Rapid Pyrolytic Method to Determine Total Mercury in Fish Raymond J. Thomas, Richard A. Hagstrom, and Edward J. Kuchar Olin Corporation, Central Analytical Department, Chemicals Group, 275 Winchester Avenue, New Haven, Conn. 06504
A rapid pyrolytic procedure to determine the total mercury content in fish is described. A weighed amount of homogenized fish tissue is combusted in a flowing air stream at 900 OC. Elemental mercury vapor i s expelled into the carrier stream and, after passing through absorbent traps to remove possible interfering gases, is detected and measured in an ultraviolet photometer. Relative error is approximately =klOoJ, for inorganic and organic mercury over a linear response range of 0.05 to 3.0 ppm at the sample size used. Approximately 8 minutes are required for a single determination. Comparison with other mercury analytical methods is described. THE PHOTOMETKIC DETERMINATION of elemental mercury by ultraviolet light absorption at 253.7 nm has become the basis of many modern methods, replacing the older colorimetric dithizone techniques for trace mercury determination. Dynamic systems described by Hatch ( I ) , Lindstadt (2), Rathje (3), and others make use of the ease of reducing mercury from the mercuric state to elemental mercury with complete removal from solution by a gas stream into an absorption cell of an ultraviolet photometer. Tissue analyses have been cumbersome because of the methods used to isolate mercury. These types of samples are usually chemically treated to digest the tissue and convert all mercury compounds to an inorganic form prior to reduction for measurement. Jacobs ( 4 ) used this method with isolation of mercury by dithizone, volatilization of elemental mercury, and photometric detection. Ulfvarson (5) applied this technique, but used a gold collector instead of dithizone. Mercury was volatilized from the gold by heating and passed through a cell of an ultraviolet detector. Total mercury analysis of fish by acid digestion and permanganate oxidation has been described by Uthe (6). Mercury is then released by chemical reduction and detected photometrically. This is essentially the method used in this laboratory for total mercury analysis of fish until it was found that consistently low and erratic results were obtained on fish containing fatty tissue. Mercury can be isolated from tissue by direct combustion at high temperatures in air or oxygen. Lidums (7) has combusted biological samples and concentrated released mercury vapor on a noble metal. Heating of the collector allowed release of the trapped mercury and detection was made photometrically. Pappas and Rosenberg (8) have combusted biological samples, fish included, in a Schoniger flask, concentrated on cadmium sulfide pads, with burning of the pads to release mercury vapor. Again, mercury was detected by ultraviolet detection. (1) (2) (3) (4)
W. R. Hatch and W. L. Ott, ANAL.CHEM. 40, 2085 (1968). G. Lindstadt, Analyst. 95, 264-71 (1970). A. E. Rathje, Amer. Ind. H y g . Ass. J., 30, 126-32 (1969). M. B. Jacobs, S. Yamazuchi, L. J. Goldwater, and H. Gilbert,
ibid., 21,475 (1960). ( 5 ) U. Ulfvarson, Acta Chem. Scand., 21, 641 (1967). (6) J. F. Uthe, A. J. Armstron, and M. P. Stainton, J . Fish. Res. Bd. Can. 27,805-1 1 (1970). (7) V . Lidums and U. Wlfvarson, Acta Chem. Scand., 22, 2150-6 (1968). ( 8 ) E. G. Pappas and L. A. Rosenberg, J . Ass. Ofic.Anal. Chem., 49,782-93 (1966). 512
ANALYTICAL CHEMISTRY, VOL. 44, NO. 3, MARCH 1972
Lindstrom (9) was able to analyze urine samples photometrically by burning in any oxyhydrogen flame. Interfering substances were filtered out of the gas stream before the vapor passed into the optical cell of the ultraviolet detector and measurement was made without the need of concentration. This paper describes a dynamic approach by which the total mercury in fish is pyrolytically converted to the elemental form and quantitated photometrically. The samples are burned in a flowing air stream of 900 "C and the vapors are passed over copper oxide at 850 "C to ensure complete combustion. Possible interfering gases that would absorb in the ultraviolet region of 253.7 nm, such as oxides of sulfur and nitrogen, are removed by silver wire heated at 450 "C, a caustic scrubber, and an ascarite filter. Halides which could recombine with the mercury formed are also removed with the heated silver wire (10). The flowing air stream containing the elemental mercury is then passed through an ultraviolet photometer. Electronic integration of the detector signal is employed to improve analysis statistics. EXPERIMENTAL
