Anal. Chem. 2011, 83, 152–156
Evaluation of Radiochemical Neutron Activation Analysis Methods for Determination of Arsenic in Biological Materials Rick L. Paul* Analytical Chemistry Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, United States Radiochemical neutron activation analysis (RNAA) with retention on hydrated manganese dioxide (HMD) has played a key role in the certification of As in biological materials at NIST. Although this method provides very high and reproducible yields and detection limits at low microgram/kilogram levels, counting geometry uncertainties may arise from unequal distribution of As in the HMD, and arsenic detection limits may not be optimal due to significant retention of other elements. An alternate RNAA procedure with separation of arsenic by solvent extraction has been investigated. After digestion of samples in nitric and perchloric acids, As(III) is extracted from 2 M sulfuric acid solution into a solution of zinc diethyldithiocarbamate in chloroform. Counting of 76As allows quantitation of arsenic. Addition of an 77As tracer solution prior to dissolution allows correction for chemical yield and counting geometries, further improving reproducibility. The HMD and solvent extraction procedures for arsenic were compared through analysis of SRMs 1577c (bovine liver), 1547 (peach leaves), and 1575a (pine needles). Both methods gave As results in agreement with certified values with comparable reproducibility. However, the solvent extraction method yields a factor of 3 improvement in detection limits and is less time-consuming than the HMD method. The new method shows great promise for use in As certification in reference materials. Neutron activation analysis (NAA) has played a key role in the certification of As in biological Standard Reference Materials (SRMs) at NIST.1 Because nuclear analytical methods share few common sources of uncertainty with non-nuclear methods, such as inductively coupled plasma mass spectrometry (ICPMS), NAA is invaluable to the certification process where two or more independent methods are needed. Also, nuclear methods do not suffer from the chemical blank problem encountered with conventional methods, so lower detection limits are often achievable. In NAA, arsenic is quantified by counting of the principal γ-ray line (559 keV) emitted from 76As (t1/2 ) 1.093 79 ± 0.000 45 d) formed upon neutron capture of 75As, based on comparison with As standards prepared from high-purity materials irradi* To whom correspondence would be addressed. Phone: (301)975-6287. Fax: (301)208-9279. E-mail:
[email protected]. (1) Zeisler, R.; Mackey, E. A.; Lamaze, G. P.; Stover, T. E.; Spatz, R. O.; Greenberg, R. R. J. Radioanal. Nucl. Chem. 2006, 269 (2), 291–296.
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ated in the same manner.2 Instrumental neutron activation analysis (INAA) is typically used to determine As mass fractions of g1 mg/kg in biological materials, though lower amounts may be determined, depending on the matrix; e.g., INAA was used to determine As in SRM 1575a Trace Elements in Pine Needles, 39 ± 2 µg/kg and SRM 2670 Trace Elements in Urine, 60 ± 8 µg/kg. However, the presence of significant 24Na (t1/2 ) 15 h), 82Br (t1/2 ) 35.3 h), or 32P (t1/2 ) 14.3 d) results in high count rates, high dead time, and elevated spectrum baseline due to Compton scattering, hence decreased signalto-noise ratio and poorer detection limits. The effect of 24Na can be minimized by optimizing the decay time after irradiation (typically 3-5 days) to allow significant decay of 24Na yet retain sufficient 76As to give good counting statistics. However, 82Br and 32P have longer half-lives than 76As, so longer decay times do not improve detection limits if significant amounts of these nuclides are present. When significant amounts of these elements are present, the best results for arsenic (highest reproducibility, lowest uncertainties, best detection limits) are obtained using radiochemical separation. Radiochemical neutron activation analysis with retention of arsenic and other elements on hydrated manganese dioxide (HMD) has been used to measure As at submilligram/kilogram levels in SRMs.3,4 This method has been used in the certification of As mass fractions in SRM 1548a Typical Diet, SRM 1570a Trace Elements in Spinach Leaves, SRM 1573a Tomato Leaves, and SRM 1577c Bovine Liver as well as several SRM soils and sediments. Although this method provides very high and reproducible yields, and detection