Gas Chromatographic Determination of Beryllium in Biological Materials and in Air Madbuli H. Noweir' and Jacob Cholak Kettering Laboratory, Department of Environmental Health, University of Cincinnati, Cincinnati, Ohio 45219
w The gas chromatographic determination of ultratrace quantities of beryllium in urine, blood, tissue, and air-borne dust is described. Except for air-borne dust, the procedure involves a doiible extraction, first with acetylacetone in benzene and then with a benzene solution of trifluoroacetylacetone. Beryllium in air-borne dust is extracted only by trifluoroacetylacetone in benzene. Levels as low as 1 x 10-10 gram (0.OOOl pg.) of beryllium per sample can be determined by this method. Except for Fe3+ and Ala+, none of the ions usually present in air-borne dust or biological material interferes with the analysis. Fe3+ is removed in a magnetic mercury cathode. Ala+ can be precipitated with 8-quinolinol in chloroform. Recovery of beryllium ranged from 70 to 90% at levels from 1 X 10-9 gram (0.001 pg.) to 1 X lo-' gram (0.10 pg.) per sample. The method is reliable, as shown by the results of analysis of air-borne dust and lung tissues.
B
eryllium has been determined in air-borne dusts and biological materials using colorimetric, fluorometric, or spectrographic methods (Cholak, 1959; Smythe and Florence, 1963). Of these methods the fluorometric morin method is the most sensitive, the detection of as little as 2 X 10-5 pg. of Be per ml. being reported by Sill, Willis, et al. (1961) and Meehan and Smythe (1967). One of us (JC) has used the spectrographic method for many years, chiefly because of the greater reliability of the specificity of detection of ultratrace quantities of beryllium than is possible with wet chemical methods (Cholak, 1959). The sensitivity of the spectrographic method used in this work is 3 X 10-3 pg. per ml. when only 0.2 ml. of solution is evaporated on the electrode, but can be improved to 5 X 10-4 pg. per ml. when 1 ml. is evaporated. However, the sample sizes that make it possible to report the presence of 0.01 pg. of beryllium per 100 grams of tissue, per liter of urine, or per cubic meter of air, to achieve a sensitivity of 3 X pg. per ml. would have to be 33 ml. of urine, 33.3 grams of lung or other tissue, and 333 liters of air (Cholak, 1959). In a search for a method for determining beryllium in as little as 1 gram of tissue, where interference would be a minimum (or could be completely eliminated), the feasibility of the gas chromatographic method described by Ross and Sievers (1968) was considered. These authors, working with solutions of pure beryllium salts, were able to detect as little as 4 X l O - l 3 1 Permanent address, High Institute of Public Health, University of Aiexandria, Alexandria, Egypt (U.A.R.)
gram of beryllium. The above procedure apparently had not been successfully applied to the analysis of biological material. [Since the preparation of this paper, a gas chromatographic method was reported by Taylor, Arnold, et al. (1968).] This paper, therefore, describes a gas chromatographic procedure which has been employed to analyze a wide variety of biological material and dusts or particulate matter removed from air. The sensitivity is such that only one thirtieth of the sizes of sample used in the spectrographic method is required to attain the sensitivity of 0.01 pg. per liter of urine, per 100 grams of tissue, or per cubic meter of air. Experimental
Special Reagents. Acetylacetone, freshly distilled, with the fraction boiling between 134" and 135' C. being used. (Ethylenedinitri1o)tetracetic acid, disodium salt (Na2EDTA), freshly prepared 0.05M solution in distilled water. Trifluoroacetylacetone (K and K Laboratories, Inc., Hollywood, Calif.), redistilled as described by Scribner, Treat, et af. (1965). The fraction boiling between 105" and 106' C. is dissolved in redistilled benzene to form a 0.005M solution. Standard Beryllium Solution. Place 0.1 gram of high purity beryllium metal in 50 ml. of concentrated hydrochloric acid; dilute with 100 ml. of distilled