For the cathodes described in this study, as well as for Cu and T a cathodes run separately, weak cathodic ion signals correlated with strong residual gas signals (N+, 0+, NHz+, H20+) which in some cases became as intense as the cathode matrix element.

DISCUSSION Since the HCIS ionizes most cathode materials with approximately the same efficiency ( I , 2), the 5sFe+ intensity represents the total cathodic ion intensity. Furthermore, since the background noise in most mass regions is independent of mass, the iron intensities shown in Figures 2 to 4 are inversely proportional to the minimum detectable concentration; the exceptions are volatile elements such as Zn or Hg which are selectively evaporated from the cathode and ionized ( 2 ) . It is apparent from Figures 3 and 4 that optimization of the bore diameter and depth is essential for best sensitivity and, indeed, is much more critical than optimizing the discharge current. The anode-to-cathode distance may also have indirect importance in determining the maximum discharge current tolerated before the cathode begins to short to the anode. Extrapolating the results of Figures 3 and 4 suggests that a flat cathode with no bore should produce the most intense ion beam. Instead, such a cathode produced no ion beam, apparently because the discharge did not occur near the anode orifice. The cathodes employed were 0.250 inch wide and separated from the anode by 0.008 inch while the orifice was 0.020 inch in diameter; thus, for the flat cathode, the pressure close to the orifice was significantly less than in other sections of the source, and the discharge occurred preferentially in other sections of the source. To some extent, this situation can be corrected by using a greater anode-to-cathode distance. The data described in this study are entirely consistent with the numerous investigations of the optical properties of the hollow cathode discharge (7). Mitchell, for example, has shown that in a hollow cathode discharge the concentration ratio of charged to uncharged species increases with decreasing bore depth and increasing bore diameter ( 4 ) .To minimize emissions from charged species, hollow cathodes

for lamps are thus drilled to a depth of several bore diameters. With such a geometry, most sputtering occurs near the center of the bore and cathodic material is deposited near the open end (8) suggesting that the mouth of the bore is an area depleted in ions. It is also well known from optical studies that when the negative glow regions corresponding to different parts of the cathode are forced to coalesce (e.g., by cathode geometry), the current density and optical brightness increase manyfold (9). This phenomenon is known as the hollow cathode effect. From the data available in optical studies as well as from the studies described here, it thus appears that the ideal bore geometry for the HCIS is short and wide to enhance the formation of ions but also sufficiently confined to maintain the hollow cathode effect with its associated high current densities. While a check of absolute sensitivity was not undertaken in this study, an increase in sensitivity over that already reported (2) can be expected. The previous authors showed that a detection limit of 10 to 150 ppma is possible for those elements examined. The present studies indicate that a gain of IO2 or more is possible through the use of high bore diameter to depth ratios. Consequently, detection limits for HCIS mass spectrometry should be in the ppba range.

LITERATURE CITED (1) (2) (3) (4) (5)

(6) (7) (8) (9)

W. W. Harrison and C. W. Magee, Anal. Chem., 46, 461 (1974). E. N. Colby and C. A. Evans, Jr., Anal. Chem., 46, 1236 (1974). C. J. Belle and J. D. Johnson, Appl. Spectrosc.,27, 118 (1973). K. 8. Mitchell, J. Opt. SOC.Am., 51, 846 (1961). J. R. Wallace, "The Chemical and Physical Characterization of Airborne Particulate Matter", Thesis, School of Chemical Sciences, University of IIlinois, Urbana, Ill., 1974. C. A. Bennett and N. L. Franklin, "Statistical Analysis in Chemistry and the Chemical Industry", John Wiley and Sons, New York, 1954, p 29. R. Mavrodineanu, "Bibliography on Flame Spectroscopy", Nat. Bur. Stand. (US)Misc Pub., 281, U.S. Government Printing Office, Washington, D.C.. 1967. A . D.White, J. Appl. Phys., 30, 71 1 (1959). P. F. Little and A. von Engel, Proc. RoyalSoc. London, 224, 209 (1954).

RECEIVEDfor review June 10, 1975. Accepted October 6, 1975. This work was supported in part by the National Science Foundation Grants DMR 72-03026 and MPS 7405745.

Sequential Determination of Arsenic, Selenium, Antimony, and Tellurium in Foods via Rapid Hydride Evolution and Atomic Absorption Spectrometry John A. Fiorino,' John W. Jones,* and Stephen G. Capar Bureau of Foods, Food and Drug Administration, Washington, D.C. 20204

Analysis of acid digests of foods for As, Se, Sb, and Te has been semiautomated. Hydrides generated by controlled addltlon of base stabilized NaBH4 solution to acid digests are transported directly into a shielded, hydrogen (nitrogen diluted), entrained-alr flame for atomic absorption spectrophotometric determination of the individual elements. The detectlon limits, based on 1 g of digested sample, are apPresent address. C h e m i s t r y D e p a r t m e n t , V i r g i n i a Polytechnic I n s t i t u t e a n d State L'niversity, Blacksburg. Va. 24060.

