Arlctl_vticcr Chinrico Actct, 73 ( 1974) 33-38

,(Y Elscvicr Scientific Publishing Company. Amsterdam - Printcd in The Ncthcrlands 33

SUB-MICROGRAM PER GRAM CONCENTRATIONS OF MERCURY IN ORCHARD LEAVES DETERMINED BY ISOTOPE DILUTlON AND SPARK-SOURCE MASS SPECTROMETRY

ROBERT ALVAREZ

The difficulty of reliably determining sub-microgram per gram (146 g-‘) concentrations of mercury in biological materials has been reported’.‘. In 1967, an interlaboratory comparison was made of elcmentai determinations in a kale powder by different analytical techniques ‘. For mercury, the neutron activation results from two laboratories could be poofed to give a value of 0.15O~O.OO8 116 g- ’ for the mean concentration and standard deviation but the colorimctric value reported by a single laboratory was 0.0122 rt: 0.0024 rcg g- ‘. In 1971, recent analytical methods for mercury developed in Scandinavia and in the United States were reviewed under the principal headings of neutron activation. atomic absorption, and gas chromuto- graphy*. Reliable chemical analyses having a sensitivity of at least 0. i ccg g- 1 and an accuracy, if possible, to within + lO% were cited as desirable for regulatory purposes.

The availability of biological Standard Reference Materials with certified values for trace amounts of mercury would enable an analyst to verify the .accuracy of existing methods in his laboratory and aid him to develop new procedures. As a first step in this certification, an analytical program was undertaken by the National Bureau of Standards to provide a reliable value for the mercury content of the ground Orchard Leaves, SRM 1571 3. Three independent analytical methods were used: atomicabsorption spectrometry, neutron activntion, and stable-isotope dilution with the spark source mass spectrograph (i.d.-s.s.m.s.). The Iast method is the object of this investigation.

In previous applications of i.d.-s.s.m.s. methods to standardization problems at NBS’*‘*“. the trace elements being determined were not appreciably volatile under the experimental conditions employed. For these determinations, relatively simple procedures and apparatus sufficed to avoid losing the trace elements and added enriched isotope spikes bofore’equilibration. Once this prerequisite has been achieved, trace element losses do not invalidate the results because only isotope ratios are measured. The volatility of mercury required a morccomplex procedure and apparatus to ensure its retention before equilibration and determination.

EXPERiMENTAL

Deionized water was prepared by flowing distilled water from the laboratory distribution system through a mixed-bed, ion-exchange resin coIumn. High-purity

34 R. ALVAREZ

nitric and perchloric acids, which had been prepared previously for other trace element determinations by double distillation of the reagent-grade acids in a sub- boiling quartz still were used’.

Isotopically enriched mercury(II) oxide, containing 81.53 at.‘x 201Hg and 1 1.10 at.‘%; “‘Hg for the isotopes of interest, was obtained from the Oak Ridge National Laboratory. Isotopically enriched mercury containing 99.11 at.‘:(, 19sHg, <0.003 at.‘%, 201 Hg, and 0.004 at.% 202 Hg was obtained from Atomic Energy of

Canada Limited for use as a carrier. Solutions of these materials were prepared by weighing 5 mg of the element 02 equivalent to +O.Ol mg, dissolving in 1 ml of 4 M nitric acid. and diluting to volume with water in 5-ml volumetric flasks.

A natural mercury solution was prepared by weighing 100 mg of high-purity mercury (99.99+ ‘;/,) to k 0.05 mg, dissolving in 4 ml of 4 M nitric acid, and diluting to volume with water in a 50-ml volumetric flask.

The apparatus used in this study (Fig. 1) is similar to that described by Bethge” for wet-ashing organic matter. The borosilicate glass components, assembled with standard taper 24/40 joints, consist of a 250-ml flask, a refiux collector, a 300- mm water-cooled (West) condenser, and a bubbler. For the present application, the modified apparatus also serves for chemically and isotopically equilibrating the mercury of the sample with the enriched isotope spikes without losses. The joints

Fig. I. Apparatus for wet-ashing sample.

MERCURY IN ORCHARD LEAVES 35,

are provided with polytetrafluorethylene (PTFE) sleeves for better sealing of the components. The lower portion of the condenser tube is indented immediately above the inner joint to support an 80-mm height of 4-mm diam. quartz Raschig ringsg. Connected to the top of the condenser is an inner joint sealed at the top and having a side tube. A bubbler containing 4 M nitric acid is attached to this side tube with a short section of PTFE tubing. This arrangement prevents mercury vapor that may be present in the laboratory environment from being drawn into the system during the cooling periods. The flask and side tube of the reflux collector are electrically heated with a heating mantle and insulated resistance wire respectively. Variable transformers are used to regulate the voltage to the heaters.