The combustion train is typical micro-analytical equipment used for carbon-hydrogen analysis. Assembly of the apparatus is shown in Figure 1. In this study, a single, three-section furnace, each section separately temperature controlled, was used. While three individual furnaces may be used, care must be exercised to eliminate cold zones between furnaces. The amount of sample to be pyrolyzed must be balanced with the air flow rate and length of effective oxidant. For the geometry described, it has been found that 50 to 150 mg of a fish samples can be completely pyrolyzed at 900 "C in a 9 Ipm air stream using a 10-inch length of copper oxide heated at 850 "C. An insufficient length of copper oxide will not permit complete combustion, resulting in low mercury recoveries in organic based materials. A 7-inch length of fine silver wire heated at 450 "C will effectively remove halides and oxides of sulfur from th.: air stream. The dimensions of the remaining part of the train are not critical. A 100-ml round bottom flask is used for the caustic bubbler. The added volume lends stability to the system and reproducibility is increased. A four-way stopcock added before the magnesium perchlorate-ascarite tube enables a constant flow to be maintained through the combustion tube without passing through the optical cell when samples are not being pyrolyzed, thus extending the life of the absorbers. With the geometry described, the solid absorbers should be changed after 4 hours of operation. The detection of mercury vapor in an air stream by ultraviolet spectrometry is convenient, and sensitivity was obtained by using a Laboratory Data Control Mercury Monitor. The unit has a full scale response of 0.005 pg of mercury. For this study, an attenuated range giving a linear response from 0.01 to 0.10 pg of mercury was used. A digital integrator was connected directly to the photometer, and the recorder was powered from the integrator output. (9) 0. Lindstrom, ANAL.CHEM.31,461-7 (1959). (10) A. Steyermark, "Quantitative Organic Micro Analysis," Academic Press, New York and London, 1961, p 234.
jf==
-
VACUUM PUMP
ROTAMETER
4-WAY VALVE
PERCHLORATE INTEGRATOR
Figure 1. Combustion apparatus with ultraviolet mercury detector 1. First furnace, 900 "C combustion zone 2.
Second furnace, 850 "C packed with 10-in. copper oxide
3. Third furnace, 450 "C packed with 7-in. silver wire
It became evident in initial work that mercury was not released from fish in the same manner as from water standards. Therefore, peak height measurements could not be used and an integrator was required to obtain peak areas for adequate measurement precision. The recorder serves as a useful operational guide, and is not strictly required for analysis. Apparatus. A Laboratory Data Control Mercury Monitor was used as the ultraviolet photometer. This instrument has an absorption cell length of 30 cm and an internal volume of 14 ml. The output of the photometer was connected to a Perkin-Elmer Model 165 recorder but any suitable 10-mV recorder with a 1-second pen response is suitable. For the digital readout system, both an Infotronics Model CRS-11 HSB and a n Autolab 6300 Integrator have been used successfully. The combustion tube was a 36-inch, 8-mm, i.d. quartz tube; the combustion boat was micro-nickel, 2 inches long by a//l8 inch wide by 6/32 inch deep. The heating ovens were a multiple unit from the HeviDuty Electric Company; three units are respectively 9 inches, 12 inches, and 5 inches long. Reagents. Copper oxide (Mallinckrodt), A.R. wire form, was water washed to remove fines. Fine silver wool, size No. 8, 0.005 inch, (Engelhard Minerals and Chemicals