essary to boil the samples for a longer time in the pretreatment step. It is believed, however, that these levels are unlikely to be experienced in the analysis of natural waters and that the 15 minutes specified for the time of boiling is adequate for natural waters analysis.
RECEIVED for review November 26, 1973. Accepted March 19, 1974. This paper was presented at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, by P. D. Goulden, March 5, 1974 (Paper No. 153).
Hot Atomic Absorption Spectrometry Method for the Determination of Mercury at the Nanogram and Subnanogram Level Haleem J. Issaq' and W. L. Zielinski, Jr. NCI Frederick Cancer Research Center, Frederick, Md. 2 170 1
Atomic absorption spectrometry having a heated graphite atomizer was used to observe a 50-fold Hg signal enhancement when hydrogen peroxide was added ( 1 % v/v) to aqueous mercury solutions. The sensitivity and detection pg/1% absorption and 1.6 X pg, limits were 2 X respectively. A precision of 1.3 % and signal-to-noise ratio of 88 were obtained. Parameters which influence the analytical sensitivity were evaluated. There was no change in the mercury signal when hydrogen peroxide was varied from 0.5% to 4 % (v/v). The optimum drying and atomizing temperature ranges were 115-140 O C and 2150-2300 OC, respectively, and an optimum drying time range of 50 to 120 seconds was observed. The method described here is faster, simpler, and more sensitive than the flameless vapor method, and is less prone to contamination and interference effects.
mining mercury, which is now widely used. This method is based upon the generation of a mercury vapor which is continuously recirculated until a maximum signal is attained. Rains and Menis (8) studied the vapor method, discussed its advantages and disadvantages, and introduced some improvements. The use of the technique for the determination of nanogram quantities of mercury in different samples, using a 30-cm long absorption tube was reported. Despite improvements, the method has interference and contamination problems, and the use of ultra-high purity reagents was recommended (8). Recent developments in atomic absorption (tantalum strip, HGA, and carbon rod atomizer) were either not fully utilized or failed to give sufficiently high sensitivities at the nanogram and subnanogram levels. This is principally attributed to the high volatility of mercury (0.001 mm at 18 "C which rises to 0.27 mm at 100 OC (2). Recently, the determination of mercury in air samples by flameless atomic absorption using gold plated graphite cups was reported
Mercury and many of its compounds exhibit a high mammalian toxicity which can be cumulative. In the natural environment, mercury is found broadly distributed. Gibbs et al. ( I ) reported an average daily intake of 0.02 mg mercury per day by adult man. Recent analyses have shown mercury to be present in the lungs, kidneys, hair, teeth, nails, and skin ( 2 ) . Trace mercury levels have been determined by neutron activation (3, 4), but this method is not widely used because of its complexity and high cost. In contrast, the application of atomic absorption to mercury analysis is a fast and inexpensive technique. The sensitivity of flame atomic absorption is in the order of 10 Mg/ml ( 5 ) .The method of choice for mercury determinations a t the microgram and submicrogram level has been the flameless method. A review of these methods was presented by Manning (6). Hatch and Ott (7) used a cold vapor technique for deter-
(9).
1 Author
to whom correspondence should be sent.
(1) 0. S. Gibbs, H. Pond, and G. A. Hansmann, J. Pharmacol. Exp. Ther., 72, 16 (1941). (2) E. J. Underwood, "Trace Elements in Human Nutrition." 3rd ed., Academic Press, London, 1971. (3) C. Kellershohn. D. Comar, and C. Lopeoc, J. Lab Chim. M e d . , 66, 168 (1965). (4) H. Smith and J. M. A. Lenihan. in "Methods in Forensic Science," A. S. Curry, Ed., Wiley. New York, N.Y., 1964. (5) I. Rubeska and 6.Moldan, "Atomic Adsorption Sepctrophotometry," 2nd Impression, CRC Press, Cleveland, Ohio, 1971, p 135. (6) D. C. Manning, At. Absorption Newslett., 9, 97 (1970). (7) W. R. Hatch and W. L. Ott, Anal. Chem., 40, 2085 (1968).
1436
In this paper, the use of the hollow graphite atomizer for the determination of mercury at the nanogram and subnanogram level is reported. Hydrogen peroxide is used for stabilization of mercury. Several parameters which affect the sensitivity of the method were evaluated and are discussed.
EXPERIMENTAL A p p a r a t u s . A Perkin-Elmer Model 403 atomic absorption spec-
trophotometer equipped with a deuterium background corrector, a rapid-response strip chart recorder, and a Westinghouse mercury hollow cathode lamp was used for this study. The burner assembly was replaced by a Perkin-Elmer HGA-2000 graphite furnace without further modification. The containers were precleaned with concentrated nitric acid followed by five rinses with deionized water. Eppendorf microliter pipets having disposable plastic tips were used for sample introduction. R e a g e n t s a n d M a t e r i a l s . All reagents were of analytical grade. Deionized water was used for all sample preparations. The stock mercury solution was a 1000 ppm (1000 Fglrnl) certified atomic absorption standard obtained from Fisher Scientific. All mercury solutions used experimentally in which H2 0 2 and " 0 3 were used, were (v/v). I n t e r m e d i a t e S t a n d a r d s . These were prepared by pipetting the required amount of the stock solution into the appropriate container which was then accurately brought to volume with deionized water. The sample containers were volumetric flasks of (8) T. C. Rains and D.Menis, J. Ass. Offic. Anal. Chem., 5 5 , 1339 (1972). (9) J. F. Lech, D. D.Siemer. and R. Woodriff, Spectrochim. Acta, 288, 435 (1973).