Electrolysis cell The electrolysis cell consists ofa 30-ml beaker with a PTFE cover from which

are suspended a OSI-mm diam. platinum wire anode and two 0.9-mm diam. gold wire cathodes in a tubular holder. The beaker is made of a translucent fluorinated copolymer. The wires had been examined previously by spark source mass spectro- metry and found to be of high purity. The portion of the 7-cm length of platinum wire that dips into the solution is coiled into a small loop. The cathode wires, cu. 10 cm in length, are forced through undersize holes in a PTFE plug, which fits tightly into a 6-mm diam. PTFE tube. Approximately 2 mm of each wire projects through the plug for exposure to the solution being electrolyzed. A PTFE- encapsulated stirring bar is used’for magnetically stirring the solution.

Mass spectrogrqdt mtl ntic~oplrotoineter. The double-focussing Mattauch-Herzog design spark-source mass spectro-

graph hnd the microphotometer with its analog computer have been described previously4* 5.

Proceclitre Determine the moisture content by drying l-g samples of the orchard leaves

material in an oven at 90°C for 24 h. Weigh a 5-g sample to the nearest mg, transfer to a 250-ml flask, and spike with .a solution containing 0.370 clg ‘OIHg and 50 c(g lg”Hg as a carrier. At the same time, mi,x an aliquot with a known volume of the natural mercury solution for future verification of the “‘Hg con- centration in the spiking solution. The 201 Hg addition to the sample should be calculated to yield an altered isotope ratio of almost unity, which is desirable to minimize the effect of photographic emulsion calibration errors. After a preliminary spiking, the experimental procedure must be followed to determine the approximate mercury content. The general isotope dilution equation was used in these cal- culations4* 5.

After assembling the apparatus with water flowing through the condenser, and adding 20 ml, of 8 M nitric acid to the sample through the condenser, heat the flask gently until the initial reaction subsides. Add 20 ml of nitric acid-per- chloric acid (1+ 1) through the condenser, and heat the flask and side tube of the reflux collector to accumulate the acid condensate in the collector. When the organic material in the flask has been oxidized, allow the acid in the collector to flow back into the cooled flask. Add several portions of water through the con-

36 H. ALVAREZ

denser, swirl the collector, and allow the water to flow into the flask. Then remove the collector and attach the flask to the condenser. After draining the condenser water, heat the flask to distil the acid through the condenser until l-2 ml of perchloric acid remains. Add CN. 5 ml of water in small portions through the condenser to the cooled flask. Transfer the solution to the electrolysis cell and electrolyze while stirring for UI. 16 h at an applied voltage of 2.2 V.

Spark the gold wire-cathodes in the mass specirograph and prepare a graded series of exposures.

The ion-sensitive photographic plates (Ilford Q-2) were processed with the bleach and internal image developer MK-7 described by Cavard lo. They were placed in the dichromate bleach for 4 min, rinsed in water for 0.5 min, developed for 7 min, fixed for I min, and washed for 15 min. The intensity-areas of 201Hg and ‘02Hg on the photographic plate were measured and the altered isotopic ratios were determined. The mercury concentrations were calculated from these ratios and other data, by means of the general isotope dilution equation.

For evaluation of the method blank, the entire analytical procedure was performed with the same quantities of acids and 100 mg of bovine liver, NBS- SRM 15773. The liver sample, which has a mercury content approximately ten times lower than that of the orchard leaves, was used in simulating the spiking operation.

The solution that had been set aside for conlirming the concentration of ‘O*Hg in the spi king solution was electrolyzed after adding a solution con- taining 50 ~18 of high-purity copper. The conditions for electrolysis were the same as described in the experimental procedure for the sample.

Because of the low source-pressures that are routinely obtained in the mass spectrograph, the possibility of electrodeposited mercury on gold wire cathodes being lost through volatilization was investigated. A solution of mercury and copper, both present in approximately the same amounts as found in a 5-g sample of orchard leaves, was spiked with radioactive 203Hg and electrolyzed with gold wire cathodes as in the chemical procedure. The wires were loaded into the mass spectrograph, which was evacuated for the same period used in a normal analysis. The unsparked wires were removed from the instrument, counted, and found to have greater than 95”/;; of the original activity. These results indicated that no significant mercury loss occurred from the electrodes while they were in the evacuated instrument.

Recovery qf’ tnercwy irl the cltemiccrl procedwe A 5-g sample was spiked with radioactive 203Hg and a solution containing

50 jig of natural mercury- the amount of *98Hg carrier added in the procedure. After assembling the apparatus, the analytical procedure was followed to the point of oxidizing the sample and allowing the accumulated acid in the reflux collector to flow back into the flask. The nitric acid from the bubbler was counted with a single-channel scintillation y-counter. No activity above background was found, indicating that ionic. mercury was not lost through the bubbler during oxidation.

At this stage in the actual analytical procedure, the mercury would have been

MERCURY IN ORCHARD LEAVES 37

equilibrated and losses would not invalidate the results. The trace experiment was then continued. After evaporation of the acid through the condenser and addition of water to the flask, the activity of the solution in the flask was counted. The results indicated that over 90(x, of the mercury remained in the flask at this stage.