limits at low microgram/kilogram levels, counting geometry uncertainties may arise from an uneven distribution of As in the HMD. Furthermore, although the separation does eliminate the bulk of 24Na and other interfering nuclides, a number of other elements are retained on HMD (Ag, Cr, Mo, Sb, and Se), and other elements may be incompletely separated, resulting in increased background from Compton scattering. The method is also somewhat laborious and time-consuming if only measurement of As is desired and produces a large amount of radioactive HMD as waste. Methods are being investigated to minimize the uncertainties associated with counting geometry, improve arsenic detection (2) Lindstrom, R. M.; Blaauw, M.; Fleming, R. F. J. Radioanal. Nucl. Chem. 2003, 257 (3), 489–491. (3) Greenberg, R. R. Anal. Chem. 1986, 58, 2511–2516. (4) Greenberg, R. R.; Zeisler, R.; Kingston, H. M.; Sullivan, T. M. Fresenius Z. Anal. Chem. 1988, 332, 652–656. 10.1021/ac102073h Not subject to U.S. Copyright. Publ. 2011 Am. Chem. Soc. Published on Web 12/02/2010
limits, and decrease the time needed for separation (important because of the short half-life of 76As). The use of 77As tracer to correct for differences in yield and counting geometry has been investigated and used in conjunction with the HMD procedure. A solvent extraction procedure with counting of 76As in the liquid phase has also been developed and used in conjunction with the 77As tracer. EXPERIMENTAL SECTION Preparation of 77As Tracer Solution. A solution containing 77 As (t1/2 ) 1.6179 d) was used for the purpose of correcting for differences in chemical yield (fraction of arsenic recovered from the sample) and counting geometry from sample to sample and between samples and standards. The tracer was prepared using a variation of the method described by Byrne.5 Approximately 50 mg of high-purity GeO2 was added to each of two polyethylene vials. The vials were placed in a polyethylene irradiation vessel (rabbit) and irradiated in the NIST reactor pneumatic tube irradiation facility, RT-2, for a total time of 6 h at a reactor power of 20 MW, at a thermal neutron fluence rate of approximately 3 × 1013 cm-2 s-1. To compensate for a nearly linear drop-off of flux as a function of distance along the length of container, i.e., distance from the reactor core, the rabbit was rotated 180° at the midpoint of the irradiation and reinserted. Approximately 2 d after irradiation, the rabbit was opened and the contents of each polypropylene vial transferred to a Teflon beaker.6 Each 50 mg GeO2 portion was dissolved by heating in 5 mL of 1 mol/L potassium hydroxide solution. Each dissolved portion was then processed in the following manner to separate 77As from 77Ge: 1 mL of 2.5 mol/L H2SO4 was added to neutralize the solution, followed by addition of 5 mL of H2O, 4.5 mL of concentrated H2SO4, and 5 mL of 2 mol/L potassium iodide solution. The 20 mL total solution was transferred to a 125 mL separatory funnel, and As was extracted into 10 mL of toluene. The aqueous phase was discarded, and the toluene phase washed with two 20 mL portions of a 4 mol/L H2SO4/0.5 mol/L KI solution. Arsenic was then back-extracted into 10 mL of a 10% (volume fraction) H2SO4 solution. The 10 mL portions from the GeO2 dissolutions were combined and heated to near boiling on a hot plate while 30% H2O2 was added to convert iodide to I2. After the solution had cleared, and no more purple I2 vapor was observed with addition of H2O2, the solution was evaporated to approximately 2 mL, allowed to cool, transferred to a 125 mL polypropylene bottle and diluted to the desired volume (usually 15-25 mL depending on the number of samples and standards to be analyzed) with deionized H2O. Preparation and Irradiation of Samples and Standards. For verification of the method, SRMs were chosen which were certified for arsenic and which represented a variety of matrixes (both animal and vegetable matter). Biological reference materials were prepared by pressing approximately 0.2-0.3 g of powdered sample into 12.7 mm diameter pellets using a stainless steel die and hydraulic press. The pellets were then sealed into bags made (5) Byrne, A. R. Fresenius Z. Anal. Chem. 1986, 326, 733–735. (6) The identification of certain commercial equipment, instruments, or materials does not imply recommendation or endorsement by the National Institute of Standards and Technology. These identifications are made only in order to specify the experimental procedures in adequate detail.
