(5) R. K. Munns and D. C. Holland, J . Assoc. Off. Anal. Chem., 54, 202 (1971). (6) M. J. Fishman, Anal. Chem., 42, 1462 (1970). (7) I. Okuno, R. A. Wilson, and R. E. White, J . Assoc. Off.Anal. Chem., 55, 96 (1972). (8) W. J. Herrmnn,Jr., J. W. Butler, and R. G. Smith, in "Laboratory Dhgnosis of Diseases Caused by Toxic Agents", F. W. Sunderman and F. W. Sunderman,Jr., Ed., Warren H. Green, Inc., St. Louis, Mo., 1970, p 379. (9) R. J. Thomas. R. A. Haastrom, and E. Kuchar. Anal. Chem., 44, 512 (1972). (IO) G. Thiiliez, Chem. Anal. Part., 50, 226 (1966). (11) V. Lidums and U. Ulfvarson, Acta Chem. Scand., 22, 2150 (1968). (12) 0. W. Kalb, At. Absorp. News/., 9, 84 (1970).
(13) T. Ukita, T. Osawa, N. Imura, M. Tonomura, Y. Sayato, K. Nakamura, S. Kanno, S. Fudui, M. Kaneko, S. Ishlkura, M. Yonaba, and T. Nakamura, J . Hyg. Cbem., 18, 258 (1970). (14) 0. I. Joensuu, Appl. Spectrosc., 25, 526 (1971). (15) H. E. Knauer and 0. E. Mllliman, Anal. Chem., 47, 1263 (1975). (16) W. L. Hoover, J. R. Mehon, and R. A. Howard, J. Ass@. Off. Anal. Chem., 54, 860 (1971).
RECEIVED for review February 15, 1977. Accepted June 1, 1977* This research was supported by the National of Science (Republic of China) NSC-65B-0409-18(02).
Dry Ashing of Animal Tissues for Atomic Absorption Spectrometric Determination of Zinc, Copper, Cadmium, Lead, Iron, Manganese, Magnesium and Calcium E. E. Menden, D. Brockman, H. Choudhury, and H. G. Petering" Kettering Laboratory, Department of Environmental Health, Universily of Cincinnati College of Medicine, Cincinnati, Ohlo 45267
In the course of a study of the toxicity of heavy metal ingestion to the offspring of pregnant rats, we were faced with the task of analyzing a single sample by flame atomic absorption for zinc, copper, iron, manganese, magnesium, calcium, and cadmium or lead in a difficult matrix containing bone in addition to other tissues. Wet ashing was initially employed. It was observed then that a large part of the sample residue resulting from ashing and evaporation of the remaining acid could not be dissolved in hot 10% or concentrated nitric acids prior to dilution of the samples for atomic absorption spectrophotometry. This prompted a decision to investigate dry ashing as a suitable alternate method of sample preparation. A survey of literature indicated that there were several dry ashing methods with possible application to the kind of samples being investigated, although none described the determination of all of the eight metals of interest to us ( I ) . It was also evident that the recovery of metals, such as cadmium (2), lead ( 3 ) ,iron and calcium ( 4 ) and to a lesser degree zinc and copper, could be affected by sample matrix composition, temperature of ashing, interaction with the sample vessel and by incomplete solubility of the ash. Therefore, in the dry ashing method which was subsequently developed and which is described in this publication it was necessary to incorporate optimal dry ashing conditions and an effective solubilization procedure so that these obstacles were overcome and maximum recoveries of all metals were achieved.