Corporation) was used without treatment. The magnesium perchlorate used was anhydrous, reagent grade, purchased from Fisher Scientific Company. Ascarite, 8 to 20 mesh, was purchased from Arthur H. Thomas Company. Sodium hydroxide (0.5N) was aerated to remove possible mercury contamination. Mercuric acetate, ACS, reagent grade, B& A, 1.595 grams, was dissolved in distilled HzO containing 5 ml of concentrated nitric acid and diluted to 100 ml. Dilute 1/100 for 100 pg/ml solution and then 5/100 for a 5 pg/ml working solution. Procedure. After the apparatus has been allowed to warm up, pull air through the combustion train at a rate of about 9 liters/minute. It is critical that air flow rate be kept constant. Prepare a standard curve with 5 to 20 ~1 of a 5 pg/ml standard solution of mercuric acetate. Combust each standard and record the area measurement from the integrator. The recorder is used to make a permanent trace of the values obtained. A calibration curve relating integrator area units us. micrograms of mercury is prepared. A straight line relationship is obtained from 0.01 to 0.10 pg of mercury. Fish to be analyzed are received frozen and are fileted,
homogenized, and kept frozen prior to analysis. Approximately 50 to 150 mg of frozen fish homogenate are weighed into a combustion boat. The boat is pushed into the hot combustion zone and left there until the readout system indicates that pyrolysis is complete. The amount of mercury in micrograms contained in the weighed portion of fish is obtained from the standard curve, and mercury concentration in ppm of the fish is calculated. RESULTS AND DISCUSSION Since the advent of the crisis, this laboratory had obtained the total mercury concentration in fish by a chemical digestion technique with hot sulfuric acid, followed by permanganate oxidation to convert mercury to an inorganic form. Excess permanganate was reduced with hydroxyl amine hydrochloride prior to reduction of mercury with stannous chloride in a flowing nitrogen stream. Quantitation was effected using an ultraviolet photometer at 253.7 nm. Methyl mercury was extracted using the procedure proposed by Rudling (11). Quantitation was obtained using a gas chromatographic separation with electron capture detection. A statistical analysis of results accumulated over several months indicated the possibility of a method bias. As a result, methyl mercury concentrations were occasionally higher than total mercury concentrations. A further review of the data indicated that this trend was more apparent in fish of relatively high fat content. Discrepancies of this type have also been reported by Wood (12). To test both analytical methods in routine analysis, a fish filet was uniformly homogenized and analyzed repeatedly by both methods. This was a selected Buffalo specimen that had previously given poor method agreement. The results are shown in Table I. As can be seen for this fish species, the methyl mercury concentration was more than twice the total mercury concen(11) L. Rudling, Swedish Institute of Air and Water Pollution Research, unpublished data, 1971. (12) J. M. Wood, Chem. Eng. News, 49(27), 22-30 (1971).
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513
Table I. Comparison of Total Mercury and Methyl Mercury Methods Total Methyl mercury, mercury, -
X
(.t)
Coefficient of variation, %
PPm
PPm
0.44 0.07
1.10 0.10
16
9.1
Table 11. Methyl Mercuric Iodide Recovery Total mercury Mercury as methyl recovery" Hg recovery Added, Recovery, Recovery, fig Pg % Pg % 0.0 0.1 ... 0.06 ... 0.47 0.94 1.41 1.88 2.34 2.81 3.28 3.75 4.70
0.24 0.49 0.91 0.81 1.51 1.78 2.18 2.79 3.68
53 51 64 48 54 63 66 74 78
0.51 1.05 1.54 1.96 2.39 2.94 3.24 3.50 4.80
108 112 109 104 102 105 99 93 102
Potassium permanganate used as oxidant.
0.0 0.22 0.61 0.94 1.17 1.51 1.82 2.04 2.59 2.95
... 55 78 80 75 78 78 75 83 76
0.0 0.35 0.75 1.08 1.52 1.80 2.64 2.47 2.99 3.44
1.34 0.123
=
Coefficient of variation,
92 36
N = ~~
Comparison of Mercury Analysis in Various Fish Specimens Combustion Digestion Mercury as method method methyl total Hg, total Hg, Hg, Fish ppm ppm PPm Carp 2.7 1.5 2.4
Table V.
