Determination of Sulfur as Arsenic Monosulfide Ion by Isotope Dilution

The ionization efficlency Is about 0.1 % and the precision of the a2S/34S ... I D-07cm. I D -I 3cm. 0 D - I9cm. OD-O9cm. 1- 5 cm-/------. 20 3 cm. -I...
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708

Anal. Chem. 1984, 56,708-713

phenols and last the n-alkylpyridines. These provisionally identified components are most certainly affected differently depending on temperature, pressure, type of catalyst, etc., and as such may serve as very sensitive monitors of retort and processing conditions. Measuring the presence and distribution of such components may be the best way of tracking the processing environment of feeds. Registry No. 2,4-Dimethylphenol, 105-67-9; 3,5-dimethylphenol, 108-68-9;2,4,6-trimethylphenol,527-60-6; 3,4-dimethylphenol, 95-65-8; 2,3,6-trimethylphenol, 2416-94-6; 2,3,4-&methylphenol, 526-85-2; 2,3,5-trimethylphenol, 697-82-5; 3,4,5trimethylphenol, 527-54-8.

LITERATURE CITED (1) Richardson, J. S.; Mlller, D. E. Anal. Chem. 1982, 5 4 , 765-768. (2) Gallegos, E. J. J . Chromatogr. Sci. 1981, 79, 177-182. (3) Budzlklewlcz, H.; Djerasiz, C.; Williams, D. H. "Interpretation of Mass Spectra of Organlc Compounds"; Holden Day: San Francisco, CA, 1964; p 242. (4) Stenhagen, E.; Abrahamsson, S.; McLafferty, F. W. "Atlas of Mass Spectral Data"; Interscience: New York, 1969; Vol. 2. (5) Budzlklewlcz, H.; Besler, U. Org. Mass Spectrom. 1978, 7 7 , 398-405. (6) Baker, E. W.; Yen, T. F.; Dlckle, J. P.; Rhodes, R. E.;Clark, L. F. J . Am. Chem. SOC. 1987, 89, 3631.

RECEIVED for review June

27, 1983. Accepted January 10,

1984.

Determination of Sulfur as Arsenic Monosulfide Ion by Isotope Dilution Thermal Ionization Mass Spectrometry P. J. Paulsen and W. R. Kelly* Center for Analytical Chemistry, National Measurement Laboratory, National Bureau of Standards, Washington, D.C. 20234

A new procedure has been developed for the determlnatlon of mlcrogram quantltles of sulfur In metals by isotope dliutlon thermal lonlzatlon mass spectrometry. Typically 1% metal samples are splked with 34Senrlchedsplke and dlssolved In a closed system to prevent loss of volatile S compounds uslng a mixture of HCVHNO, acids whlch oxidizes all S to sulfate. The S Is reduced to H2Sand the sulfide precipitated as As#,. The As2S3Is dlssolved In an ammoniacal As3+ solullon to yleld an As/S atom ratlo of two. A small portlon of thls solution, equlvalent to 1.5 pg of S, Is placed on a Re-flat fllament with silica gel and the 32S/34Sratlo Is measured at 950 OC as the thermally produced "Asa2S+ and " A S ~ ~ Smolecular ' Ions. The ionization efficlency Is about 0.1 % and the precision of the a2S/34Sratio measurement is about 0.1 % (1s). Thls procedure has been applled to the determlnatlon of S In 11 Cu base and Fe base alloys ranging In S concentratlon from 2.8 f 0.2 to 81 f 1 pg of S/g (fs, 95% confidence Interval). The chemlcal blank Is the major source of uncertainty at these levels.

The sulfur content of materials has an important effect on their physical properties. One of the most important properties of low alloy steel is toughness, particularly in subfreezing environments. This property can be increased by lowering the S content since toughness increases exponentially below 100 pg of S/g (1). To meet the toughness specification for the Trans-Alaskan pipeline, the Japanese steel companies reduced the S concentrations in their pipe steel to 20-130 pg of S/g. In copper, S concentrations above 25 pg of S/g cause casting problems in static molding. Conductivity and mechanical properties are adversely affected by S at concentrations of a few parts per million (2, 3). Therefore, the accurate determination of S below 100 pg of S/g is of considerable industrial importance. The most common methods of measuring S are gravimetry and combustion using iodate titration or infrared detection. Gravimetric determination of S by BaSOl precipitation is unreliable below 50 pg of S/g ( 4 ) and the accuracy of combustion techniques depends on This artlcle not subject to