EXPERIMENTAL Apparatus. A Perkin-Elmer Model 403 atomic absorption spectrophotometer was used as equipped with a strip chart recorder, air-acetylene and nitrous oxide-acetylene burners, and a deuterium arc lamp for background correction. A large hot plate with regulated temperature settings was used for evaporating and dissolving. Dry ashing took place in a Thermolyne Model F-6020muffle furnace equipped with a close tolerance temperature controller (Furnatrol 133). Reagents and Standards. Reagent grade Fisher Scientific hydrochloric and perchloric acids and potassium sulfate, and J. T. Baker nitric acid were sufficiently pure for direct use. Ash-aid (6) consisted of potassium sulfate dissolved in hot concentrated nitric acid (0.25 g/mL). Atomic absorption mixed metal standards, covering the range of 0.01 to 100 ppm were made in a Bolution consisting of 10% nitric acid, 0.85% hydrochloric acid, and 2.5 g of potassium sulfate per liter. 1644
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Single metal standards, from which aliquots were added to tissue samples for recovery tests, were prepared in a solution containing 2.4% nitric acid, 0.18%hydrochloric acid, and 0.05% perchloric acid. An equal volume of this blank solution was added to each tissue serving as control. Procedure. Bodies of day-old rat pups of 5-7 g fresh weight, one-half of which were from cadmium-exposedmothers, were used in the experiments. Variable surface contamination of the bodies was removed by soaking them for several minutes in 1% EDTA (6)solution and rinsing with deionized water. Chromium plated surgical instruments also immersed in the EDTA solution, were used to cut up and mince the tissues. Each tissue sample was then distributed in equal weights between two 50-mL metal-free Pyrex beakers, one serving as the control and the other as the recovery test sample. The duplicate samples were oven dried until constant weight was attained and weighed. Standard aliquots containing 70 pg of zinc, 26 pg of copper, 3 pg of cadmium and lead, 300 pg of iron, 10 Ng of manganese, loo0 pg of magnesium, and 8OOO pg of calcium were added to the test samples. An equal volume of the solution in which single standards were prepared was added to each control sample and all samples were taken to dryness at ca. 120 OC. The added metals amounted to between 40 and 200% of the metal levels usually contained by such tissue samples (except for manganese, which was in 9-fold excess). The dried tissue was subjected to charring in the furnace at 300 "C for 5 h. The samples were placed in a cold furnace and the charring temperature was reached in approximately 40 min. One milliliter of ash-aid and 3 mL of concentrated nitric acid were added to each sample and the char was broken up and roughly ground with a thick glass rod while in contact with the liquid. The samples were taken to dryness at the ca. 120 "C temperature, which was used for all evaporation and dissolution treatments on the hot plate. The ashing took place at 400 "C in the furnace, over 20-24 h. Then the samples were treated with 3 mL of concentrated nitric acid and the acid was evaporated. Ashing was completed at the same temperature over 2-4 h, resulting in a white to yellow ash. At this point the samples were divided into groups of 5 test/control pairs per group, each group including 2-3 pairs of tissue samples from cadmium exposed animals. A different ash solubilization method was tested on each group. One method which produced good results for all metals and two other ones which gave poor or inconsistent recoveries of calcium only, are further described. Method A consisted of successive additions and evaporations of two 5-mL volumes of aqua regia. The ash residue was then dissolved by adding a third volume of aqua regia, heating until vapors appeared, agitating the beaker contents, adding 5 mL of water, reheating and again mixing the contents. Water was
Table I. Recoveries of Added Metals, Mean Standard Deviation (Percent, n = 5) Metal Zn Cu Cd Pb Fe Mn Mg
Ca
Method A
100.1 i 99.9 f 95.9 i 96.0 94.7 f 99.8 * 98.7* 96.0 i
6.8 6.3 2.2 8.5 8.2 3.5 7.6 7.3
f
Relative
Method B
Method C
95.1 f 4.4 95.6 * 7.7 97.4i 3.6 96.4 * 8.1 96.2 f 9.0 102.8 r 5.9 99.6~ 7.1 51.0 i 24.0
100.2 * 6.1 96.5 i 7.0 99.8f 3.7 98.9 f 9.8 97.8 f 5.9 102.1 f 7.1 102.3 f 6.2 101.8 * 26.8
replenished if a volume reduction occurred upon longer heating, and the contents were transferred to a 10-mL volumetric flask and readjusted to 10.0 mL with water upon cooling. Method B was somewhat similar to Method A. The first two treatments were with concentrated hydrochloric acid and the third one was with concentrated nitric acid. The nitric acid was evaporated to ca. 1.5 mL. Water was added not to exceed a total volume of 10 mL, and the contents reheated and transferred to a 10-mL volumetric flask. Method C followed the procedure of Method B, except for two treatments with aqua regia after the hydrochloric acid ones, and before the final nitric acid one. Background compensation was used with the atomic absorption instrument when determining cadmium, lead, manganese, magnesium, and calcium, and 100-fold dilutions were required to read magnesium and calcium.
