Atomic Absorption Determination of Nanogram Quantities of Arsenic in Biological Media R . M. Orheim and H. H. Bovee Department o f Environmental Health, University of Washington, Seattle, Wash. 98 795
A variety of procedures have been developed to prepare samples for the analysis of arsenic (1-7). Some are cumbersome and time-consuming; others involve the problems encountered with perchloric acid digestion. Quantitation has been accomplished in a variety of ways, ranging from colorimetric methods to deuterium-corrected double-beam atomic absorption spectrophotometry ( 1 - 2 1).This paper describes an improved method for the preparation of organic samples and a sensitive procedure for arsenic analysis using a single-beam atomic absorption spectrophotometer (US). EXPERIMENTAL A p p a r a t u s . A Perkin-Elmer AAS Model 103 equipped with a hollow cathode Intensitron arsenic lamp was combined with a Honeywell Electronik Model 19 multi-range recorder. The 2-mV full-scale span range was used with t h e chart speed of the recorder set a t 0.025 inch per second. T h e flame parameters were: hydrogen. 12 psig, (about 10 l./min), and argon, 20 psig, (about 15 l , / m i n . ) , T h e nebulizer was adjusted t o aspirate 150 cm3 air/min. An arsine generator and collection train was assembled from a standard laboratory 125-ml filter flask a n d a 4-way glass stopcock. A “ U ” shaped loop had been fused to two arms of the stopcock. R e a g e n t s . Primary standard grade Asz03, 1.320 grams. was dissolved in 1 liter of 107c HzS04 to give a 1000-ppm arsenic stock solution. Other chemicals used were 1570 reagent grade KI in distilled water, 20% SnC12 in 8-V HC1. 20-mesh zinc, 4-mesh anhydrous CaC12, nitric acid. sulfuric acid, and hydrochloric acid. S a m p l e Digestion. Organic samples containing 5 t o 500 ng of arsenic were weighed in 125-m1 filter flasks. T e n milliliters of conand 1 ml H2S04 were added. These mixtures centrated ” 0 3 were charred on a hot plate a t 175-200” until SO3 was evolved. Ten-milliliter portions of 30% Hz02 were added and evaporated t o SO3 evolution until darkening of t h e solution no longer occurred. Table I lists typical sample size and quantities of 30% of H ~ 0 needed 2 for complete digestion. Arsine Generation. T h e filter flask was connected by its sidearm to t h e 4-way valve with a %inch piece of Tygon tubing ( 5 A 6 - h . i.d.) filled with 4-mesh CaC12. T h e closed loop of t h e valve was immersed in liquid nitrogen (Figure 1 ) . A half-inch Tefloncovered magnet was placed in the flask and positioned over a magnetic stirrer. T h e following reagents were added sequentially with slow mixing: 25 ml glass distilled water, 15 ml concentrated HC1, and 1 ml KI solution. Stirring was interrupted while the 4-way valve loop was opened to the reaction flask; 1 ml of SnC12 solution was added, followed by 1.5 grams of 20-mesh zinc: immediately after addition of the zinc, a No. 5 rubber stopper was rapidly positioned into the neck of t h e flask and stirring was resumed for 5 minutes of arsine generation. At the end of this period, the pressure in t h e open loop was reduced to about Ii’ atmosphere G R Kingsley and R R Schaffert Anal Chem 23, 914 (1951) R J Evans and S L Banderner Anal Chem 26. 595 (1954) J I Morrison J Ass O f f ~ cAgr Chem 44, 740 (1961) L R Stone J ASS Offic Anal Chem 50, 1361 (1967) J I Morrison and G M George J Ass Offfc Ana/ Chem 52 930 (1969) H K Hundley and J C Underwood J ASS Offic Ana/ Chem 53 1176 (1970) R C Chu G P Barron and P A W Baumgarner Anal Chem 44, 1476 (1972) V Vasak and B Sedivek Chem Listy 46, 341 (1952) G W Powers R L Martin F J Piehl and J M Griffin Anal Chem 31, 1569 (1959) Walter Holak Anal Chem 41, 1712 ( 1969) F J Fernandez and D C Manning A t Absorption Newsleft 10, 86 (1971)
~~
Table I. Typical Sample Size and Hydrogen Peroxide Requirement Medium
Blood Milk Hair Tissue
Fat Urine
G r a m digested
ml 30% H ? 0 ?