accurate standards for instrument calibration. Watanabe (5-7)has developed an isotope dilution procedure to determine S in steels using conventional SO2 gas mass spectrometry. He has reported values lower than the certified values on a number of international standards. Following these reports, Paulsen et al. (8)and Burke et al. (9) developed an isotope dilution procedure for sulfur determination by using a spark source mass spectrometer (ID-SSMS). In this procedure, samples are dissolved and oxidized in a sealed tube to prevent loss of volatile sulfur compounds and ensure complete equilibration of S isotopes from sample and spike. At high levels the ID-SSMS data were in good agreement with the existing certified values; however, at lower levels large differences were observed. For example, SRM 342a, Nodular Cast Iron, was certified at 60 pg of S/g but was found to be 23 f 2 pg of S/g by ID-SSMS (8). Determinations by IDSSMS yielded blanks of less than 1kg of S and measured the altered 32S/34Sratio with a precision of f 3 % (Is). Thus with a 1-g sample, concentrations of a few to several hundred parts per million can be determined with an accuracy comparable to the measurement precision of the spark source mass spectrometer. Recently, Kelly et al. (10) observed an isobaric interference in the Ag mass region while measuring Ag isotopic ratios, using silica gel as an emitter. This interference was positively identified as 7sAs32S+and 7sAs34S+which occur at the two Ag masses with a 107/109 ratio of about 22. Based on this discovery that S can be thermally ionized as the ASS+ molecular ion, we have developed a procedure to measure sulfur concentrations by isotope dilution thermal ionization mass spectrometry (ID-TIMS) in a wide variety of materials by measuring the ASS+molecular ion. In our procedure the sulfur in the sample is oxidized to sulfate in a sealed tube. The sulfate is reduced to H2Swhich is trapped in an As3+-NH3solution and precipitated as AszS3. The As2SBis dissolved in NH3, an aliquot containing 1.5 pg of S is loaded onto a Re-flat filament with silica gel, and the 32S/34Sratio is measured at 950 O C as 75As32S+ and 75A~34S+ with a precision of 0.1%. Since As is mononuclidic, the ion currents at mass 107 and 109 are proportional to the 32Sand 34Sabundances. This procedure makes it possible for all

US. Copyright. Published 1984 by the Amerlcan Chemlcal Soclety

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5 cm-/------

20 3 cm

@E I

3

7

c

-I

-

IO

r

n

Flgure 1. Diagram of modlfied Carius tube for sample dissolution (top)

and steel shell used for heating sealed tube (bottom).

I +SO; HC I Flgure 2. Diagram of reduction apparatus used for generating and trapplng H,S. The scale bar equals 2 cm. As'3

- NH

W

I

H

thermal ionization mass spectrometers, which exist in relatively large numbers, to make precise S isotopic measurements. The high precision of this technique allows precise concentration determinations over a wide concentration range. At low concentration the uncertainty is limited by variability of blank which is below 1pg of S. At high concentrations the uncertainty is limited by the measurement precision which is 0.1 70. This procedure also allows the natural variability of the 32S/345ratio to be determined on small samples to 0.1% (1s).

EXPERIMENTAL SECTION Apparatus. Digestion tubes (modified Carius tube) were fabricated from thick wall borosilicate tubing (see Figure 1). Solid samples were transferred to the Carius tube with a glass boat funnel. The sealed Carius tube was encased in a steel shell (11). Standard 50-mL beakers with cover glasses were used for sample evaporation. A glass cover was used to provide a N2 atmosphere during evaporation (8,9).The reduction apparatus is shown in Figure 2. Centrifuge tubes were 15-mL graduated tubes made of borosilicate glass. Pipets were standard Pasteur pipets. Sample loading was performed with a 5 cm length of 0.030 in. i.d. intramedic polyethylene tubing attached to a 21 gauge hypodermic needle affixed to a syringe. Ratio measurements were made on a single sector 12 in. radius NBS designed mass spectrometer equipped with a Faraday cage collector. Filament power supply was dc with current control. The temperature was read with an optical pyrometer. Sample loading was performed in a small glovebox in a dry NZatmosphere. Re flat filament were fabricated from zone refined Re of 0.001 in. thickness and 0.030 in. width. Reagents. High-purity HCl, "OB, and HzS04acids were prepared by subboiling distillation (12). An ammonia solution was prepared by bubbling high-purity NHs through a water scrubber and into quartz distilled HzO chilled with ice (12).Two As-NH3 solutions were prepared from SRM 83c (Asz03) and NH6 solution no. 1 was 1000 pg of As/mL; solution no. 2 was 312 pg of As/mL. The reduction solution was prepared according to Thode et al. (13)from ACS Reagent Grade HI (125 mL), HCl(205

ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984

709

mL), and H3P02(61 mL) which was refluxed with a stream of Nz (0.2 L/min) at 120 "C for 2 h to reduce all S and remove it as HzS. The %enriched sulfur spikes were purchased from Oak Ridge National Laboratory (ORNL) and Mound Facility, Monsanto Research Corp. High-purity dry Nz was delivered to the four reduction flasks through Tygon tubing and a valved manifold. Silica gel was prepared from high-purity quartz that was fused with NaZCO3and washed with quartz distilled water (14). Phosphoric acid was prepared from high-purity Pz06and highpurity water (subboiling quartz distilled). A mixture of silica gel and H3P04acid was prepared which contained 20 pg of silica gel/pL in 0.4 M H3P04. This mixture was neutralized with aqueous NH3. Solutions of known sulfur concentration were prepared from high-purity NazS04,KzSO4, and three different bottles of NBS HzS04acid. Preparation of the S Spike. The @Senriched spike in the form of elemental S was converted to sulfate by oxidation with 10 g of 16 M HN03 and 4 g of 11M HC1 in a sealed Carius tube at 240 OC. After dissolution the tube was opened and the solution transferred to a 50-mL beaker. Sufficient high-purity Na2C03 was added to yield a Na/S atom ratio of 4. Excess Na was used to prevent loss of S as sulfuric acid during heating since sufficient countercations were absent. The solution was evaporated to dryness and the nitrates were destroyed by repeated additions of HCl and evaporation. The dried spike was dissolved in 2 M HC1 to a volume of 200 mL and transferred to a storage flask of 250 mL capacity. Two different S spike solutions were prepared one of 100 pg of S/g and one of 300 pg of S/g. Digestion of Spiked Samples. Aliquots of the spike solution were added by weight (k0.1mg) to the Carius tube using a 5-mL plastic syringe that had been modified to cover the rubber plunger with a TFE Teflon sheet. Approximately 1pg of 34Sspike was added per pg of S estimated to be in the sample. This was followed by the addition of 10 g of 16 M HN03 and 4 g of 11M HC1. The solution was then frozen in a COZ(s)-CCl4-CHCl3slush. After the solution was completely frozen, typically 1-g samples in chip or rod form were added to the tube and the tube immediately sealed with a gas-oxygen torch. The tubes were placed in sealed steel sheels (Figure 1)along with -50 g of solid COz to equalize the pressure on the glass tube when heated. The samples were heated to 240 "C for 16-24 h in an oven. The steel shells were cooled to room temperature and the Carius tubes removed behind an explosion shield. The tubes were then cooled to -0 OC and the neck was heated with the torch until a small vent opened and released the internal pressure. The tube was scored 5 cm below the shoulder and fire opened. The top of the tube was removed and the contents were transferred to a 50-mL beaker, which was covered with a watch glass. The sample was immediately placed on a hot plate under a flowing Nz atmosphere (2 L/min) and evaporated to dryness. Two milliliters of 11M HC1 was added with a pipet to rinse the side of the beaker and the solution was evaporated to dryness again. Two more additions of 1mL of HCl each followed by evaporation to dryness were performed. These HCl additions eliminated all nitrates which would interfere in the reduction step. The dried sample was dissolved in 5 mL of HC1. Reduction of Sulfate to HzS. Twenty-five milliliters of the reducing solution was refluxed for 45 min at 120 "C with N2 gas injected through the side arm (0.12 L/min) and then cooled. The contents of the sample beaker were transferred to the reduction flask (see Figure 2) and refluxed for 45 min. The HzS formed was flushed from the system first through a 5-mL distilled water trap and then trapped in a 15-mL centrifuge tube to which had been added 1 mL of aqueous NH3 solution containing 1000 pg of As3+(solution no. 1). During the collection step the As-NH3 solution was cooled in an ice bath to retard the loss of NH3. The trapped sulfur was precipitated as As& by acidifying the solution with HC1. As little as 5 pg of S can be recovered by this procedure. The precipitate was centrifuged for 10 min to ensure that all particles of AszS3 were removed from the solution. The supernatant, which contains a large (>lo0 mg) amount of NH,Cl, was removed with a pipet. The precipitate was washed and centrifuged five times with 5-mL portions of distilled water to remove imsolution purities. The washed Asz& was dissolved in the As-", no. 2 to yield a sulfur concentration of 100 pg of S/mL and an As/S atom ratio of 2.

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ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984