RESULTS AND DISCUSSION Table I depicts the recoveries of zinc, copper, cadmium, lead, iron, manganese, magnesium, and calcium, expressed as a percentage of the added standard metal amount. The dry ashing procedure is identical for the three methods, which only differ at the ash solubilization stage. Of these methods, only Method A produced good results with calcium, since Method B had low recovery and Method C a relative standard deviation of ca. 27%. The average total micrograms of the reagent blank metal levels for zinc, copper, cadmium, lead, iron, manganese, magnesium, and calcium were, respectively, for Method A 0.11, 0.06, 0.03,0.10, 0.22,0.01,0.60, and 0.37. For Method B,they were 0.12,0.05, 0.03, 0.10,0.25,0.01, 0.63, and 0.40, and for Method C, they were 0.16, 0.08,0.05,0.15, 0.34,0.02, 0.85 and 0.64. The somewhat large relative standard deviations of the recoveries of most metals can be attributed to the fact that since the amount of recovered metal is obtained as the difference between two determined values, experimental errors reflected in both are cumulative. Such effect is especially pronounced in cases where the control tissue sample metal level is high in comparison with the amount added for the recovery test. See Table I. Dry ashing conditions were selected with the aim of minimizing metal losses and achieving complete oxidation at the same time. Since no losses occur at 300 “C (7), this temperature was employed to reduce through charring the amount of organic matter in the sample, and thus eliminate the often vigorous reaction during treatment with nitric acid. Since the chloride salts of some metals (8) are prone to losses during dry ashing, the chloride anion was eliminated
by heating with nitric acid and evaporating it to dryness. The generated hydrogen chloride volatilizes (9) before all nitric acid is evaporated. The addition of potassium sulfate permits the formation of nonvolatile cadmium sulfate and possibly other low volatility metal sulfates, which minimizes their loss at the ashing temperature. Moreover, potassium sulfate provided a matrix with a large air-exposed surface facilitating oxidation during the mild ashing a t 400 “C and reduced any chemical interaction (10) of metals such as copper and iron with the vessel surface by limiting their contact with it. The low ashing temperature may also have contributed to reduction in metal-vessel surface interaction. Although such temperature necessitated longer ashing, the furnace was conveniently left in operation overnight. Further work CM then be continued the next day with little extra time needed. A second treatment with nitric acid is necessary to complete the ashing. The acid is evaporated and the sample heated at 400 “C in the furnace, which completes the oxidation. Evidently, occluded carbonaceous particles are exposed and further oxidation during heating in the furnace is accelerated. The ash, due to the presence of sulfates, phosphates, and pyrophosphates, did dissolve completely only in aqua regia, after two pretreatments with it-as indicated by the calcium results. The above described dry ashing method has also been found to be useful in our laboratory for the preparation of microsamples of tissue and milk for anodic stripping voltammetry. Here, the ash is not dissolved in acids but is directly taken up in hot, pH 9 citrate-based supporting electrolyte (11)for the determination of cadmium, lead, zinc, and copper. Preliminary results indicate that with suitable scale-up, larger samples or samples with more bone in them can be prepared by Method A. Similarly, we have also found that by heating the ash in aqua regia, adding sufficient water and reheating, the samples can be solubilized and the first two treatments with aqua regia used in Method A can be eliminated.
ACKNOWLEDGMENT We thank Leslie W. Michael and Bernard Meiners of the Analytical Section for a helpful discussion.
LITERATURE CITED T. T. Gorsuch, “The Destruction of Organic Matter” Pergamon Press, New York, N.Y., 1970, pp 60-79, 93-99, 116-117, 121-129. Society for Analytlcal Chemistry, Analytical Methods Commlttee, Analyst (London), 94, 1153 (1969). T. T. Gorsuch, Analyst (London), 84, 135 (1959). J. E. Allen, “Agricultural Analysis ’, Varlan Techtron Fty. Ltd., Springvale, Victoria, Australia, 1970, p 6. D. W. Yeager, J. Cholak, and E. 0. Meiners, J. Am. Ind. Hyg. Assoc., 34, 450 (1973). E. E. Menden, L. Murthy, and H. G. Petering, unpubllshed data (1971). Ref. 1, pp 34-35, 78-79. Ref. 1, pp 35-36, 75-77, 97-99, 128. E. E. Menden and H. G. Petering, unpublished data (1976). Ref. 1, pp 35-39, 62-65, 77, 117, 128. E. E. Menden and H. G. Petering, unpublished data (1976).
RECEIVED for review April 15,1977. Accepted June 13,1977. Financial support for this work was provided by US.Public Health Service Grant No. ES-00159.
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