flame was not visible a t the ends of the furnace. The flow of air introduced to the precombustion tube was to support the combustion of the contaminants. Recovery. The retention times established from the authentic compounds were used to identify the selenium compounds. The efficiency of the sample trap was assessed by comparing the peak areas of dimethyl selenide (10 ng as Se) and dimethyl diselenide (16 ng as Se) spiked in an air sample and that directly injected onto the GC column without the use of the sample trap. The recovery for the two compounds for the trap was satisfactory (95-98%). As atomic adsorption was used as the detector of the GC, the response was directly related to the amount of selenium atomized. Both dimethyl selenide and dimethyl diselenide gave the same response expressed as Se. When peak areas were plotted vs. the absolute weight of both compounds expressed as Se, the calibration curve was linear up to a t least 50 ng. The accuracy of this method was tested by duplicate analyses of a synthetic air sample (250 ml) containing 10 ng and 16 ng (as Se), respectively, of dimethyl selenide and dimethyl diselenide. The results were 10 f 0.5 and 16 f 0.7 ng, respectively, for these two compounds. The precision of the method was evaluated by running replicate (8) analysis of synthetic air samples spiked with 10 ng and 16 ng as Se, respectively, of dimethyl selenide and dimethyl diselenide. The relative standard deviation of these levels was 8% and 7%, respectively. Figure 2 illus-
trates a typical recorder tracing of the two dimethyl selenides. The lower limit of measurement of the method depended on the scale expansion of the AA instrument. Since the conventional flame was not used, the Se hollow cathode energy could be operated with relatively low amplifier gains, which enabled higher scale expansion of the instrument. Under the present instrument parameters, 0.1 ng Se or lower could be detected with certainty.
LITERATURE CITED F. Challenger, Adv. Enzymol. Relat. Areas Mol. Bioi. 12, 429-491
(195 1). R. W. Fleming and M. Alexander, Appi. Microbial., 24,424-429 (1972). L. Barker and R. W. Fleming, Bull. Environ. Contam. Toxicol., 12, 308-
311 (1974). C. S. Evans, C. J. Asher, and C. M. Johnson, Aust. J. Biol. Sci., 21, 13-20 (1968). V. Vladkova, J. Ben& and J. Parizek. Radiochem. Radioand. Lett., I O , 251-258 (1972). C. S.Evans and C. M. Johnson, J. Chromatogr.,21, 202-206 (1966). E. G. Lewis, C. M. Johnson, and C. C. Delwiche, J. Agric. Food Chem., 14,638-640 (1966). J. Benes and V. Prochazkova,J. Chromatogr., 29,239-243 (1967). P. D. Goulden and P. Brooksbank,Anal. Chem.. 46, 1431-1436 (1974). R. C. Chu, P. Barron, and P. A. W. Baumgarner, Anal. Chem., 44, 1476-1479 (1972). V. Cantutl and G. P. Cartoni, J. Chromatogr. 32, 641-647 (1968). Y. K. Chau, P. T. S.Wong, and H. Saitoh, J. Chromatogr. Sci.. in press.
RECEIVEDfor review June 9, 1975. Accepted August 19, 1975.
Modification of a Graphite Tube Atomizer for Flameless Atomic Absorption Spectrometry Haleem J. Issaq’ and Walter L. Zlelinski, Jr. NCI Frederick Cancer Research Center, Frederick, Md. 2 170
The analysis of trace metals a t the nanogram and subnanogram levels in microsamples of clinical and biological nature (whole blood, serum, urine, tissue, etc.) is of great interest because of their toxicity, essentiality, and other factors (1-3). Nonetheless, the determination of trace metals in such samples by flame atomic absorption has been, and still is, fraught with the problems of size and complexity of the sample, sample pre-treatment, standard preparation, background effects, and sensitivity of the technique. Clearly, a need existed for the development of new microsampling systems and atomizing techniques to achieve the required sensitivity for microliter samples having minimum pre-treatment. This has been approached by the introduction of the graphite furnace (4-7), the heated graphite cell ( 8 ) ,the carbon rod atomizer (9, I O ) , the graphite cup (II), the gold plated graphite cup (12), and the tantalum strip (13, 14). Although the use of the above microsampling systems achieved about a 1000-fold improvement in sensitivity over the flame, their use did not eliminate all the problems involved in analyzing clinical and biological samples without pre-treatment. Some are restricted in the size of sample used (9, l o ) , while others have inherent background problems. For tube-like furnaces, spectral interferences due to light-scattering and molecular absorption are observed when complex matrix samples are used. The light scatterAuthor to whom correspondence should be addressed.