Mass Spectrometry. Flat rhenium filaments were outgassed for 0.5 h at 1500 "C at lo-' torr and stored overnight before use. Sample loading was performed in a plastic box flushed with Nz to purge room air. The silica gel-H3P04 solution was shaken vigorously to disperse the silica gel and 5 p L was drawn into a 5 cm length of intramedic tubing and placed in the center of the filament. A current of 0.9 A was passed through the filament until the silica gel was dry. It was then heated an additional 15 s. Fifteen microliters (equivalent to 1.5 pg of S) of the Aspsssample solution was drawn into the tubing and added as single drops to the dried silica gel. After the first drop was added the filament current was adjusted to 1.6 A. The silica gel will break up and disperse within the sample drop. The remainder of the sample was added without allowing the solution on the filament to evaporate to dryness between additions. After the sample was dry it was heated for 15 s. The current was increased and the sample dried at approximately 700 OC for 5 s. No fumes were observed in this final heating step. The sample was loaded into the mass spectrometer with a total exposure to room atmosphere of 2 min or less. The pressure in the source was reduced below 2 X lo-' torr with the aid of a liquid Nz cold finger. The filament was then initially heated to 800 O C which commonly gave an ASS+ beam of 2 to 5 V with a 10" fl feedback resistor. At this temperature the signal was decaying rapidly. The filament temperature was increased to 950 "C in 50 OC increments at 5-min intervals. The source was focused for maximum intensity after each temperature adjustment. The ion signal at 950 "C was stable and commonly in the 10-V range. Larger ion currents can be obtained above 950 "C, but they are unstable. The signal increased and then decayed after each temperature increase. Data collection commenced 30 min after reaching 800 "C. The major isotope was on either the 10-V or 3-V scale and decaying at a moderate rate (-5%/min). Three data sets were collected. Each data set consisted of six integrations of mass 107 current and five integrations of mass 109 current using an integration time of 15 s. A delay time of 14 s and 10 s was used for ratios of 20/1 and 1/1, respectively. Background signals and electronic offsets were read before each set. The precision for a single set was about 0.05% (Is). During the 21 min of data collection, the observed decrease in the 107/109 ratio was less than 0.1% as a result of isotopic fractionation. Different As/S ratios were explored for ion production ranging from the stoichiometric As2S3to an As/S ratio of 8. Although all mixtures gave ASS+beams, an As/S ratio of 2 appears to give optimum signal stability and intensity. It was obbserved that freshly prepared As&13-NH3sample solutions gave a visible precipitate of As& on the Re filament. These samples yielded unstable ion currents and hence poor precision. If, however, the sample solution was aged for 16 h for more before loading, a uniform mat gray silica gel surface was obtained with no observable Asz& precipitate. These aged samples gave uniformly stable signals with precisions of *0.1% (Is) for three data sets. Caution. As with all pressure vessels, extreme care should be exercised in handling the pressurized tubes. The tubes must be stress-relieved at 560 "C and checked for fire cracks and leaks before use. A small hole in the tube is potentially very dangerous because when the tube is placed in the pressurized shell and heated to 240 "C, the external COz will equilibrate with the internal portion of the tube. When the tube is removed from the shell at room temperature, the internal C02pressure alone is in excess of 60 atm since liquid C02is visible. The most common leak stems from improper sealing of the neck of the tube. All handling of the Carius tubes after sealing and heating should be done behind a blast shield. Before opening with a torch, the tube should be cooled to at least 0 "C. As an additional precaution the contents of the tube can be frozen in COz(s) slush or in liquid Np The H,P02 reducing solution and some metals can form an explosive mixture if taken to near dryness. The refluxing of our reductive mixture has never resulted in any measurable loss of volume, but this could occur during prolonged heating or rupture of the flask. RESULTS AND DISCUSSION Isotope dilution is based upon the equilibration of a known amount of an enriched isotope with the isotopes of the element being determined and the measurement of the isotopic ratio

of the resulting mixture. For S the measured ratio in the resulting mxture is given by the following relation in units of moles:

(2)

(32S)sample

+

(34S)sample

+ (34S)tracer

=

m

(32S)tracer

(1)

which on rearrangement gives

and the S concentration is given by

" Of s/g =

(32S),(at. wt) (32s)(&),

x 106

(3)

where the numerator equals the moles of 32Stimes the S atomic weight and the denominator equals the 32Sfractional abundance times the sample weight in grams. It is obvious from eq 2 that only ratios need be determined and therefore the accuracy of isotope dilution is independent of chemical yields provided blanks are relatively small. Equation 2 can be written in the following form for calculation of concentrations: gg Of '/g

=

WK(A,, - B s p w M(BR - A)

(4)

where W is the weight of spike in pg, K is the ratio of natural atomic weight to atomic weight of spike, A,, and B,, are atom fractions of isotopes a and b in the spike, A and B are the atom fractions in the sample, R is the measured altered ratio of a / b , and M is the sample weight in grams. For the vast majority of elements the isotopic composition is invariant in nature. However, S is one of the few elements which exhibits variability in nature as a result of isotopic fractionation (15). Therefore, the isotopic composition of each sample was determined on an unspiked sample to preclude bias from this effect. Silica Gel-H3P04-NH3Mixture. The amount of silica gel used per loading (- 100 pg) was a larger than the amount commonly used for Pb. The common silica gel method uses an excess of H3P04and then fumes off this excess in the last drying step. However, for ASS+generation, close control of the silica gel/H3PO4-AszS3ratio must be maintained if stable signals are to be obtained. At low H3P04content, low signal levels and poor signal stability result. As the relative amount of &Po4 increases, both signal level and stability show improvement. However, if excess H3P04is used, it causes the decomposition of As&, freeing the sulfur as H2S. For these reasons we premix the silica gel and H3P04(neutralized with NH3) in a ratio designed to maximize both signal level and stability while avoiding excess amounts of H3P04that could destroy the As2S3sample. Blanks. Three samples and one total chemistry blank were processed together. The total chemical blank was determined by adding H N 0 3 and HC1 plus about 10 pg of 34Sspike to a Carius tube and processing in the same manner as a sample. The measured blank was subtracted from the total S measured. The uncertainty due to blank assigned to the three determinations was conservatively estimated to be equal to the blank value itself. The range of more than 60 blanks ww 0.07-0.9 pg of S with a mean value of 0.27 f 0.22 pg (Is). In Table I are listed the S content of reagents. Note the very high concentration of S in the unheated reduction solution. Because of its high value this mixture is reduced for 2 h before use which results in a marked reduction in S blank. AS an

ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984

Table I. Sulfur Concentration in Reagents reagent S concn reduction solution (unreduced) 792 kg125 mLa 803 pg/25 mLa reduction solution (prereduced) 0.043 pgi25 y L NBS HCl 0.003 pg/mL NBS HNO, 0.003 pg/mLC 0.003 pg/mL distilled H,O a Two independent determinations on same solution. Mean value of 25 deterThe values are lower limits. minations, range was 0.018 to 0.097 k g of s. Tvpical values, individual bottles may be higher. added precaution, the 25-mL aliquots are prereduced just before use to remove any S in the solution and the apparatus. Also given in Table I are typical S concentrations in the other reagents used in the analysis. The contribution from all the reagents used is small relative to the total chemistry blank. The blank obtained during reduction, H2S collection, precipitation, washing, and mass spectrometric loading was determined by direct addition of 34Sspike into the reduction apparatus. Twenty-five measurements gave a mean value of 0.05 with no value exceeding 0.1 pg of S. We conclude that the major source of blank occurs before these steps and may include S as gas or particulates from laboratory air. Chemical Yields. The chemical yield of the reduction and collection step was measured by adding a known amount of SO:- (50-100 pg of S) to the reduction flask followed by reduction to HzS. The trapped H2S was precipitated as A&, washed with distilled water, transferred to a Carius tube, and spiked with a known amount of %. The samples were treated as described in the chemical procedure. The mean value for eight determinations was 82% with a range of 7340%. Some of the loss is a result of physical loss of As2S3 during the mechanical transfer of the precipitate to the Carius tube. These high yields and the small blank component in the reduction step indicate that the blank amplification in this step is very small. Calibration of 34SSpike. The 34Sspike was calibrated against gravimetrically prepared solutions of K2S04 and Na2S04plus three different lots of coulometrically assayed sulfuric acid solutions. The sulfuric acid solutions were calibrated by coulometric titration of H*. Ion chromatographic analyses of the sulfuric acid demonstrated that negligible amounts of C1- and NO3- were present. The isotopic composition of S was determined in all of these in order to calculate accurately the spike concentration. All five of these solutions showed measurable differences in their S isotopic composition. Accurately weighed portions of a natural solution and %-enriched spike solution were added to a Teflon beaker, mixed thoroughly, and added directly to the reduction flask. Blanks were determined concurrently with the calibration mixtures and resulted in corrections of less than 0.02% relative in all cases. The results of these calibrations are given in Table 11. There is good agreement between the six sulfuric acid mixes and the four salt mixes. The close agreement of all ten mixes as reflected in the 0.14% relative standard deviation demonstrates the high precision capabilities of this technique. For samples below 100 pg of S/g the uncertainty in the spike calibration is a minor source of error relative to the blank uncertainty. Sulfur Concentrations in Copper-BasedMaterials. To demonstrate the reproducibility of this technique on an actual sample, a 2.5-g sample of SRM 184 was spiked and dissolved. After dissolution the sample was divided into three equal aliquots and each processed separately. The results are given in Table I11 and show excellent agreement among the aliquots. The mean value is in good agreement with the mean of sam-

711

Table 11. Calibration of 34SEnriched Spike Solution concn determined in spike, p g of S / g a natural sulfur H,SO,-135 mix 1 mix 2 H,SO,-137 mix 1 mix 2 H,SO,-139 mix 1 mix 2 mean of 6 acids

313.47 313.63 313.60 313.39 314.30 314.26 313.78 i 0.40 (IS)

&SO,

mix 1 313.98 mix 2 312.88 Na,SO, 313.27 mix 1 313.73 mix 2 mean of 4 salts 313.47 i 0.49 (1s) grand mean of 10 measurements 313.65 i 0.44 (1s) The three a Total pg of S per gram of solution. H,SO, acid natural solutions were prepared from three different lots of acids. Table 111. Reproducibility Experiment on SRM 184 (Leaded-Tin Bronze) sample 1