water; heat to boiling to dissolve the metal and, after allowing the solution to cool, make up to 1 liter with distilled water. This solution, containing 100 p g . of beryllium per ml., was used for preparing more dilute solutions by subsequent dilution. Instruments and Operating Conditions. A Loenco Model 70 Hi-Flex chromatograph (Loe Engineering Co., Altadena, Calif.) provided with an electron-capture detector using a tritium source at 22.5 volts, was used. The injection port was modified by inserting a borosilicate glass liner to reduce the likelihood of sample decomposition. Samples were introduced into the instrument with a Hamilton microsyringe. A Servoriter I1 portable 0- to 1-mv. range recorder, Model PSOI W6A (Texas Instruments, Inc.), was used to record the chromatographs. The gas chromatographic column consisted of 4 feet of Du Pont Teflon, 0.06-inch inside diameter. The column packing was composed of 60- to 80-mesh Gas Chrom Z (Applied Science Laboratories, State College, Pa.) impregnated with 5 weight of SE52 (rnethylphenylj silicone gum (Analabs, Hamden, Conn.). The column was operated at a temperature of 80" C. with nitrogen provided at an inlet pressure of 24 p.s.i.g. and at a rate of 50 ml. per minute being used as the eluent gas. The temperatures at the injection port and at the detector were maintained at 160' and 200' C., respectively. A high-speed magnetic mercury cathode was used to remove iron from solutions to be analyzed. Volume 3, Number 10, October 1969 927
A mechanical shaker equipped with a homemade holder for 20-ml. culture tubes was used in the trifluoroacetylacetone chelating procedure. Procedures
Preparation of Samples. URINE. Samples of urine (10 to 100 ml.) are wet-digested and beryllium is isolated with acetylacetone and transferred into hydrochloric acid essentially as described by Cholak and Hubbard (1952), except that 5 ml. of 0.05M EDTA are added, the pH is adjusted with a pH meter, and freshly distilled acetylacetone is used to extract the beryllium. The hydrochloric acid solution containing beryllium is removed to a 50-ml. borosilicate beaker and evaporated gently to dryness on a hot plate. After cooling, the residue is wetted with concentrated sulfuric acid (5 drops are usually sufficient), 2 ml. of concentrated nitric acid are added, and the beaker is heated gently to fumes of sulfur trioxide. The heat is then increased steadily for complete evaporation of the sulfuric acid, after which the sample is ashed for 20 minutes in a muffle furnace maintained at 500' C. The ash is redissolved in 20 ml. of distilled water with constant stirring and heating at the boiling point until the final volume of the sample reaches approximately 10 ml. BLOODAND ANIMALTISSUES.Samples of blood (1 to 10 grams) or animal tissues (1 to 25 grams) are digested as described by Cholak and Hubbard (1948). After oxidation is complete, all but approximately 0.5 ml. of the sulfuric acid is evaporated off. On cooling, the contents of the beaker are washed out quantitatively into a cell of the mercury cathode electrode for removal of iron as described by Center, Overbeck, et af. (1951). This step may not be necessary if the iron is present at concentrations below 10 times that of the beryllium in the sample or in the diluted sample in the case of high concentrations of beryllium. After the iron is removed, the solution, along with the aqueous washings of the cell, is transferred to a beaker and evaporated to approximately 10 ml. The beryllium is isolated and treated as described for urine. In digesting samples of bone or other samples where large amounts of calcium are thrown out as the sulfate, the calcium sulfate is removed by filtration or centrifugation before the iron is removed. Samples which are suspected of having a high content of aluminum may be treated with a solution of 8-quinolinol in chloroform in the presence of an acetate buffer solution to precipitate the aluminum for its removal (Lundell and Hoffman, 1938). AIR-BORNEDUST. Samples of air-borne beryllium dusts may be collected from the air by