5 5 1 2 1 2
10-30 10 10 10 20-40 10
Figure 1. Arsine generator w i t h liquid nitrogen trap
( a ) 125-ml filter flask, ( b ) Magnetic stirrer, ( c ) CaC12 drying tube, ( d ) Stoppered U-tube arsine trap, ( e ) Liquid nitrogen/Dewar. ( f ) CaC12 drying tube
to a s p i r a t o r Q
Q
0 050 A Time
Figure 2. Strip c h a r t print-out of 400 ng
B
of arsenic
with house vacuum to avoid excess pressure. T h e loop was then closed and warmed to room temperature, Analysis. T h e closed valve was connected to the AAS aspirator with a reducer made with short sections of plastic or rubber t u b ing. The recorder chart was started and t h e valve opened to the flame. A sharp peak was noted within several seconds. Neither a flow interruption interference nor a hydrogen surge was evident (Figure 2). Aqueous standards containing 0 to 500 ng of arsenic were analyzed by the arsine generation procedure. T h e results are plotted in Figure 3 to give a working standard curve. T h e precision of nine replicate runs of 100 ng of arsenic was within 5 7 ~ . Five-gram aliquots of whole blood were spiked with added As and analyzed to determine the recovery and precision of the procedure: 0, 10, 20. 50. 100, 200, 300. and 500 ng of arsenic were added to successive samples. T h e method of standard additions was used to calculate t h a t 30 ng As/sample or 6 ng As/gram whole blood was present. This compares favorably with cited levels of “normal” blood arsenic content (12). The accuracy of the procedure for recovered arsenic varied from 95 to 10570. “Advances in Forensic and Clinical Toxicoiogy,” Chemical Rubber Company Press. Cleveland, Ohio, 1972, p 169
(12) A . S. C u r r y ,
ANALYTICAL CHEMISTRY, VOL 4 6 , NO. 7, JUNE 1974
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0
0 I00
200
300
400
500
Nanograms arsenic
Figure 3.
Arsenic standard curve
The sensitivity of the procedure described in this paper is compared with two other AAS methods in Figure 4. The double beam flameless method ( 7 ) is approximately three times more sensitive than the deuterium-corrected hydrogen flame technique (11). The concentrating effect of t h e liquid nitrogen trap increases the sensitivity of the single beam HZflame procedure until it is comparable with the flameless technique over t h e critical 0 to 100-ng range.
RESULTS AND DISCUSSION
IO0
200
300
Nanograms arsenic
Figure 4. Comparison of sensitivity with other A A S methods 0 ,By flameless AAS ( 7 ) ; 0 , By single beam argon/hydrogen-entrained air AAS; A , By double beam argon/hydrogen-entrained air AAS, deuterium correction ( 17 )
Quantitative recovery of nanogram amounts of added arsenic from whole blood has been demonstrated. Similar results were obtained with other biological samples, including milk, hair, and tissue. These results are in preparation for publication (13). The single beam AAS arsine response is essentially linear to 100 ng of arsenic, and a useable curve to 500 ng can be plotted. The minimum detection level is about 5 ng arsenic. The use of a 2-mV recorder scale gives a 5X expansion of absorbance over the normal 10-mV output. With this
increased sensitivity, flame noise becomes a critical factor a t the arsenic wavelength of 1937 A. To reduce this problem, a 20-mesh Tyler standard screen sieve (20-cm diameter) was centered over the flame. A 10-inch 3-sided chimney of sheet metal was designed to shield, additionally, the front and sides above the hydrogen flame from air currents. The burner undercarriage was surrounded with asbestos cloth. With the added shielding, flame noise was reduced to 0.005 absorbance unit. The applicability of this technique to other hydrides is being investigated.
Orheim, L. Lippman, C. J Johnson, H . H . Bovee, "Lead and Arsenic Levels of Dairy Cattle in Proximity to a Copper Smelter," in preparation.
Received for review August 27, 1973. Accepted January 14, 1974.
(13) R M .
Gas Chromatographic Determination of Barbiturates after Extractive Methylation in Carbon Disulfide Hans Ehrsson' Pharmaceutical Centre, Sodersjukhuset, 100 64 Stockholm 38, Sweden
Determination of barbiturates by gas chromatography is complicated by adsorption to the chromatographic support ( I ) . Methylation has in several cases been used to overcome this problem. The reaction has been performed prior to the gas chromatographic procedure using dimethyl sulfate (2, 3 ) , diazomethane ( 4 ) , and methyl iodide ( 5 ) Present address, Department of Pharmacology, Medical University of South Carolina, Charleston, S.C. 29401 ( 1 ) B. J. Gudzinowicz and S. J. Clark, J. Gas Chromatogr., 3, 147 (1965), (2) E. Mary Baylis, D E. Fry, and V. Marks, Clin. Chim. Acta, 30, 93 (1970). (3) J. T. Stewart, G . B. Duke, and J. E. Willcox. Anal. Lett., 2, 449 (1969). (4) J. G . H . Cook. C. Riley, R . F . N u n n . and D. E. Budgen, J. Chromatogr., 6, 182 (1961). (5) W. Dunges and E. Bergheim-lrps. Ana/. Lett., 6, 185 (1973).
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or in the injector of the gas chromatograph by flash methylation with tetramethylammonium (6) or trimethylanilinium ( 7 ) hydroxides as reagents. Both techniques have certain disadvantages. The former requires, in general. long reaction times. and with the latter there is a risk of incomplete methylation (8) and decomposition by the strongly alkaline reagent solutions (9, I O ) . In both techniques, such solvents have been used that large tailing fronts are obtained and the sensitivity of the flame ionization detector cannot normally be utilized to its full extent. Alkylation of acids by an extractive technique was used ( 6 ) G . W. Stevenson, Anal. Chem., 38, 1948 (1966) ( 7 ) E. Brochmann-Hanssen and T . 0 . Oke, J. Pharm. Sci.. 58, 370 (1969). (6) H . J. Kupferberg, Ciin. Chim. Acta, 29, 283 (1970). (9) J C Van Meter and H . W . Gillen, Clin. Chem., 19, 359 (1973). (10) R . J Perchalski and B. J. Wilder, Clin. Chem., 19, 788 (1973).