2 3 4 aliquot A aliquot B aliquot C

sample wt, g 0.678 66 1.440 07 1.323 99 mean 2.472 79

P g of sig 17.3 17.7 17.4 17.5 i 0.4a

17.7 17.8 17.7 mean 17.8 a Total uncertainty was computed as the linear sum of the uncertainty ( t s , 95% confidence interval) in the spike calibration and sample reproducibility, plus the absolute value of the blank divided by the sample weight, -0.8 -0.8 -0.8

Table IV. Sulfur Concentration in Cu Base Materials sample /Jg of Slgapb SRM 52c (cast bronze) 19.8 5 0.5 SRM 184 (leaded-tin bronze) 17.5 i 0.4 12.0 i 0.3 SRM 394 (unalloyed Cu, CUI) 2.8 i 0.2 SRM 1034 (unalloyed Cu, CuA) SRM 1035 (leaded-tin bronze) 22.3 * 0.3 SRM C1253 (phosphorized Cu) 54.7 i 0.5 a Total uncertainty was computed as the linear sum of the uncertainty ( t s , 95% confidence interval) in the spike calibration and sample reproducibility, plus the absolute value of the blank divided by the sample weight. Mean of three different bottles. ples 1, 2, and 3. The small differences between the two sets of data may reflect sample inhomogeneity and blank variability. Sulfur concentrations determined by this procedure are given in Table IV. Samples were 1 g or less for all SRMs. The blank correction to these data was about 1% except for SRM 1034. SRM 1034 was found to have a sulfur concentration of 2.8 pg of S/g with an uncertainty of 0.2 kg of S/g. Even for this low S concentration the blank correction was less than 10%. The relative uncertainty for samples above 10 pg of S/g was 1-2.5%.

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ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984

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Table V. Replicate Sulfur Determinations for Two SRM's bottle no. SRM 348 1 2 3 4 5 6

sample wt PLg of s/g (HT Alloy, A286, 26 Ni-14.5 Cr) 0.9569 24.2 0.9823 23.8 0.9923 24.2 0.8766 23.2 0.8684 23.6 1.1977 24.2 mean 23.9 i 1.4a SRM l O l f (AISI 304L, 18 Cr-10 Ni steel) 1 0.5055 80.69 2 0.4549 80.95 3 0.6608 80.84 4 0.7119 80.55 5 0.4276 80.69 6 0.6738 80.64 mean 80.73 i 0.9ga a Total uncertainty was computed as the linear sum of the uncertainty ( t s , 95% confidence interval) in the spike calibration and sample reproducibility, plus the absolute value of the blank divided by the sample weight. The relative contributions from these three sources were 0.35%, 4.45%, and 1.08% for SRM 348 and 0.35%, 0.46%, and 0.42% for SRM 101f. Table VI. Sulfur Concentration in Fe Base Materials sample SRM 50c ( W-Cr-V Steel) SRM 68c (high carbon ferromanganese) SRM 101f (18 Cr-10 Ni Steel AISI 304L) SRM 1036 (high Si steel) SRM 348 (high temp alloy A286, 26 Ni-14.5 Cr)

P g of s/ga 63.7 i. 2.2b 29.8 i 3.0b 80.73 i 0.99 6.3 i 0.Bc 23.9 i 1.4b

a Total uncertainty was computed as the linear sum of the uncertainty (ts, 95% confidence interval) in the spike calibration and sample reproducibility, plus the value of the blank divided by the sample weight. Mean of six different bottles. Mean of three different bottles.

Sulfur Concentrations in Iron Based Materials. The replicate values for two Fe-base materials are given in Table V. SRM 348 was found to have a mean value of 23.9 pg of S/g with an uncertainty of 5.9%. This relatively large uncertainty may reflect sample inhomogeneity among the bottles sampled or blank variability. In the case of SRM l O l f with a mean value of 80.73 pg of S/g the total uncertainty is 1.2% relative. The excellent agreement among the replicates as reflected by the relative standard deviation of 0.18% for the mean of the six determinations indicates uniform S concentration and the high precision obtainable by isotope dilution thermal ionization mass spectrometry. A summary of the S results obtained for Fe-base materials is given in Table VI. These results cover more than a 10-fold concentration range. The S contents of these samples are in the lowest concentration range normally encountered in steels, and these samples should be valuable for the calibration of automated instruments. We have compared the S values determined in this study by using isotope dilution thermal ionization mass spectrometry with the values on existing certificates in Table VII. The values given in column three were determined by a variety of techniques which included BaSOl precipitation, combustion followed by SO2titration, and combustion followed by infrared detection of SOz. In all but two cases the existing certified values are in good agreement with those determined in this study. For S R M s 50c and 68c we found significantly lower

