(14)P. S.Papavasiliou and G. C. Cotzias, J. Bioi. Chem., 236,2365 (1961). (6) R. T. Ross and J. G. Gonzalez, Bull. Environ. Contam. Toxicol., 12, 470 (1974). (15) M. Suzuki and W. E. C. Wacker, Anal. Biochem., 57,605 (1974). (16)L. Ebdon, G.F. Kirkbright, and T. S.West, Anal. Chim. Acta, 58,39(1972). (7)J. P. Mahoney, K. Sargent, M. Greland, and W. Small, Clin. Chem. (Winston-Salem, N.C.) 15,312 (1969). (8) H. J. M. Bowen, J. Nuci. Energy, 3, 18 (1956). (9)G. c. Cotzias, S.T. Miller, and J. Edwards, J. Lab. Ciin. M e d . , 67,836(1966). RECEIVEDfor reviewjanuary 22, 1976. ~ ~ M~~ 17, ~ (10)E.L. Kanabrocki, L. F. Case, L. Graham, T. Fields, E. B. Miller, Y. T. Oester, 1976. T h i s work was supported in p a r t by grants from t h e and E. Kaplan, J. Nuci. Med., 8, 166 (1967). (11) J. Versieck, F. Barbier, A. Speecke,and J. Hoste, Acta Endocrinol., (CO- United Parkinson Foundation, the Boothroyd Foundation, penhagen), 76,783 (1974). (12)A. A. Fernandez, L. Sobel, and S. L. Jacobs, Anal. Chem., 35,1721(1963). and the Michael Reese Medical Research Institute Council, Chicago, Illinois. (13)G. C. Cotzias and P. S. Papavasiliou, Nature (London), 195,823 (1962).
Determination of Platinum and Palladium in Blood and Urine by Flameless Atomic Absorption Spectrophotometry Antrim H. Jones Analytical Chemistry Department, Research Laboratories, General Motors Corporation, General Motors Technical Center, Warren, Mich.
48090
A rapid method for the decomposition OF whole blood and urine with HN03 and HC104has been developed for the determination of platinum and palladium by flameless AAS. The minimum detectable concentrations in 5-9 samples of blood are 0.03 pg Pt/g and 0.01 pg Pd/g, while in 50-g samples of urine, they are 0.003 pg/g for both platinum and palladium.
Some industrial hygiene questions arose related t o t h e ingestion of platinum and palladium by workers in t h e assembly of catalytic converters for automobiles. The monitoring of airborne particulate matter at t h e facility showed that, because of t h e precautions established for catalytic converter assembly, very little, if any, of these metals a r e available for ingestion. However, a program was established for t h e determination of platinum and palladium in blood and urine as a precautionary measure. The analytical technique must be sensitive, a n d t h e sizeable number of samples precluded t h e use of a method that involves much time and attention by t h e analyst. Atomic absorption spectrophotometry (AAS) is well-known for its rapidity. Also, it is sensitive and subject to a minimum of interferences ( I ) ; flameless AAS techniques are several times more sensitive than the AAS flame technique (2). Therefore, AAS in conjunction with t h e heated graphite furnace was selected as t h e method of analysis. This report describes a rapid, simple procedure for preparing the sample solution and t h e precautions necessary in t h e application of flameless AAS t o t h e determination of platinum a n d palladium.