ing is caused by condensation of the vapor escaping from the carbon ends of the tube and formation of inorganic molecules ( 1 5 ) . With the carbon rod atomizer, similar effects are observed because of evaporation from the heated graphite surface into a cool inert gas atmosphere. When the inert gas atmosphere is replaced by a diffusion flame (9), condensation and recombination are prevented. When flameless devices are used, smoke from residual organic materials or salts can enter the sample beam, resulting in a severe nonspecific background absorption. With such devices, the need for background correction becomes the rule rather than the exception (16). If factors giving rise to smoke, recondensation, and recombination are eliminated, the use of background correction should become the exception, not the rule. In the present study, the effects of using new designs of the hollow graphite tube (U.S. Patent disclosure submitted), in which smoke and recondensation problems are substantially reduced, are discussed. As an evaluation of these new tube designs, preliminary results are given for the determination of Cu in whole blood and serum, and for the determination of P b and Hg in aqueous solutions.
EXPERIMENTAL Apparatus. A Perkin-Elmer Model 403 atomic absorption spectrophotometer with a strip chart recorder and a Cu, Pb, or Hg hollow cathode lamp (Westinghouse) was used. The 324.8-mm line was employed for the detection and measurement of the copper
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
2281
Top View
A
2 Srnrnaia
(dl
0.71
I
Side Vjk:
Figure 1. Top and side views of the modified tube design I
Figure 4. Comparison of the copper signal in whole blood at different charring temperatures using the design I: ( a ) 399 OC, ( b ) 460 OC, and (c)521 OC; and the unmodified tube: (d)410 OC, (e) 460 OC, (0 485OC, (g) 520 OC, and (h) 600 OC
I
I
Figure 2. Top and side views of the modified tube design I1 0 4-
Figure 5. Calibration curve for Pb in aqueous solution using the design l ( a )and the unmodified tube ( b )
A
0.71
I
la)
Figure 3. Comparison of the copper signal in serum at different charring temperatures using the design I: (e) 400 OC, ( b ) 500 OC, (c) 650 OC; and the unmodified tube: (d)400 OC, (e)500 OC, and (0650 OC
absorption signal. A Perkin-Elmer HGA-2000 hollow graphite atomizer was used with modification. Four 10-mm by 7-mm rectangular open windows (two on each side) were cut on opposite sides of the graphite tube a t 9 mm each from the center hole (enlarged from 1.5-mm to 2.5-mm i.d.). An alternate design was made by enlarging the windows to 13 mm by 7 mm, thus eliminating the four standard holes. Eppendorf microliter pipets having disposable plastic tips were used for sample introductions into the tubes. An alumel-chrome1 thermocouple was used to measure the temperature of the tube by inserting the thermocouple through the center hole of the graphite tube until it contacts the opposite internal wall of the tube. Reagents and Samples. Deionized water was used in all sample preparations. Whole blood and serum were obtained from normal mice. Lead nitrate and mercuric chloride (1000 ppm Fisher certified atomic absorption standards) were used after dilution with 2% (v/v) Hz02 to the desired concentration. Procedures. Ten +l of whole blood diluted 1:2 (v/v) with deionized water, or whole serum without dilution, were introduced directly into the tube without pre-treatment. Samples were dried a t 100 "C for 30 seconds, charred a t a selected temperature for 60 seconds, and atomized a t an optimum temperature for seven seconds. Since whole blood is quite viscous, dilution was necessary to ensure repeatability of pipetting, providing more reproducible results. The instrumental conditions for P b and Hg were those reported previously (17, 18). 2282
TIME
Figure 6. Comparison of 10 ng Hg in aqueous solution using the design l (a)and the unmodified tube (b)
RESULTS AND DISCUSSION The top and side views of two related modifications of the hollow graphite tube are shown in Figures 1 and 2. All analyses reported in this paper were carried out using the design shown in Figure 1. Samples of mouse whole blood and serum were analyzed for native Cu content at relatively low charring temperatures. Figures 3 and 4 show the copper signals obtained from serum and whole blood samples at different charring temperatures, comparing the performances of the modified and unmodified tube. The blood analyses illustrate the absence of a smoke peak for the modified tube at charring temperatures which result in such a peak for the unmodified tube. The tube illustrated in Figure 2 also showed the absence of a smoke peak at the same temperatures. It is likewise observed that this interference peak, attributed to smoke arising in the atomiza-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
1
(bl 2100
2300
0.7
2000
2100 2450
0.3.
0.1.