Table VII. Comparison of Previously Certified Sulfur Values with Those Determinated in This Study value currently SRM date of on certificate value determined S) in this study (% S) no. certificate 184 1/26/73 0.0020a 0.00175 i 0.00004 394 1/20/78 0.0015 i. 0.0003 0.00120 i. 0.00003 50c 6/25/57 O . O I O a 0.0064 ? 0.0002 68c 8/15/79 0.008 .r 0.002 0.0030 i 0.0003 lOlf 5/19/70 O.OOBa 0.0081 lr 0.0001 348 lO/l/Bl 0.002 i 0.001 0.0024 i 0.0001 C1253 9/16/80 (0.0050)b 0.00547 i 0.00005 a No uncertainty given on certificate. Information only value.

values than certified. At these low levels many of the chemical techniques used for these certifications were near their detection limit and hence may have been subject to bias. The sensitivity of this technique is well below the magnitude of our blanks. For example, direct reduction blanks of 0.05 pug of S can be measured with a precision of 0.5%. Since our mass spectrometric technique has submicrogram detection and all blanks were precisely measured at below 1 pg of S, we conclude that our determinations are accurate. In the low level S determinations (-100 pg of S/g) presented in this paper the blank is the main source of imprecision. The high precision inherent in this technique, as demonstrated by the precision of the spike calibration (Table 111, can be used to its full potential for high accuracy determinations at high S concentrations where blanks are negligible. We are currently determining S at levels above lo00 pg of S/g in a variety of materials including coals and biological samples.

ACKNOWLEDGMENT The authors thank Fouad Tera of the Carnegie Institution of Washington for his generosity in giving us silica gel that he prepared from his own recipe, NBS colleagues George Marinenko and W. F. Koch for preparation, ion chromatographic analysis, and calibration of the sulfuric acid standards, and Carol Anthony for the drawings of our apparatus. Registry No. Sulfur, 7704-34-9; arsenic, 7440-38-2.

LITERATURE CITED Pickering, F. B. I n "Proceedlngs of an International Symposium on Hlgh-Strength, Low Alloy Steels"; Unlon Carbide Corp. Danbury, CT, 1977; pp 9-30. Tuddenham, W. M. I n "Encyclopedia of Chemical Technology", supplemental volume, 2nd ed.; Wiley: New York, 1971; pp 249-270. Smart, J. S., Jr. I n "Copper-The Science and Technology of the Metal, Its Alloys and Compounds"; Butts, A., Ed.; Hafner Publishing Co.: New York, 1970 pp 410-416. Proc. Am. SOC. Test. Mater. 1074, E350. Watanabe, K. Anal. Chlm. Acta 1975, 80,117-123. Watanabe, K. Talanta 1070, 26,251-253. Watanabe, K. Anal. Chlm. Acta 1083, 147, 417-421. Paulsen, P. J.; Burke, R. W.; Maienthal, E. J.; Lambert, G. M. I n "ASTM STP 747"; Javelr-Son, A., Ed.; American Soclety for Testing and Materials: Philadelphia, PA, 1981; pp 113-120. Burke, R. W.; Paulsen, P. J.; Maiental, E. J.; Lambert, G. M. Talanta 1082, 29, 809-813. Kelly, W. R.; Tera, F.; Wasserburg, G. J. Anal. Chem. 1078. 5 0 , 1279-1286. Gordon, C. L.; Schlecht, W. G.; Wichers, E. J. Res. Natl. Bur. Stand. ( U . S . ) 1944. 33, 457-470. Kuehner, E. C.; Alvarez, R.; Paulsen, P. J.; Murphy, T. J. Anal. Chem. 1972, 4 4 , 2050-2056. Thode, H. G.; Monster, J.; Dunford, H. 0. Geochlm. Cosmochim. Acta 1061, 25, 159-174. Tera, F., Carnegie Instltution of Washington, DC, personal communication, 1980. Krouse, H. R. I n "Handbook of Environmental Isotope Geochemistry"; Fritz, P., Fontes, J. Ch., Eds.; Elsevier: Amsterdam, 1980; Vol. 1, The Terrestrial Environment A; pp 435-471.

RECE~VED for review September 26,1983. Accepted December 21,1983. Partial funding for this work was provided by the NBS Office of Standard Reference Materials and the

Anal. Chem. 1984, 56,713-719

DOE-Pittsburgh Energy Technology Center under Interagency Agreement No. DE-AI22-82PC53141. Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental proce-

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dure. Such identification does not imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

Determination of Serum Urea by Isotope Dilution Mass Spectrometry as a Candidate Definitive Method Michael J. Welch,* Alex Cohen, Harry 5.Hertz, Fillmer C. Ruegg, Robert Schaffer, Lorna T. Sniegoski, and Edward White V

Center for Analytical Chemistry, National Bureau of Standards, Washington, D.C. 20234