EXPERIMENTAL Sample Preparation. Blood is collected in evacuated test tubes, Le., Becton-Dickinson “Vacutainer” vials (Catalogue No. BD4751), containing sodium heparin. The sample volume is approximately 5 ml. Enough urine is collected to provide a 50-ml sample. Since several days may elapse between collection and analysis, the samples are preserved by freezing. Certain considerations are necessary for sample preparation, especially in the analysis of blood. Because so little is known about the physiological effects of platinum and palladium and their distribution in blood, it is considered necessary to analyze whole blood instead of just the serum as is the case with many metals. This stipulation makes the processing of the blood samples more difficult than if the red cells could be separated from the serum. When the sample is thawed, clotting occurs in spite of anticoagulants, thus rendering the sample inhomogeneous. 1472
0
Direct injection of well-mixed whole blood into the graphite tube furnace for the determination of platinum and palladium was tried in our laboratory without success. Samples “spiked” with up to 1pg of metal per g gave no signal, or one so small that proper measurement could not be obtained. The direct atomization of urine gave consistent results which, however, showed that this approach was not sensitive enough for the expected concentration in the samples. In a recent publication ( 3 ) ,a method is described for the determination of platinum and palladium in tissue samples; lyophilized tissue is wet-ashed with nitric acid, sodium chloride, and aqua regia, and the platinum and palladium are determined by AAS with a graphite tube furnace. Another wet ashing technique involving “ashing acid” ( 4 ) also was investigated for adaptation to our analytical problem. These and some dry ashing techniques that were investigated were rejected because they involved too much elapsed time and/or manipulative detail. An extraction technique similar to that using diethyldithiocarbamate for the determination of other metals in whole blood and urine with flame AAS (5) was studied, but the application to platinum and palladium in the concentration involved proved unsuccessful for the graphite tube furnace. A method for the decomposition of blood or urine with HC104 in a closed container (6) was investigated and showed promise. However, because of the clotting that occurs when the sample is thawed, the small sample size (200 pl) caused doubt that it would be representative of the whole sample; therefore, the method was not considered further. The procedure for sample preparation that was finally adopted involves a minimum of elapsed time and attention by the analyst. A batch of 10 samples can be carried through the decomposition process in less than 2 h, most of that time without attention. The whole sample is decomposed with “ 0 3 and HC104. In the case of urine, the metals which has been said are concentrated by a factor of 10. The “03, t o depress the sensitivity of the method for the metals ( 3 ) ,is eliminated by fuming with HC104 which is also used to eliminate organic matter. The ratio of acid volumes to sample volume precludes any danger of an explosion. Procedure. The sample, at room temperature and well mixed, is transferred to a 125-ml Erlenmeyer flask. In the case of blood, its container is weighed before and after emptying to obtain the sample weight. For urine, the 125-mlErlenmeyer flask is weighed before and after the sample is added. Twenty-five ml of 16 M ” 0 3 and 2 ml of 16 M HC104 are added, plus one boiling bead to minimize bumping. The solution is heated to fumes of HClOd and fuming continued for 5 min with sufficient heat to produce refluxing of fumes halfway up the side of the flask. At the end of this time, approximately 1ml of HClOd remains. To the cooled solution, 5 ml of 1.2 M HCl is added by pipet, and the flask is swirled occasionally for 5 min or until all salts are in solution, whichever is earlier. In the case of urine, insoluble KC104 salts are removed by centrifugation. The solution is transferred to a 2-dram (7.4-ml)vial from which a 50-pl aliquot is taken for injection into the graphite tube furnace.
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
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~~
Table 11. Relative Standard Deviation (96 s)n Blood pg Pt/g
0.4 0.8 1.2 1.6 2.0
Urine
% s pg Pd/g 3.7 0.4 4.9 0.8 6.5 1.2 4.5 1.6 4.3 2.0
%s
pg Pt/g
76 s
,pg Pd/g
%s
1.3 1.1 1.0 1.5 1.2
0.04 0.08 0.12 0.16 0.20
3.5 0.7 2.7 3.8 3.1
0.04 0.08 0.12 0.16 0.20
3.3 3.4 1.8 1.5 1.4
aBased o n a 5-g sample of blood and a 50-g sample of urine.
0 Acid Standards 0 Pd i n U r i n e
Pd in Blood 0 P I in Urine
Table 111. Minimum Detectable Concentration I
0
I
’
l
l
I
/
0.4
0.M
0
0.08
l
l pq!q in Blood
pg1g
l
I”
Urine
l
l
l
OJ2
l
/
I
l
i
/
1.6
2.0
Sample
0.16
0.20
Blood Urine
Figure 1. Typical calibration curves
Table I. Effect of Acid Treatment upon Concentrationa
Platinum (pg/g) Palladium (pg/g)
0.03 0.003
Table IV. Day-to-Day Precision and Accuracya
Urine
Urine
Platinum
Palladium
Untreated
Treated
Untreated
Treated
0.040 0.080 0.120 0.160 0.200
0.042 0.090 0.125 0.164 0.210
0.040 0.080 0.120 0.160 0.200
0.039 0.079 0.119 0.153 0.199
Blood Platinum Untreated
Palladium Treated
Untreated
Treated
Amount added Amount determined First day Second day Third day Fourth day Fifth day Sixth day Av %S
0.40 0.80 1.20 1.60 2.00
0.39 0.81 1.17 1.48 1.92
0.01 0.003
0.40 0.80 1.20 1.60 2.00
0.40 0.79 1.20 1.58 1.99
a All values are in pg/g sample.