from polyethylene film. Each bag was subsequently sealed into a second polyethylene film bag in order to ensure encapsulation during irradiation. A solution containing (54.36 ± 0.31) µg/g of arsenic (expanded uncertainty), prepared by gravimetric dilution of SRM 3103a Arsenic Standard Solution, was used to prepare As standards for analysis. Standards were prepared by using a disposable pipet to deposit 2-3 drops of the solution onto Whatman 41 filter paper. The pipet was weighed before and after dispensing to determine the mass of solution deposited. The filter papers were allowed to dry for several days in a clean Teflon hood and were then formed into disks using a stainless steel die and hydraulic press. Each disk was double encapsulated in polyethylene film. Biological samples, standards, and iron foil neutron flux monitors (each ≈6 mg) were placed into rabbits, each rabbit typically containing 5-10 samples along with 4-6 standards and 2 or 3 flux monitors. Each rabbit was irradiated in the NIST reactor pneumatic tube irradiation facility, RT-1, for a total time of 2-3 h at a reactor power of 20 MW (thermal neutron fluence rate of approximately 1.0 × 1014 cm-2 s-1). The rabbit was rotated 180° at the midpoint of irradiation and reinserted. Rabbits were allowed to sit for 2-4 days after irradiation before processing in order to allow decay of 24Na. Dissolution of Targets. Figure 1 depicts the experimental scheme used for determination of arsenic in biological materials. Biological SRMs and standards were digested individually in Teflon beakers containing: 10 mL concentrated HNO3, 10 mL H2O, 0.1 mL of a solution containing approximately 1 mg/g of As carrier, and 1 mL of the 77As tracer solution. About 0.2-0.3 g of an unirradiated biological matrix material was added to each standard to minimize differences in chemical processing between samples and standards arising from matrix effects. Beakers were covered with Teflon lids and placed on a hot plate with a surface temperature of approximately 200 °C for 4-5 h. Lids were removed and the surface temperature increased to approximately 250 °C. Each beaker was removed from the hot plate after volumes were reduced to approximately 1-2 mL. An additional 10 mL of concentrated HNO3 and 10 mL of concentrated HClO4 were added to each beaker, and the solutions were evaporated on the hot plate until volumes were reduced to about 1-2 mL. These were cooled to room temperature, and then an additional 10 mL of concentrated HNO3 and 10 mL of concentrated HClO4 were added and each beaker was covered and placed on the hot place at a surface temperature between approximately 150 and 180 °C overnight. Lids were removed, and the surface temperature of the hot plate increased to approximately 250-280 °C. Each beaker was removed from the hot plate when the volume was reduced to between 1 and 2 mL. After digestion, arsenic was separated from the matrix using one of the two procedures outlined below. Method 1: Separation on HMD. In preparation for separation of the As from the digests onto HMD, 10 mL of 1 mol/L HNO3 was added to each digest, and the beaker was covered and heated at 100-150 °C for approximately 30 min to ensure dissolution of the residue. After cooling, each digest was added to a column containing about 5 mL of HMD conditioned in 1 mol/L HNO3, with a flow rate of approximately 0.25-0.5 mL/ Analytical Chemistry, Vol. 83, No. 1, January 1, 2011
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Figure 1. Experimental scheme for determination of arsenic showing the two different separation methods.
min. When the digest had passed into the HMD, an additional 5 mL of 1 mol/L HNO3 was used to rinse the beaker and the rinse was added to the column. After that 5 mL had passed into the HMD, two 15 mL additions of 1 mol/L HNO3 were added to each column sequentially. After all column rinsings were completed, the HMD portion from each column was transferred to a 20 mL plastic scintillation vial by inverting the column and rinsing with 1 mol/L HNO3. Each vial was centrifuged to separate the HMD from the excess 1 mol/L HNO3, and the liquid was discarded. Method 2: Separation by Solvent Extraction. The method was adapted from a procedure given by Kucera and Soukal.7 A volume of 5 mL of concentrated H2SO4 was added to the digest in each beaker. The beakers were heated uncovered at a temperature of 250-275 °C for about 30 min in order to expel traces of HClO4. Each beaker was removed from the hot plate and cooled to room temperature, followed by dropwise addition of 15 mL of H2O and 1.5 mL of 0.2 mol/L KI solution to reduce As5+ to As3+. Beakers were again covered and heated to boiling for 10-15 min, during which time the solution turned a yellow or pale orange color due to the formation of I2. Lids were removed, 1.5 mL of 0.8 mol/L ascorbic acid was added to complete the conversion of As to As3+ (solution turned clear), and the beakers were removed from the hot plate and allowed to cool for 30 min (color changed to pink or light brown). Each solution was transferred to a 60 mL separatory funnel and extracted with 10 mL of 0.025 mol/L zinc diethyldithiocarbamate (hereafter abbreviated as Zn(DDC)2) in chloroform for about 2 min. The chloroform layer (bottom layer) was drained (7) Kucera, J.; Soukal, L. J. Radioanal. Nucl. Chem. 1993, 168, 185–199.
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off into a beaker, and the aqueous phase again extracted for 2 min with 5 mL of Zn(DDC)2 solution. The chloroform phase was drained off, the aqueous phase discarded, and the combined 15 mL chloroform phase transferred back to the separatory funnel. The chloroform phase was next washed with 5 mL of 2 mol/L H2SO4 containing 100 mg of ZnSO4 for 2 min, and the chloroform layer was drained into a 20 mL plastic liquid scintillation counting vial (aqueous phase discarded). Counting and Data Reduction. γ-Ray spectroscopy was used to measure 76As and 77As in the HMD and Zn(DDC)2/ chloroform phases. Vials were counted using a high-purity intrinsic germanium detector (efficiency ) 25-35%) with associated electronics. Vials were counted at the face of the detector (detector-to-sample distance ) 0 cm). Counting times for samples generally ranged from about 1 to 8 h; standards were usually counted for