electrostatic precipitation, by means of an impinger, and by filtration on paper or on membrane filter media. The collector tubes of the electrostatic precipitator are rinsed and policed down with 5 % by volume nitric acid. The rinse solutions, caught in 250-ml. borosilicate beakers, are then evaporated to dryness. Filter media used to remove dust from the air are digested in the same manner as animal tissues and the solutions are taken to dryness. The dried residue from any of the above samples is treated with sulfuric acid, fumed to dryness, and then ashed in a muffle furnace at 500' C. The ash is dissolved with constant stirring in 20 ml. of boiling distilled water. The heating is continued until the solution is reduced to approximately 10 ml. by evaporation. Chromatographic Determination. The solution containing the beryllism is adjusted to between pH 5 and 6, using dilute ammonia solution (1 to 10) if necessary; then is transferred quantitatively to a 20-ml. borosilicate glass culture tube 928 Environmental Science & Technology
provided with a Teflon-lined cap. Two milliliters of 1Msodium acetate buffer (Ross and Sievers, 1968) and 1 ml. of 0.0005M trifluoroacetylacetone solution in benzene are then added. The Teflon-lined cap is applied and the tube is shaken in a mechanical shaker for 2 hours. If an emulsion is present, it may be broken up by centrifuging. An aliquot (0.5 ml.) of the benzene layer is transferred to a clean 8-ml. borosilicate vial, and 0.5 ml. of 0.005M aqueous sodium hydroxide is added. The mixture is immediately shaken manually for 15 seconds, and the organic layer free of the uncomplexed ligand is allowed to separate from the aqueous phase. Depending on the expected concentration of beryllium, 1.0- to 5-p1. portions of the benzene layer are injected into the gas chromatograph by means of a microsyringe. Concentrations of beryllium are determined from a calibration curve prepared by analyzing solutions containing beryllium concentrations ranging from 1 X gram per ml. (0.OOOl pg. per ml.) to 1 x gram per ml. (0.1 pg. per ml.). Results and Discussion
Recoveries of beryllium added at levels of 0.001, 0.01, and 0.1 pg. to 100-ml. portions of urine (simulated or actual) or to 10-gram portions of blood are shown in Table I. Recoveries ranged from approximately 70 to 90% at the different levels of concentration, the lower recoveries occurring at the lower concentrations. Since the recoveries were reproducible at each concentration range, it appears possible to apply a loss correction factor to determine the actual quantity of beryllium present in a sample. In our hands, the relative standard deviations for the heights and areas of the peaks obtained on the repetitive analysis of the same solutions of beryllium chelate in benzene were found to agree with each other to within 1 % at concentrations of 0.1 and 0.01 pg. of beryllium per ml. of solution. At a concentration of 0.001 pg. of beryllium (as chelate) per ml. of benzene, the relative standard deviation was 3.5 %. This increased error in precision was partially due to the slight electronic instability of the instrument at lower attenuations and most likely to the error in measuring the lower peak heights and smaller peak areas at the extreme low concentration range. The method has been applied to the analysis of tissues, fluids, and air-borne dusts, especially when only small quantities of materials were available or when it was suspected that the quantities of beryllium present were likely to be less than,
Table I. Recovery of Beryllium Added to Samples of 100 M1. of Urine and 10 Grams of Blood Beryllium Added,
Urine (synthetic) Urine Urine Blood Blood Urine (synthetic) Urine Blood Urine (synthetic) Urine Blood
0.1 0.0 0.1 0.0 0.1 0.01 0.01 0.01 0.001 0.001 0.001
Beryllium Recovered,
0.09, 0.09 0, 0, 0 0.08, 0.09 0, 0, 0 0.08, 0.09 0.008, 0.009 0.008, 0.009 0.008, 0.008 0.0008, O.OOO8 O.OO07, 0.0008 0.0007, G . GOO7