Atomization. A Perkin-Elmer Model 403 atomic absorption spectrophotometer with a deuterium background corrector, a 10-mV 0 . 5 s full scale recorder, and an HGA-70 graphite tube furnace were used to obtain our data. The 50-pl sample injected into the furnace is dried for 20 s a t 100 “C and “charred” for 20 s a t 500 “C. With the furnace tilted out of the optical path, the temperature is increased to 1900 O C t o volatilize the HC104, which otherwise would interfere with platinum and palladium absorbance. The furnace is then tilted back into place, the platinum and palladium are atomized at 2700 “ C , and their absorbance is recorded as a sharp peak on the recorder. Calibration. Standard solutions of platinum and palladium are prepared by dissolving platinum and palladium of 99.9% purity in aqua regia, evaporating the solution to eliminate most of the “ 0 3 , and then redissolving in HCl. Calibration solutions are prepared by adding aliquots of the standard solutions to a series of 125-ml Erlenmeyer flasks containing either 5 g of platinum- and palladium-free blood or 50 ml of platinumand palladium-free urine. The solutions are carried through the procedure just as if they were samples. The use of blood or urine, as the case may be, in the calibration solutions is necessary to compensate for the inhibiting effect produced by these substances in the final solution. (The terminology “platinum-free” and “palladium-free’’ refers t o human blood and urine from sources that had no unusual exposure to these metals. Thus, these samples of blood and urine were found to contain less than the minimum detectable levels of platinum and palladium.) The amounts of platinum and palladium are 2,4,6, 8, and 10 fig, producing the concentrations for blood and urine shown in Figure 1. The calibration solutions are atomized before and after each batch of samples. Solutions more than 1week old are not used. The curves in Figure 1are presented to indicate the extent of adherence to Beer’s law.
%d a
Blood
Pt
Pd
Pt
Pd
0.100
0.100
1.00
1.00
0.105 0.098 0.102 0.107 0.100 0.103
0.097 0.097 0.103
0.98 1.02
0.100 0.102 0.105
0.91 1.02 1.05
0.98 1.06 1.01 0.97 1.03 1.04
0.103 3.2 3.0
0.101 3.3 1.0
1.00 4.9 0
1.02 3.5 2.0
1.00
All values are in p g per g of sample
RESULTS Since n o blood or urine samples containing known concentrations of platinum and palladium were available, samples of blood and urine to which known amounts of platinum and palladium had been added were used to evaluate the method for precision and accuracy. To determine if any platinum or palladium is lost during the treatment of the samples with ” 0 3 and HC104, the following study was made. First, a series of urine and a series of blood “synthetic” samples were prepared as described for calibration solutions. A duplicate series of samples was prepared, but, in this case, no platinum and palladium was added before the treatment with HNO3 and HC104. Instead, the 5.0 ml HCl (1 + 9) added to each flask after the acid treatment contained the proper amount so that each series, one for blood and one for urine, contained 2,4,6,8, and 10 yg of platinum and palladium that had not been exposed to the acid treatment. The solutions were then paired with their counterparts in which the platinum and palladium had been exposed to the acid treatment. Each pair was injected into the furnace 10 times, first one solution, then the other. Table I shows the effect of acid treatment by comparing platinum and palladium values for treated and untreated “synthetic” sample solutions; these values are the averages of 10 determinations and, therefore, any error due to imprecision of the method is minimized. Table I shows that little, if any, metal is lost in the treatment with “ 0 3 and HClO4. The precision of the atomization portion of the method is shown in Table 11. As mentioned previously, a single solution
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
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of “synthetic” sample at each level of concentration was injected 10 times into the graphite tube furnace for each element, and the relative standard deviation (% s) was calculated from the results. Table I1 shows that, with the possible exception of palladium in urine, the relative standard deviation (% s) appears to be unaffected by the concentration. The minimum detectable concentration of platinum and palladium in blood and urine, shown in Table 111,was calculated as two times the variation, calculated from the relative standard deviation a t the lowest concentration for which this value was determined, namely, 2 pg total in the solution. To establish day-to-day precision and accuracy, a “synthetic” sample of blood containing 1.00 Fg of platinum and 1.00 pg of palladium per g of sample and a “synthetic” sample of urine containing 0.100 pg of platinum and 0.100 F g of palladium per g of sample were prepared and then analyzed according to the procedure on each of six separate days. Table IV shows the results, which show precision in terms of relative standard deviation (% s) and accuracy in terms of the average deviation (% d ) from the added concentration.