Shiner
0.39
Chub
0.19
Buffalo
Carp
0.33 0.53 0.44 0.54 0.64 0.52 0.25 0.26 0.52 0.47 0.25
Crappie
0.14
Crappie
0.20
Carp Blue Cat
1.6 0.33 0.28 0.10 0.09 0.14 0.12
2.3 0.33 0.35 0.16 0.10 0.41
0.28 0.29
0.47
0.21 0.27 0.37 0.55 0.31 0.34 0.12 0.12 0.19 0.14
0.21 0.42 0.26 0.22
0.09 0.11 0.13 0.11
... 91 96 92 97 92 113 91 96 88
tration. While a definite method bias was indicated, a further test was undertaken to ascertain which of the two procedures was in error. In this test, synthetic mixtures of 99f % purity methyl mercuric iodide were prepared covering a concentration range of 0 to 4 pg in 0.5-pg increments. The samples were extracted using the methyl mercury procedure, then split so that one half of the sample was analyzed using the total mercury method and the other half analyzed by the methyl mercury procedure. For the total mercury determinations, potassium permanganate was used as an oxidant. The results are given in Table 11. From a regression analysis of the data, the slope indicated a methyl mercury recovery of 96.7%. At the concentration levels employed, this was considered adequate. Poor recoveries were obtained for all concentration levels with the total mercury method. However, recovery decreased with decreasing mercury concentration. A second series of analyses was obtained similar to the first, except that a combination potassium permanganate-potassium persulfate oxidant is employed. The results are shown in Table 111. In this series, a 91.3% recovery of methyl mercury was obtained. While persulfate appears to improve 514
x= u(*)
Channel Cat
Table 111. Methyl Mercuric Iodide Recovery Total mercury Mercury as methyl mercury recovery ~ recovery _ _ _ _ Added, Recovery, Recovery, fig fig % P8 % 0.0 0.39 0.78 1.17 1.56 1.95 2.34 2.73 3.12 3.90
Table IV. Total Mercury Recovery Combustion Method
ANALYTICAL CHEMISTRY, VOL. 44, NO. 3, MARCH 1972
the total mercury recovery, the overall improvement is less than adequate for routine testing. It was concluded, therefore, that the methyl mercury method was adequate for routine testing, and that method bias was attributable to the poor recovery of the total mercury method. While considerable effort was expended in testing improved digestion and oxidation procedures for the total mercury procedure, any improvements were made at the expense of lengthening an already long analysis time. In addition, with chemical treatment, each use of a new chemical enhanced the possibility of sample contamination with trace mercury from a secondary source. The pyrolytic approach eliminated many of the problems associated with the chemical procedure. As described, no prior treatment of the sample is required. Further, a single analysis of a homogenized fish sample can be obtained in approximately 8 minutes. An analysis of the same Buffalo species studied gave the results shown in Table IV for 36 separate determinations over a 4-day period. As can be seen, the results are higher than the methyl mercury results shown previously. Westoo (13) has reported that methyl mercury concentration values range from 52 to 9 7 z of the total mercury concentration with a mean value of 7 6 % of the total mercury. A comparison of mercury by the three analysis methods is given in Table V for various species of fish. It is evident that the pyrolytic system gave, on the average, more reasonable results. The combustion temperature of 900 "C was sufficient to burn carbon with the aid of copper oxide and also to pyrolyze (13) G. Westoo, Acta Chem. Scand., 21, 1790-1800 (1967).
CONCLUSIONS
organic and inorganic forms of mercury. Standards prepared from mercuric acetate, mercuric chloride, methyl mercury chloride, and methyl mercury iodide were pyrolyzed and areas obtained for mercury measurement were equivalent. Acid gases which would give positive readings with the ultraviolet detector were effectively scrubbed from the gas stream, while mercury vapor passed through. One-ml quantities of SO2 and NOz gas introduced into the open end of the combustion tube gave no reading on the detector. Area measurements obtained for pyrolyzed mercuric acetate standards were equivalent to area measurements obtained by the stannous chloride reduction method. It became evident early in the studies that recorder peak heights were not adequate for mercury quantitation. The cause was traced to the nonreproducible rate of mercury release from various specimens in the combustion zone of the furnace. Measurement of peak area with a digital integrator overcame this problem and, in effect, further decreased the overall analysis time.
A rapid and convenient analysis scheme for the determination of total mercury in fish has been presented. While not tried, its possible application for mercury analysis in other materials is obvious. Measurement precision appears to be adequate for present requirements and further increases in sensitivity are inherent to the system if required. ACKNOWLEDGMENT
The authors thank R. D. Householder for the data provided in the chemical procedures for total mercury, w. A. Nichols for the methyl mercury analytical data, and R. C. Rittner for assistance in establishing the micro-elemental conditions. RECEIVED for review September 3, 1971. Accepted October 29,1971.