or near, the limit of detection by the more variable spectrographic method. However, the method also performs satisfactorily for higher concentrations of beryllium, as can be seen from Table 11, where the findings for animal tissues are compared with those by the spectrographic method. The analyses shown are from aliquots of the same solutions prepared by the procedure described above. Ross and Severs (1968) working with beryllium solutions reported that the lower limit of detectability was 4 X gram of beryllium. With the instrument available to us, we were able to detect only 1 X 1O-I2 gram of beryllium, or in our procedure the equivalent of 1 x 10-9 gram (0.001 pg.) per ml. of the final benzene solution of chelate. The effective sensitivity, however, can be increased tenfold to O.OOO1 pg. per ml. by carrying out the extraction with 0.5 ml. of benzene and injecting 5 pl. of the benzene solution of the chelate into the gas chromatograph. Ions normally present in urine, blood, and tissues, as well as metallic ions expected to be present in the urine of industrial populations, have been tested for interference at the levels expected in 100 ml. of urine, 10 mi. of blood, and 1 or 20 grams of lung tissues. Na+, K+, Mg*+,Ca2+,Cr3+,Cu2+,Mnz+, and Zn2+ do not interfere (Table 111). Fe3+, NHr-, and A13+
P
Table 11. Beryllium Determination by Spectrography and Gas Chromatography in Lungs of Monkeys Exposed to Beryllium
Spectrographic 0.03 0.04 0.07 0.12 0.15 0.36 0.39
(rg. Be/gram)
0.44 1.04 1.04 1.52 1.57 1.72 2.15 3.13
Chromatographic 0.03 0.02 0.06 0.22 0.12 0.39 0.40 0.38 0.97 0.77 1.28 1.40 1.68 1.44 2.22
Table 111. Ions Tested for Interference Ion Concentration per Analyzed Ions Tested Sample Interference Na+ 350.0 mg. 180.0 mg. Kf Mg2+ 25.0 mg. 11 .O mg. Ca 2+ POc895.0 mg. Fe a+ 5 . 0 mg. NH4+ 200.0 mg. 0 . 5 pg. Cr 8+ c u 2+ 1 . 5 pg. Mn2f 1 .o pg. 50.0 pg. Zn2+
+ + +
~13+
i
10.0 pg. 1 . o pg. 0 . 1 pg.
+ +-
>
~
I
(
0 2 4 6 8 1 0 0
2
4 6 8 Minutes
1
0
Figure 1. Effect of Fe3+ ion on beryllium chelation A . 0.1 pg. of beryllium chelated
in 1 ml. of ligand B. 0.1 pg, of beryllium plus 5 mg. of Fe3+ (amount present in 10 ml. of blood) C. Same as B after washing with 0.01NNaOH
Minutes Figure 2. Effect of A13+ ion on beryllium chelation A . 0.1 pg. of beryllium chelated in 1 ml. of ligand B. 0.1 pg. of beryllium plus 0.1 pg. of A1 C . 0.1 pg. of beryllium plus 1.0 pg. of A1 D. 0.1 pg. of beryllium plus 10 p g . of A1
ions showed interferences, as is evident from Table I11 and Figures 1 and 2. In the case of aluminum the interference appeared to be in raising the base line (Figure 2). Repeated injections of aluminum trifluoroacetylacetone, whether from commercial sources or freshly prepared in the laboratory, yielded the same results, The slight peak at approximately 6 minutes in A is not due to aluminum but to an unidentified organic product which was present because the acid-ashing treatment of the hydrochloric acid extract of the acetylacetone chelate was omitted. A retention time of' approximately 7 minutes was obtained for aluminum trifluoroacetylacetone. Beryllium in digested samples of air-borne dust could be directly chelated with the trifluoroacetylacetone, but because of the phosphate generally present in biological material, the latter required the double extraction procedure for deterrnining its content of beryllium. Figure 3 shows chromatograms Volume 3, Number 10. October 1969 929
~
‘I 0
2
4
6
8
IO
Minutes
Figure 3. Effect of phosphate ion on beryllium chelation with 0.005M trifluoroacetylacetone
A. 0.1 pg. of beryllium chelated in 1 ml. of ligand B. Same as A in presence of 95 mg. of Pod3-