ANALYTICAL CHEMISTRY, VOL. 46, NO. 11, SEPTEMBER 1974
L
h
M
Time min
10 lec
Time ( m i " ,
Figure 1. Mercury signal in 2 %
"03
Figure 2. Comparison of the signal of 20 ng mercury in 2 % H N 0 3 (a),2 % "03 2 % H 2 0 2 ( b ) and 2 % H 2 0 2 (c)
+
M
10rec
(a)and 2 %
H202
(6)
Pyrex brand (Fisher Scientific), and 125-ml polyethylene bottles (Sprayon Prod. Inc., Cleveland, Ohio). Procedure. The spectrometer was operated a t an absorption line of 253.7 nm with a lamp current of 8 mA and a 1-mm slit width. An aliquot of the test sample was introduced into the graphite tube furnace. The sample was then dried for 60 seconds and atomized for 5 seconds a t the optimum temperatures. The graphite furnace was operated with a nitrogen purge and a water flow of approximately 1.0 and 3 literdminute, respectively. Standard graphite tubes were employed.
RESULTS AND DISCUSSION The use of hydrogen peroxide for the stabilization of lead in aqueous solutions has been previously reported (10). Hz02 prevented the loss of lead ions to the surfaces of borosilicate glass and polyethylene containers. Since mercury in aqueous solutions was also reported to adsorb to borosilicate and polyethlene surfaces (11, 12), we speculated that H202 might be used to prevent the adsorption of mercury. Preliminary analyses of 2-ppm mercury solutions, to which Hz02 had been added, showed an increase in the mercury signal. This was initially interpreted as a desorption of mercury from the container surface. However, when was added to 2-ppm mercury solutions, no ap2% "03 preciable change in the signal was observed. This was interesting in view of the fact that nitric acid is used as a preservative for aqueous mercury solutions (11, 12). Furthermore, the mercury signal in 2% nitric acid was far less than that in 2% H2Oz (See Figure 1).Finally, it was observed that an addition of 1 ml of Hz02 to 50 ml of a 2-ppm Hg solution in 2% "03 resulted in a signal enhancement equal to that of 2 ppm Hg in 2% HzO2 (Figure 2). These results indicated that an adsorption/desorption phenomenon was not the operating factor. Furthermore, when solutions containing 2 ppm mercury in 2% HzOz were stored in borosilicate glass or polyethylene containers, there was no significant loss of signal after one week. A second experiment was carried out to verifyhegate adsorption contributions to reduced mercury signals. Samples of 2-ppm mercury solutions in 2% H N 0 3 and water, respectively, were stored for 24 hours in glass. The samples were then transferred to precleaned containers. The addition of 1 ml of H202 to such solutions resulted in a signal equivalent to that of 2-ppm mercury solutions containing H202. Furthermore, when H202 or 10% "0.7 was added to the rinsed glass containers used to store these solutions, no measurable Hg signal was observed. These results con(10) H . J. lssaq and W. L. Zielinski. A m i . Chem.. 46, 1328 (1974) ( 1 1) R . V. Coyne and J. A. Collins, Anal. Chem., 40, 1093 (1972). (12) C. Feldman, Ana!. Chem., 46, 99 (1974).
.
151
75
100
.
.
125
150
Drying Temperature
.
175
.
