determinedby TXRF. quotient of mx and the weighed portion of the sample material (80–100 mg).A raw recovery rate was then calculated from Open wet decomposition the ratio of the value of cx to the certified value. The recoveries of As and Bi were high but mostly ,90%. Portions of 80–100 mg of a biological sample material were To correct for the incompleteness of hydride generation and weighed in 10 ml quartz glass tubes. To soak the dry samples, collection, aqueous standard samples of As and Bi in 1 M HCl 500 ml of de-ionized H2O were added to each tube.After a few were prepared by addition of these elements and measured in minutes, 500 ml of HNO3 (65%) and 40 ml HClO4 (70%) were parallel with the real samples (the whole procedure except pipetted into the tubes. In a first step, the tubes were heated decomposition). The addition of As and Bi was made so as to in the aluminium heating block from room temperature to give concentrations in the aqueous standard samples compar- 140 °C within 60–70 min.The tubes were then allowed to cool able to the mass fractions of these elements in the real samples. outside the block and 500 ml of HNO3 together with 100 ml of The recoveries of As and Bi in the aqueous standard samples HClO4 were added. In a second step, the tubes were again were finally used as scaling factors in order to correct the mass placed in the heating block and the temperature was increased fraction cx and the raw recovery rate for the real samples by from 140 to 180 °C within 60–70 min.If the digested sample normalization. solutions exhibited a dark colour (undigested material), 400 ml of HNO3 were added and the temperature was maintained at RESULTS AND DISCUSSION 180 °C until the solutions became nearly colourless. In a third step, the temperature was increased from 180 to 210–215 °C in Optimization of Hydride Generation and Collection 10 min. This temperature was maintained for a further 10 min In view of the simultaneous multi-element capability of TXRF, taking care that the solutions did not evaporate to dryness.an attempt was made to separate and collect as many elements During the described procedure the excess of nitric acid as possible via their hydrides. The eYciency of an open wet evaporated totally. The colourless residues were dissolved in decomposition method had already been tested in earlier the small volumes of HClO4 that remained but did not interfere experiments with tea leaves.11 Therefore, this type of digestion with the following hydride generation process.The temperature was adapted to the above-mentioned biological materials with and time schedules given above had to be followed exactly to only small changes. It was necessary to optimize the conditions ensure complete mineralization without losses of As and Bi. for hydride generation of the elements and for collection of Finally, the quartz glass tubes were removed from the the hydrides in a collection solution.The usual procedure of aluminium heating block and cooled. The digestion residues drying the solutions on TXRF quartz glass carriers and the were diluted with 400 ml of 5 M HCl after which 1.6 ml of H2O standard settings for TXRF measurements could then be were added. applied. The optimization was first performed with aqueous standard Generation and collection of volatile hydrides of As and Bi (Se solutions of the elements in question (10–100 ng ml-1 As, Se and Sb) and Sb in 1 M HCl) without any digestion step.Only those reagents that could be dried on TXRF quartz glass carriers A mixture of 250 ml of HNO3 (65%), 250 ml of HCl (37%) and 20 ml of Y standard solution (4 ng Y absolute in HNO3, 65%) without leaving any interfering residues were used as collection 1252 Journal of Analytical Atomic Spectrometry, November 1997, Vol. 12Fig. 2 Influence of the temperature of the collection solution [HNO3 (65%)–HCl (37%)] on the recovery of As.solutions. The Se hydride was quantitatively collected in 0.1 M ammonia solution and 0.01 M NaOH. This reaction was con- Fig. 3 Determination of As (47 ng g-1) and Bi (50 ng g-1 spiked) in trolled by the determination of Se (electrothermal atomic Bovine Liver (SRM 1577a). (a) TXRF spectrum for direct determiabsorption