ACKNOWLEDGMENT The author expresses his appreciation to Francis W. Weir and Alexandra N. Brady of the General Motors Research Laboratories for providing technical knowledge and assistance and securing a supply of blood samples.
LITERATURE CITED (1) Walter Slavin, “Atomic Absorption Spectroscopy”, interscience Publishers, New York, 1968. (2) E. Adriaenssens and P. Knoop, Anal. Chim. Acta, 68, 37 (1974). (3) Robert G. Miller and James U. Doerger, At. Absorp. News/., 14, 66 (1975). (4) “Lead”, Cornmlttee on Biological Effects of Atmospheric Pollutants, National Academy of Sciences, Washington, D.C., 1972. (5) F. Arnore, Anal. Chem., 46, 1597 (1974). (6)I. W. F. Davidson and W. L. Secreat, Anal. Chem., 44, 1808 (1972).
RECEIVEDfor review January 9, 1976. Accepted June 10, 1976.
Background Light Losses in Analysis of Complex Alloys and Metals for Trace Elements by Flame Atomic Absorption Spectrometry J. Y. Marks,*
R. J. Spellman, and B. Wysocki
Materials Engineering and Research Laboratory, Pratt and Whitney Aircraft, East Hartford, Conn. 06 108
The analysis of complex alloys for trace elements of metallurgical interest by flame atomic absorption gives rlse to serious backgroundcorrectlon problems. Background llght losses were found to result from scattering, molecular absorption, and atomic absorption by the matrlx. Several methods of correctlng for the phenomenonhave been suggested In the literature. The analyte elements may be separated from the matrix, correction for background light losses may be made with a conilnuum light source either simultaneously wlth or sequential to the analyte absorbance measurement, or a nonabsorbing line correction may be used.
The measurement of and correction for background light losses sets the lower limits for accurate element determinations in many atomic absorption analyses. The use of modern instrumentation capable of accurate measurements of very small absorbance signals makes more valid correction methods desirable. The analysis of complex alloys for trace elements of metallurgical interest gives rise to serious background light loss correction problems. The direct analysis for many trace elements requires the use of concentrated salt solutions of alloy or metal sample. The origins of the background light loss phenomenon have been discussed in several prior publications. Evidence of molecular absorption has been reported by Koirtyohann and Pickett (1) by alkali halides using the long path hydrogenoxygen flame and air-natural gas flame. Alkaline earth oxide and hydroxide bands were observed in the hotter air-acetylene flame at wavelengths above 500 nm. Light scattering in flames was reported by Billings ( 2 )at various analytical wavelengths in air-acetylene and air-propane flames. An extensive investigation of light losses in flames was conducted by Fiorino 1474
( 3 ) .Scattering was considered to be the main source of light losses at most analytical wavelengths in flames. Winefordner et al. ( 4 ) studied light losses in flames and found that while the X4 wavelength dependence was approximated for light losses observed in their flames, the scattering could not be identified as Rayleigh scattering. Methods for solving problems of background light losses have involved either tedious separation schemes to remove and concentrate the analyte, or correction of the total absorbance signal using a continuum or nonabsorbing line source. Both methods of correcting absorbance readings have certain advantages and limitations which are important in choosing the optimum method. The following study was initiated to determine the techniques most effective in correcting absorbance signals for background light losses with flame atomization and the limitations of each in metallurgical systems.
EXPERIMENTAL Two atomic absorption spectrophotometers were used for absorbance measurements. The first instrument is a Techtron Model AA-4 which has been updated with the IM-5 log amplifier and DI-30 digital readout. Continuum source measurements were made using a Westinghouse WL 23490 hydrogen continuum lamp. The latest design of the lamp utilizes a cathode constructed from molybdenum. Earlier cathodes were constructed from nickel and significant nickel emission could be measured at sensitive lines. The second instrument used in these studies is a Perkin-Elmer Model 403 spectrophotometer equipped with light baffles, masked optics, and a side mounted photomultiplier. Continuum measurements were made using the deuterium lamp supplied with the instrument^ as the source. Spectra of metal salts in the premixed air-acetylene flame were obtained by point-by-point measurements with the Techtron instrument (0.99-nm bandpass), hydrogen continuum source, and the standard 10-cm air-acetylene slot burner. The acetylene flow rate was adjusted to give a relatively lean flame (0.97 l./min acetylene and 7.8 l./min air at 0 “C and 1 atmosphere) and the continuum beam was
ANALYTICAL CHEMISTRY, VOL. 40, NO. 11, SEPTEMBER 1976