An isotope dilution mass spectrometric (ID/MS) method for serum urea Is descrlbed. The method utlllzes urea-“O as the labeled Internal standard and Involves Isolation of urea from serum, converslon to 6-methyC2,4-bls[(trlmethylsilyl)oxy]pyrlmidine, capillary column gas chromatography for sample Introduction, and measurement of the abundance ratio of the (M 15)’ Ions from the labeled and unlabeled derlvatlve. Quantltation is achieved by measurement of each sample between measurements of two standards whose unlabeled/ labeled ratios bracket that of the sample. Results are of high precislon, wlth coetflclents of varlatlon for a single measurement of 0.17% for NBS Standard Reference Material 909, a freeze-drled human serum pool, and 0.19% overall for five frozen serum pools, and have been shown to be free of measurement Interferences. The method is therefore of Sufficient accuracy and preclslon to be considered a “deflnltlve” method.

coefficients of variation were less than 5%. In this paper we describe an ID/MS method for serum urea of sufficient accuracy and precision to be considered a definitive method. We add a known weight of urea-180 to a known weight of serum such that the measured ratio of unlabeled to labeled urea is approximately 1:1. After an equilibration period, the serum is freeze-dried. The urea is extracted with methanol, the methanol is evaporated, and the urea is sublimed. The urea is then converted to 6-methyluracil by using modifications of published syntheses (13,14). For gas chromatography, the derivative is converted to its trimethylsilyl ether, 6-methyl-2,4-bis[(trimethylsilyl)oxy] pyrimidine. For our ID/MS measurements, samples are bracketed with standards, a calibration technique shown to provide excellent precision (2,3). Ion abundance ratios are measured for both samples and standards by using the (M - 15)’ ions at m / z 255 and 257. The quantity of unlabeled urea in the sample is then found by linear interpolation. As tests for measurement bias, randomly selected samples are measured again by using two other pairs of ions.

The measurement of serum urea levels is an important clinical test for kidney function as well as for protein metabolism. As part of a program for standardization of clinical methods, we have undertaken for serum urea development of a “definitive” method ( I ) , i.e., a method of demonstrated high accuracy and precision, to provide an accuracy base to which reference and routine methods can be compared. Isotope dilution mass spectrometry (ID/MS) is a technique which has been shown to be capable of meeting the rigorous requirements of a definitive method (2-8). Although ID/MS has been employed in the determination of serum urea, none of the reported methods can be considered a definitive method. Bjorkhem et al. (9, 10) reported an ID/MS method utilizing ureaJ5N2 as the labeled analogue and derivatization of the urea to a dimethylated 5,5-diallylbarbituric acid. The coefficient of variation (CV) for a single measurement by this method was 3.6%, which the authors judged to be sufficiently precise for use as a reference method for their laboratory. Two ID/MS urea methods intended for use in studies of metabolism have been reported. Nissim et al. (11) employed urea-16N2as the labeled analogue and converted the urea to its trifluoroacetyl derivative. While the method was satisfactory for its intended purpose, its precision (CV 7%) does not meet the requirements of a definitive or a reference method. Tserng and Kalhan (12)also used urea-16N2but converted urea to the trifluoroacetyl or trimethylsilyl derivatives of 2-hydroxypyrimidine. Their method was applied to enrichments of as low as 0.1%. With higher enrichment,

EXPERIMENTAL SECTION Serum. Both freeze-dried and frozen sera were studied. The freeze-dried serum was Human Serum Standard Reference Material (SRM) 909 from the National Bureau of Standards. The serum was reconstituted in the vials in which it was supplied by addition of 10 mL of the diluent water supplied with the serum. Each vial with ita contents was weighed before and after adding the water, and later, the clean empty vial was weighed again so that the weights of freeze-dried serum and added water were known. After addition of the water, the mixture was gently swirled periodically to dissolve the serum and ensure homogeneity. The frozen sera were of bovine origin and were five different pools supplied by the Centers for Disease Control (CDC),Altanta,GA. This serum was stored at -20 O C until samples were needed, at which time the serum was allowed to thaw at room temperature. Urea. The unlabeled urea used as the primary standard was SRM 912 with a purity of 99.7 h 0.1%. Urea-180was synthesized as described below. Urea-13C,16N2 was commercially available and had an isotopic purity of 90 atom % 13C and 95 atom % lSN. Synthesis of Urea-180. The synthesis of urea-180 from cyanamide and H2180(97 atom % l80)followed published procedures (15, 16) with the following modifications. Hydrogen chloride gas was used in place of hydrochloric acid. After the crude product was extracted into ethanol and acetone, the solution was treated with aqueous silver nitrate to remove any remaining cyanamide and then with aqueous sodium chloride to remove any excess silver. The urea-180 was twice sublimed at 100 “C and 15-27 Pa (0.11-0.20 torr) with a dry ice cooled condenser and recrystallized from acetone-ethanol. The yield of product (90 atom % l80)was 0.8 g, or 28%, mp 134.0-134.3 O C .

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This article not subject to U.S. Copyright. Published 1984 by the American Chemical Society