spectrometry). However, drying the collection solu- nation in the digested solution; (b) TXRF spectrum for determination after hydride separation and collection.The preset live time for both tion on a quartz glass carrier hampered the quantitative and spectra was 300 s. reproducible recovery of the volatile Se by TXRF measurements. Pd and Hg salts were added to the collection solutions as to high recovery rates and low relative standard deviations chemical modifiers in order to reduce the volatility of Se. Such (RSDs) for As and Sb, but not for Se. additions, however, resulted in a new interfering matrix which reduced the sensitivity of TXRF determinations.Application to Biological Materials An increased recovery rate was achieved by using strong oxidizing agents such as a mixture of HNO3 and H2O2 or Reference materials (RMs) were used in order to test the HNO3 and HCl. The latter acidic mixture used as a collection separation/collection technique for biological samples. In solution provided high recovery rates combined with high addition to As, Se and Sb, the element Bi was included in the precision and accuracy.Fig. 2 demonstrates the influence of investigations. First, Bovine Liver (NIST SRM 1577a) was the temperature of this solution on the recovery of As. The chosen in order to demonstrate the advantages of the proposed highest value was found for a temperature of 110 °C in the sample pre-treatment. As (47 ng g-1) and Bi (50 ng g-1 spiked) heating block. The temperature could not be increased further were determined by TXRF (a) directly in the diluted decompobecause of the instability of the polypropylene collection vessels sition solution and (b) after the described hydride generation/ used.At 110 °C, the recovery rate for As was about 80%, for collection procedure. The diVerences between the correspond- Sb about 90%, but for Se only about 10%. The velocity at which the hydrides are transported to the Table 2 Raw recovery rates for the determination of As, Se, Sb and collection solution is just as important as the temperature.A Bi in diVerent biological RMs slow transport to the HNO3–HCl mixture increased the recovery rates of the elements. Capillaries with a small inner diameter Mass fraction/ Raw recovery produced small gas bubbles of the hydrides which were obvi- Sample material Element mg g-1 (%) ously collected in a more eVective way. Furthermore, the NIST SRM 1577a volume of the collection solution had a significant influence Bovine Liver As 0.04–0.6 60–83 on the collection eYciency.A minimum volume of 400–500 ml Se 0.7–2.0 ,10 was necessary and the greatest eYciency was achieved with a Sb 0.1–0.6 20–43 Bi 0.05 70 mixing ratio of 250 ml of HNO3 (65%) to 250 ml of HCl (37%). Yttrium was added to the collection solution as an internal NIST SRM 1571 standard before starting the hydride generation procedure so Orchard Leaves As 10 .95 that evaporation of the collection solution or even possible Se 0.1–1.0 ,10 spraying would not aVect the accuracy. The optimum eYciency Sb 1.0 30–40 was obtained with the conditions indicated under NIST SRM 1573 Experimental.As listed in Table 1, the described procedure led Tomato Leaves As 0.27 60 BCR CRM 414 Table 1 Recovery rates and relative standard deviations (RSDs) for Plankton As 6.82 60 the determination of As, Se and Sb in aqueous standard solutions of Se 1.75 ,10 1 M HCl Sb 1.0 30–40 Standard Absolute mass/ Recovery RSD NIES No. 7 solution Element ng n (%) (%) Tea Leaves As 0.04–0.6 80 Se 0.1–1.0 ,5 1 M HCl As 10 11 75 11 1 M HCl Se 10 12 6 64 Sb 0.1–0.6 30 Bi 0.05–0.7 70 1 M HCl Sb 10 12 91 9 Journal of Analytical Atomic Spectrometry, November 1997, Vol. 12 1253Table 3 Results of the determination of As and Bi in diVerent RMs Certified Corrected mass fraction/ Mean found/ s/ RSD recovery Sample material Element mg g-1 n mg g-1 mg g-1 (%) (%) NIST SRM 1577a Bovine Liver As 0.047 7 0.046 0.004 9.3 98 Bi 0.05* 4 0.044 0.005 11 88 Bi 0.06* 2 0.053 88 NIST SRM 1571 Orchard Leaves As 10 8 9.8 0.9 9.4 98 NIST SRM 1573 Tomato Leaves As 0.27 9 0.26 0.02 7.7 96 BCR CRM 414 Plankton As 6.82 4 6.35 0.91 14 93 NIES No. 7 Tea Leaves As — 6 0.042 0.004 8.8 Bi — 6 0.26 0.024 9.2 * Element, spiked. ing