200
I"C1
Figure 3. Effect of drying temperature on the signal of 20 ng mercury in 2 % H 2 0 2 at an atomizing temperature of 2200 OC
firmed the absence of mercury adsorption by borosilicate glass. When the same experiment was repeated with polyethylene containers, the results were the same. The addition of both H202 and H N 0 3 to either container type likewise produced no Hg signal. It is dually clear from the above that not only are container adsorption effects absent for aqueous mercury solutions, but that hydrogen peroxide functions as a stabilizer of such solutions, ensuring precise analytical measurements. During the course of work to establish the procedure for routine analysis, several parameters were evaluated, uiz., the effect of HZOZconcentration, drying time and temperature, atomizing temperature, and inert gas flow. Effect of Hz02 Concentration. To determine the effect of H202 on the analytical sensitivity, a mercury solution of 2 pg/ml in 0.5, 1.0, 2.0, and 4.0% HzOz was used. Samples of 10 pl were pipetted into a graphite tube. The difference in the signal between samples prepared in 0.5% Hz02 and 4.0% Hz02 is less than 1%. Effect of Drying Temperature. The effect of drying temperature on the analytical sensitivity is shown in Figure 3. This figure shows an optimum at 125 "C and above. Effect of Drying Time. Samples of 10 p1 of a solution containing 2 pg/ml mercury in 2% H202 were used a t a drying temperature of 125 O C . The drying time was varied from 40 to 120 seconds. No significant change in the signal was observed. Effect of Atomizing Temperature. Samples of 25 pl of 2 pg/ml Hg in 2% HzOz were used to determine the effect of atomizing temperature on the analytical sensitivity a t a
ANALYTICAL C H E M I S T R Y , VOL. 46, NO. 1 1 , SEPTEMBER 1974
1437
025-
020-
015-
fa 0 10-
1700
1850
2000
2150
2300
2450
Atomizing T e r n p e i m ~ rl~" C l
0 05
Figure 4. Effect of atomizing temperature on the signal of 20 ng mercury in 2 % H 2 0 2 drying time of 60 seconds and a drying temperature of 125 "C. Figure 4 shows that the optimum temperature working range lies between 2100-2300 "C Effect of Inert Gas Flow. As expected, the signal increased when the inert gas flow was decreased. This is related to the residence time of free atoms in the graphite tube. A flow rate of less than 0.5 liter per minute is desirable. Precision and Reproducibility. To measure the precision of the method, a solution of 250 ng Hg/ml was used. Six samples of 25 pl each (6.25 ng) were pipetted into the graphite tube and their absorbance was measured in terms of peak height. The mean and standard deviation was found to be 7.76 f 0.10, respectively. This gives a relative standard deviation of 1.29; hence, an analytical reproducibility within 1.3%. The day to day reproducibility of the results over a period of 3 days was within 1.0%. Signal-to-Noise Ratio. Heiftje (13) defined a signalto-noise ratio S/N, in which S is the mean value and N is the excursion from the mean and
C(S - A S ) '
112
This equation is the same as that used in computing the standard deviation ( N ) of a series ( i ) of samples whose mean is ( S ) .Thus, SIN = meadstandard deviation. Using this definition and the values found for S and N in the previous section, the signal-to-noise ratio was 88. Calibration Curve. The calibration curve for mercury a t optimum conditions is shown in Figure 5 . Peak heights were measured in centimeters and converted to absorbance units. This gave a sensitivity of 2.4 X pg/l% absorption (0.0044 absorbance unit). Detection Limit. The detection limit is defined as the concentration that produces an absorption signal equivalent to twice the magnitude in background (zero absorption) (14). In accordance with this definition, the detection limit of this method is less than 0.1 nanogram. Use of H202 for Mercury Determination by Flame Atomic Absorption. The use of hydrogen peroxide to enhance the sensitivity of the mercury signal by flame AA was investigated. Although there was a signal enhancement when the HGA was used for mercury solutions containing (13) G. M. Heiftje, Anal. Chern..44(6). 81A (1972). (14) W. Slavin, "Atomic Absorption Spectroscopy," lnterscience Publishers, New York. N.Y., 1966.
1438
00
25
50
15
100
125
Hg i n g l
Figure 5. Calibration curve for mercury in 2 % H 2 0 2 analyzed by atomic absorption spectrometry using the Hollow Graphite Atomizer (HGA-2000)
hydrogen peroxide, no signal enhancement was observed in the flame mode. When 80 kg/ml Hg solutions in 2% H 2 0 2 were nebulized, comparable signals and or in 2% "03 were obtained. The observation of signal enhancement by H202 in the HGA mode and the absence of such an effect in the flame mode is believed to be due to the formation of a mercury-peroxy complex (15). It may be hypothesized that when Hg samples in H202 are pipetted into the graphite tube, excess H202 is vaporized along with the solvent during drying, leaving a stable mercury peroxy complex which restricts mercury vaporization losses. When the atomizing temperature is reached, free mercury atoms are liberated. It should be noted that the graphite tube furnishes a favorable reducing atmosphere. Evidence of a stable complex formation is provided by the observation that a smaller signal results at 1700 "C than a t 2300 "C. In the flame mode, the presence of H202 has no effect. The method presented here is a hot method for mercury determination at the nanogram and subnanogram level using microliter samples and the graphite tube atomizer. This method is extremely simple, rapid, and free of interferences encountered in the vapor method. Microliter samples can be analyzed within seconds or minutes depending on the nature and matrix of the sample. The application of this method to clinical and environmental samples is presently under investigation.
ACKNOWLEDGMENT The authors offer their appreciation to T. C. Rains of the National Bureau of Standards and to L. P. Morgenthaler of Fisher Scientific for their helpful comments. RECEIVEDfor review November 26, 1973. Accepted May 14, 1974. This Research was sponsored by the National Cancer Institute under contract NIH-NCI-E-72-3294 with Litton Bionetics, Inc. (15) L. P. Morgenthaler, Fisher Scientific, Waltham, Mass., personal communication, 1973.
ANALYTICAL C H E M I S T R Y , VOL. 46, NO. 11, SEPTEMBER 1974