RESULTS AND DISCUSSION

In the procedure, separation of most of the. acid from the equilibrated mercury, although time-consuming, does not require much attention. If necessary, the time for this step could be decreased by leaving the apparatus in Fig. 1 assembled after the wet-oxidation step, heating the flask, and collecting 20 ml of the distillate through the stopcock at the side of the collector. However, by using radioactive mercury, it was found that further distillation resulted in appreciable loss of mercury; appr.oximately 65O/” of the total mercury has been lost when 1-2 ml of perchloric acid remained in the flask. Therefore, when 20 ml of distillate has been removed, the distillation should be continued through the packed con- denser as before.

The individual mercury concentrations are listed in Table I in the order they were determined. They were corrected for the moisture content and the method blank, 0.004 /lg. The second and third determinations were made by spiking

TABLE I

MERCURY CONTENT OF ORCHARD LEAVES, SRM 1571. DETERMINED BY ISOTOPE DILUTION AND SPARK-SOURCE MASS SPECTROMETRY

0.133 0.131 0.144 0.134 0.142 0.160 Avcragc concentration 0.141 957; Confidence limits 3_0.009

TABLE II

CQMPARlSON OF MERCURY RESULTS BY ISOTOPE DILUTION WITH OTHER ANA- LY’TICAL METHODS FOR ORCHARD LEAVES. SRM 1571

0.141 kO.009 6 Isotopic dilution 0.160+0.012 15 Atomic absorption 0.155 4 0.006 11 Neutron activation 0.145~0.014 5 Neutron activation 0.148+0.010 4 Neutron activation

NBS NBS NBS A B

-

38 R. ALVAREZ

the solutions from the wet-oxidation step to determine whether this change would affect the equilibration and results. These determinations were compatible with the other results obtained by spiking the samples.

The Dixon Criterion” was used to reject an aberrant value, 0.199 /lg g- ‘, which may have been caused by contamination; its inclusion would have resulted in an average concentration and 9576 confidence limits of 0.149+0.018 clg g- ’ instead of 0.14 1 + 0.009 clg g- l listed in Table II. Independent results obtained by atomic absorption and neutron activation are also shown in this table. A con- sideration of methods and data led to a certified value of 0.155+0.015 clg g-l for the mercury content of the Orchard Leaves, SRM 157 1.

The author gratefully acknowledges the assistance rendered by P. J. Paulsen in performing the mass spectrographic measurements and by H. L. Rook in evaluating possible losses of mercury in the mass spectrograph.

SUMMARY

A stable isotope dilution procedure in conjunction with the spark source mass spectrograph was developed for determining sub-jig g-l concentrations of mercury in orchard leaves. A 5-g sample was spiked with a solution of mercury, isotopically enriched in “‘Hg and lS)‘Hg. The 198Hg served as a carrier. After wet-ashing the sample with nitric and perchloric acids under reflux and distilling most of the acid, the isotopically equilibrated mercury was electrodeposited onto high-purity gold wires for sparking in the mass spectrograph. The concentration was calculated from the altered isotope ratio, 201Hg/202Hg, and other data. The results were compared with those obtained by atomic absorption spectrometry and neutron activation, leading to a certified value of 0.155 20.015 /lg g- ’ for the mercury content of Orchard Leaves, SRM 1571 of the National Bureau of Standards.

REFERENCES

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H. J. M. Bowcn. Atdysr (Lomb). 92 ( 1967) 124. A. Wcissler, Em%-on. Rcs.. 4 ( 1971) 53. Cutnloy of Stutdcwcl Rc~feretm Mcrtwids. NBS Spcciul Pddicurior~ 260, April 1973. Oflke of Standard Refcrcnce Materklls, National Bureau of Standards. Washington. D.C. 20234. April 1973. (Available from the Supcrintcndcnt of Documents, U.S. Government Printing Oflicc. Washington, D.C. 20402.) R. Alvarez. P. J. Paulsen and D. E. Kcllchcr. A~~crl. C/IOU., 41 (1969) 955. P. J. Puulsen, R. Alvarez and D. E. Kellchcr. Specrroc*hirn. Acrtr. Parr B, 24 (1969) 535. P. J. Paulsen, R. Alvarez and C. W. Mueller. ,4ntr/. Chem. 42 (1970) 673. E. C. Kuehner, R. Alvarez. P. J. Paulsen and T. J. Murphy. And. Clrcm.. 44 (1972) 2050. P. 0. Bcthge. Ah. C/rim. Ac*ru. IO ( 1954) 317. R. K. Munns and D. C. Holland. J. Ass. O,Jfi’c-. Amd. Chem. 54 (1971) 202. A. Cavard in E. Kcndrick (Ed.), Aclcuwc.s irr Mtrss Specrronrerry. The Institute of Pctrolcum. London, 1968. pp. 4 19429. M. G. Natrclla. Espcritm~tul .S/uti.sticx. Nutimctl Bwecrrc o/’ SrtrtdurcLs Hcmlhook 91. Washington. D.C.. 1963. Section 17-3.