of the chelation products of 0.1 pg. of beryllium (as chloride) dissolved in 10 ml. of water and that of the same quantity of beryllium in the presence of 95 mg. of Pod3- in the form of NaH2POa.H20,equivalent to the phosphate present in 100 ml. of urine. The beryllium probably was bound to the phosphate to form a compound which did not chelate with the 0.005M trifluoroacetylacetone. The interference by phosphate to chelation could be overcome by using a large excess of acetylacetone or trifluoroacetylacetone. Such extracts, however, were not suitable for chromatography because of the interference by the large excess of the chelating agent, which could not be removed by increased washing with sodium hydroxide solution without decomposing the beryllium chelate. This led us to the double extraction procedure using acetylacetone in the initial extraction, because it was much lower in cost than the fluorinated compound and because of the ease of the subsequent extraction of beryllium with hydrochloric acid. Since appreciable quantities of acetylacetone entered the final benzene phase along with the beryllium trifluoroacetylacetone to produce an unclear background and extend the time required to clean the chromatographic column after each injection, it is necessary to destroy the entrained acetylacetone. For this reason the residue from the hydrochloric acid extract is treated with sulfuric-nitric acids and ashed at 500’ C. The results of the treatment are illustrated in Figure 4. The length of time that the chelate remains in contact with sodium hydroxide is critical for the stability of the chelate. Following the addition of the hydroxide, the mixture should be shaken immediately for not more than 15 seconds. If the analysis cannot be completed immediately after the addition of the hydroxide, it is necessary to remove the benzene layer, in which the chelate can then be stored for 1 or 2 days. Iron present in samples interfered because of chelation with the ligand and the partial decomposition of the chelate in the gas chromatograph (Moshier and Sievers, 1965), as demonstrated in Figure 1. Scribner, Borchers, et a f . (1966) showed that the use of 0.05M EDTA added to the sample prior to extraction with 0.25M trifluoroacetylacetone resulted in a 930 Environmental Science & Technology
0
2
4
6
8
IO 12
14 16 18 20 22 24 26 28 30
Minutes Figure 4. Effect of excess acetylacetone residue on chromatograms A . 0.1 Mg. of beryllium; extract treated with HzSO,-HNO~and ashed
as described in text E . 0.1 fig, of beryllium; extract ashed only C. 0.1 mg. of beryllium; extract without HzS04-HNOs treatment or ashing
90% decrease in the amount of femc iron extracted from the aqueous layer, but in some samples (blood) the remaining iron still interfered. Therefore, we turned to the use of the magnetic cathode for the complete removal of iron. Interference by ammonia, which might be used to neutralize excess acid, is avoided by evaporating to dryness the final hydrochloric acid extract obtained before chelation and dissolving the resulting residue in distilled water. Although interfering quantities of aluminum were not encountered in our work, the treatment, as described in the section dealing with the preparation of blood and animal tissues, will remove most of the aluminum. Literature Cited
Center, E. J., Overbeck, R. C., Chase, D. L., Anal. Chem. 23, 1134-8 (1951). Cholak, J., Arch. Znd. Health 19, 205-10 (1959). Cholak, J., Hubbard, D. M., Am. Znd. Hyg. Assoc. Quart. 13, 125-8 (1952). Cholak, J., Hubbard, D. M., Anal. Chem. 20, 970-2 (1948). Lundell, G. E. F., Hoffman, J. I., “Outlines of Methods of Chemical Analysis,” Wiley, New Ycrk, 1938. Meehan, W. R., Smythe, L. E., ENVIRON. SCI. TECHNOL.1, 839-44 (1967). Moshier, R. W., Sievers, R. E., “Gas Chromatography of Metal Chelates,” Pergamon Press, New York, 1965. Ross. W. D.. Sievers. R. E.. Talanta 15. 87-94 (1968). Scribner, W.‘G., Borchers, & J.,l. Treat,‘W. J., Anal. Chem. 38, 1779-82 (1966). Scribner, W. G.,’Treat, W. J., Weiss, J. D., Moshier, R. W., Anal. Chem. 37, 1136-42 (1965). Sill. C. W.. Willis. C. P.. Hynare. - - , J. K.. Anal. Chem. 33, 1671-84 (1961). . Smvthe. L. E.. Florence. T. M.. “Recent Advances in the kalytical Chemistry of Beryllium,” in “Progress in Nuclear Energy,” Ser. 9, (C. E. Crouthamel, Ed., Vol. 3, Chap. 6, pp 191-235) Pergamon Press, New York, 1963; C A 59, 14840d (1963). ‘Taylor, M. L., Arnold, E. L., Sievers, R. E., Anal. Letters 1 (12), 735-47 (1968). ~
~
Received for reuiew September 26, 1968. Accepted June 24, 1969. Research sponsored in part b y the Center for the Study of the Human Environment under Grant USPHS ES-00159.