Homogenization of Nonconducting Samples for Spark Source Mass Spectrometric Analysis G. H. Morrison and A. M. Rothenberg Department of Chemistry, Cornell University, Ithaca, N . Y. 14850 A method has been developed for the homogenization of powdered geological samplesfor use in spark source mass spectrometry. Uniform blending with high purity graphite has been achieved and precision of analysis of +lo% attained. In the application of the procedure to material from the meteorite Allende, 28 elements were determined with an average precision of *7%.
MANYINVESTIGATORS have evaluated the precision and accuracy of determining average elemental concentrations by spark source mass spectrometry employing photoplate detection. However, all of these studies have involved the use of fairly homogeneous metal samples containing a limited number of well-characterized impurities (1-5). Halliday et al. ( I ) have determined the reproducibility of analysis of a homogeneous aluminum sample to be 10-20x. Ahearn (2) reported standard deviations of 5-12x for CA2 copper standard and 10-20x for the CA4 sample. Vossen (3) obtained approximately 10 relative standard deviation for the aluminum standard 1791 but only 13-20z for steel sample SS53. Franzen et al. ( 4 , 5 ) made detailed studies of the precision of photoplate evaluation and presented a computer program for its improvement. However, many materials currently being studied by spark source mass spectrometry are nonconducting substances of a complex nature where sample heterogeneity is a primary source of error (6-12). In addition, these samples must be
x
(1) J. S. Halliday, P. Swift, and W. A. Wolstenholme, Aduan. Mass Spectrom. 3, 143 (1966). (2) A. J. Ahearn in “Trace Characterization, Chemical and Physical,” Nut. Bur. Stand. (US.)Monogr. 100, 1967. (3) P. Vossen, ANAL.CHEM.,40, 632 (1968). (4) J. Franzen and K. D. Schuy, 2.Anal. Chem., 225, 295 (1967). (5) J. Franzen and K. D. Schuy, Z . Natdrforsch., 219, 1479 (1966).
powdered and uniformly blended with graphite (or another suitable conducting matrix) in order to sustain the R. F. spark. Analytical precision generally achieved with these samples is A20-30x on the average (6, 12, 13). Attempts have been made to improve precision by using devices such as a rotating electrode (14) or an ion beam chopper (3). According to Nicholls et al. (9), the homogeneity of geological samples can be improved by means of successive fusions and grindings, but this method introduces the possibilities of loss of sample o r contamination, and those elements present in the flux material cannot be determined. Much work has been accomplished in this laboratory on the spark source mass spectrometric analysis of geological, meteoritic, and lunar samples blended with high purity graphite (15-17), where the homogeneity of standards and (6) D. W. Oblas, D. J. Bracco, and D. Y. Yee, in “Symposium on
Trace Characterization-Chemical and Physical,” National Bureau of Standards, Washington, D.C., 1966, p 486. (7) R. K. Skogerboe and G. H. Morrison, ibid., p 589. (8) R. K. Skogerboe, A. T. Kashuba, and G. H. Morrison, ANAL. CHEM., 40, 1096(1968). (9) G. D. Nicholls, A. L. Graham, Elizabeth Williams, and Margaret Wood, ibid., 39, 584 (1967). (10) Richard E. Honig, Aduan. Mass Spectrom., 3, 101 (1966). (11) A. Cornu, ibid., 4, 401 (1968). (12) E. B. Owens, ibid., 3, 197 (1966). (13) John Roboz, “Introduction to Mass Spectrometry,” Interscience, New York, N.Y., 1968. (14) F. Aulinger, 2.Anal. Chem., 221,70 (1966). (15) G. H. Morrison, J. T. Gerard, A. T. Kashuba, E. V. Gangadharam, A. M. Rothenberg, N. M. Potter, G. B. Miller, Geochim. Cosmochim. Acta, Suppl. I , Vol. 2, p 383 (1970). (16) G. H. Morrison, J. T. Gerard, N. M. Potter, E. V. Gangadharam, A. M. Rothenberg, and R. A. Burdo, Geochim. Cosmochim. Acta, Suppl. 11, Vol. 2, p 1169, (1971). (17) G. H. Morrison and A. T. Kashuba, ANAL.CHEM.,41, 1842 ( 1969).
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