TIME 1 ng lead in Aqueous Solution
Figure 7. Comparison of 1 ng Pb signals at different atomizing temperatures using the unmodified tube ( a )and design I (b)
Table I. Comparison of Temperatures Using the P-E Design a n d the New Design Dial set a t
P-E design,
OC
OC
100 200 3 00 400 500 600 750
104 190 290 400 510 580 671
2.5-mm i d . , makes it easier for the analyst to introduce a syringe or micropipet through the hole into the tube without touching the sides of the hole. This ensures that a portion of the sample is not lost to the hole surface in the sample introduction phase. Furthermore, the sensitivities obtained with the new tube for the analyses of P b and Hg in aqueous solutions are better than those with the old tube (Figures 5-7). In this laboratory, trace metal analysis in microsamples of biological and clinical nature was improved by use of new designs of a hollow graphite tube. Preliminary results indicate that the new design is superior to the commercial design for such analyses. I t is felt that the new tube designs should provide an improved utility for direct analyses of clinical and biological samples with little or no sample preparation. A study on the use of these tube designs for the determination of volatile metals in blood, serum, and tissues is under way and will be reported later. ACKNOWLEDGMENT
New design,
The authors express their sincere thanks and appreciation to L. Carignan for providing the blood and serum samples, and to R. Wiley for cutting the windows in the graphite tube.
OC
225 426 660 855 926 1054 1164
LITERATURE C I T E D
~
tion step due largely to incomplete tube clearance of decomposed organic matter in the charring step, diminishes and disappears at higher charring temperatures. The absence of this peak when using the modified tube designs is hypothesized as being due to a) the minimization of the internal surface area on which mobile particles may deposit during the charring step; b) the greater removal of such particles through the windows by the inert gas flow; and c) the provision of a more uniform distribution of thermal energy due to the lowering of the thermal mass of the tube. In the case of the unmodified tube, the temperature a t the center of the tube appears higher than a t the ends. It is readily observed that in the modified tube, the center area between the windows, 2 cm is glowing red a t 650 OC; while the standard design has a glowing band of less than 1 cm a t the same temperature. The presence of a temperature profile in the unmodified tube enhances the deposition of particles in the cooler regions of the tube. Table I shows that the temperature of the new design is higher than that of the unmodified tube when the same instrument setting is used. The enlargement of the center hole from 1.5-mm to
0
E. J. Underwood, "Trace Elements in Human and Animal Nutrition", 3rd ed., Academic Press, New York, N.Y., 1971. W. Mertz and W. E. Cornatzer, Ed.. "Newer Trace Elements in Nutrition", Marcel Dekker, New York, N.Y., 1971. E. L. Kothy, Ed., "Trace Elements in the Environment", American Chemical Society, Washington, D.C., 1973. A. A. Kbg. Astrophys. J., 75, 379 (1932). 9. V. L'Vov, Spectrochim. Acta,'l7, 761 (1961). B.V. L'vov, Spectrochim. Acta, Part 6, 24, 53 (1969). R. Woodriff and G. Ramelow, Spectrochim. Acta, Part 8, 23, 665 (1968). H. Massman. Spectrochim Acta, Part 6,24, 53 (1969). M. D. Amos, P. A. Bennet. K. G. Brodie. P. W. Y. Lung, and J. P. Matousek, Anal. Chem., 43, 21 1 (197 1). T. S. West and X. K. Williams, Anal. Chim. Acta, 45, 27 (1969). F. Dollmsek and J. Stupar, Analyst(London), 98,841 (1973). J. F. Lech, D. D. Siemer, and R. Woodriff. Spectrochim. Acta, Part 6, 28, 435 (1973). H. M. Donega and T. E. Burgers, Anal. Chem., 42, 1521 (1970). J. Y. Hwang, P. A. Ullucci, and S. B. Smith, Jr., Amer. Lab., 3 (8),41 (1971). J. P. Matousek. Amer. Lab., 3, (6), 45 (1971). H. L. Kahn and D. C. Manning, Amer. Lab., 4 (8),51 (1972). H. J. lssaq and W. L. Zielinski, Jr., Anal. Chem., 46, 1328 (1974). H. J. lssaq and W. L. Zielinski, Jr., Anal. Chem.. 46, 1436 (1974).
RECEIVEDfor review July 23, 1974. Accepted August 7, 1975. Presented in part at the 1975 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 1975. Research sponsored by the National Cancer Institute under Contract No. NO;-CO-25423 with Litton Bionetics, Inc.
Relative Molar Response of a Flame Ionization Detector to Some Organic Compounds Containing Sulfur W. G. Filby, K. Gunther, and R.-D. Penzhorn lnstitut fur Radiochemie, Kernforschungszentrum, Karlsruhe, West Germany
The relative molar response (RMR) to flame ionization detectors (FID) of many oxygen-containing organic compounds has been determined in recent years (1,2).Ackman
showed that, for these compounds, the RMR could be predicted reasonably well when a specific response is assigned to certain polyatomic groups (3).During the course of our
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
2283