two spectra are shown in Fig. 3. No signals of As and Bi detection limits. Following the analyte/matrix separation described in this paper, both elements can be determined by can be observed in the spectrum recorded directly [Fig. 3(a)], but both peaks are present in the spectrum recorded following TXRF down to the low ng g-1 level, which may be important for biological investigations.Unfortunately other hydride- the described procedure [Fig. 3(b)]. The given amount of about 5 ng of As and Bi in a sample mass of 100 mg may be forming elements such as Se and Sb could not be determined by this method. This was due to the incomplete separation estimated to be 3–4 times the detection limit (3s) of these elements. and trapping of the relevant hydrides under the described experimental conditions.More specific results were obtained for As, Se, Sb and Bi in the diVerent RMs. Increasing amounts of these elements were pipetted onto the samples in the form of aqueous standard This study was supported financially by the Ministerium fu� r Wissenschaft und Forschung des Landes Nordrhein-Westfalen solutions. Amounts of 4–1000 ng of each element were added depending on the concentration of the elements in the RMs.and the Bundesministerium fu� r Bildung, Wissenschaft, Forschung und Technologie (the Ministry for Science and The samples were then digested and the elements were measured by TXRF following the described hydride generation/ Research of Nordrhein-Westfalen and the Federal Ministry for Training, Science, Research and Technology). collection procedure. Raw recovery rates were calculated for the range of mass fractions given in Table 2. High recovery rates were found for As (60–95%) and Bi REFERENCES (70%). In contrast to the recoveries found for aqueous standard solutions in 1 M HCl, unsatisfactory results were obtained for 1 Prange, A., Spectrochim.Acta, Part B, 1989, 44, 437. 2 Chu, R. C., Barrons, G. P., and Baumgardner, P. A. W., Anal. Sb in the RMs (only 20–40%). This was probably caused by Chem., 1972, 44, 1476. the hampered hydride generation in the digested matrix solu- 3 Thompson, K. C., and Thomerson, D. R., Analyst, 1974, 99, 595.tion and by the incomplete collection of the hydrides in the 4 Thompson, M., Pahlavanpour, S., Walton, S. J., and Kirkbright, collection solution. The low recovery of Se (,10%) also found G. F., Analyst, 1978, 103, 568. for aqueous solutions can be explained by the high volatility 5 Nakahara, T., Prog. Anal. At. Spectrosc., 1983, 6, 163. of this element. 6 Alt, F., Messerschmidt, J., and Schaller, K.-H., in Analyses of Hazardous Substances in Biological Materials, ed. Augerer, J., and Because of their high recoveries, only As and Bi were Schaller, K.-H., VCH Verlagsgesellschaft, Weinheim, 1988, vol. 2, included in the following investigations. Individual results p. 231. found for the diVerent RMs are listed in Table 3. Since these 7 Schierling, P., Oefele, Ch., and Schaller, K.-H., A� rztl. L ab., 1982, materials are not certified for Bi, samples were spiked with 28, 21. this element. Both elements were determined in a wide range 8 HaV er, E., Schmidt, D., Freimann, P., and Gerwinski, W., of mass fractions (ng g-1 to mg g-1) with detection limits of Spectrochim. Acta, Part B, 1997, 52, 935. 9 Klockenka�mper, R., T otal-Reflection X-Ray Fluorescence 10 ng g-1 for As and 20 ng g-1 for Bi. The RSD for repeated Analysis, Wiley, New York, 1996. measurements (n=4–9) is about 10%. The mean values for 10 Klockenka�mper, R., and von Bohlen, A., Spectrochim. Acta, Part the mass fractions of As and Bi were found by normalisa- B, 1989, 44, 461. tion to the recoveries of aqueous standard solutions as de- 11 Xie, M. Y., Messerschmidt, J., von Bohlen, A., Ma, Y., scribed above (see under Procedure). By this means, corrected Pfeilsticker, K., and Gu� nther, K., Z. L ebensm.-Unters. Forsch., recoveries of 88–98% were found for the diVerent RMs. 1995, 201, 303. Paper 7/05093C CONCLUSION Received July 16, 1997 Accepted September 2, 1997 The developed method allows the simultaneous determination of As and Bi in biological materials with significantly improved 1254 Journal of Analytical Atomic Spectrometry, November 1