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proximately 10 to 20 ng/g for all four elements. Measurement precision is 1-2% relative standard deviation for each element measured at 0.10 pg. A comparison is made of results of analysis of lyophilized fish tissues for As and Se by instrumental neutron activation (INAA), hydride generation with atomic absorption spectrometry, fluorometry, and spectrophotometry. NBS standard reference materials (orchard leaves and bovine liver) analyzed for As, Se, and Sb by this method show excellent agreement with certified values and with independent NAA values.

Arsenic, selenium, antimony, and tellurium have traditionally been rather difficult elements to determine. Most analyses for these elements have been performed using absorption spectrophotometry or fluorometry (1, 2 ) . However, these classical techniques have gradually yielded to the operationally simpler atomic absorption spectrometry (AAS). Because of inadequate detection capability for these elements, AAS has been used in conjunction with separation and/or concentration techniques (3). As a means of both separation and concentration, the formation of the gaseous hydrides has many advantages, especially for AAS determinations. Minimization of interferences and possible utilization of the highly transparent hydrogen diffusion flame are two obvious advantages of hydride volatilization ( 4 , 5 ) . Generally, the hydrides are formed either by the reductive action of granular zinc in acid media (6-9) or, more recently, by reaction of the analyte(s) in acid media with sodium borohydride either as uniform pellets or in aqueous solution (10-12). Evolution and transport of the hydrides from the acid medium are governed by a number of parameters among which are the chemical nature, form, and concentration of the reductant, acid concentration, and carrier gas flow rate. With zinc or NaBH4 pellets as the reductant, it is common practice to collect the hydrides in a purgeable reservoir (e.g., a balloon) subsequent to venting them to the flame of an atomic absorption spectrophotometer. The precision of the measurements is very dependent upon the skill of the analyst since most operations involved (dosing stopcocks, injection of zinc slurries, manipulation of valves, etc.) are manual. Schmidt et al. (13) and Kan (14) have described automated methods based on the evolution of hydrides using aqueous NaBH4 reductant. These works demonstrate the excellent precision and detection capabilities obtainable, as well as the analytical speed afforded by automated chemical procedures. Both of these automated methods employ peristaltic pumping systems to provide continuous mixing of the reactants, and result is a steady-state signal. The instrument described in this paper is a semi-automatic hydride generator in which a NaBH4/NaOH solution is metered into a reaction vessel containing an acid-oxidized food sample. The hydrides are produced quantitatively and are carried directly into a shielded, nitrogen-diluted, hydrogen diffusion flame by the flow of hydrogen produced by the NaBH4 H30+ reaction. Under these conditions, a transient (peak) signal is produced rather than a steadystate signal. The apparatus is considerably simpler and less expensive than those based on peristaltic pumping systems and is capable of comparable precision, accuracy, speed, and detection limits.

+

EXPERIMENTAL Apparatus. The hydride generator (Figure 1) consists of (1) a unique reaction head constructed of acrylic plastic which incorporates two Teflon metering valves, a vent tube, and a tapered, rubber sheathed stopper for connection of an %inch, heavy-wall test tube reaction cell; (2) a 4-cam, 90-sec/rev motor driven timer; (3) two Teflon-bodied, relay-actuated solenoid valves; (4) a large diaphragm, single stage, low-pressure gas regulator; and ( 5 ) two 1-1. reagent reservoirs. (Details of construction are available upon request.) A Perkin-Elmer Model 403 Atomic Absorption Spectrophotometer equipped with a 3-slot, 10-cm Boling-type burner head was used for most of the work reported in this article. However, different atomic absorption instruments were used satisfactorily by other laboratories involved in testing the hydride generator. Both single element hollow cathode lamps (HCL) and electrodeless discharge lamps (EDL) were used as the primary atomic line sources. The extremely "soft" diffusion flame was protected from room air currents by a burner shield equipped with quartz end windows and

Figure 1. Diagram of hydride generator

single stage, low pressure gas regulator, (8)pressurized reagent bottles, (C) 4-cam motor driven timer (90 sec), (D) Teflon-bodied, relay actuated sole(A)

noid valves, (E) reaction head with Teflon metering valves, (F) reaction cell

w Figure 2. Flame shield for Boling burner head

Pyrex side plates (Figure 2). Use of the burner shield improved precision approximately twofold. The gaseous reaction products from the generator were delivered t o the burner via Latex rubber tubing and into the burner mixing chamber by replacement of the nebulizer with a simple straight-through glass adapter, identical to one described by Dalton and Malanoski (15) except that the check-valve assembly was removed. Reagents. The NaBH4 reducing solution is made by dissolving 4 g of the powder or pellets (Ventron Corp.) in approximately 50 ml of NaOH (10% w/v) solution. This solution is then diluted to 100 ml with 10% (w/v) NaOH. T h e strong base stabilizes the borohydride solution. For the arsenic and antimony determinations, a 10% (w/v) NaI prereductant solution is used. An acid diluent solution is made by mixing 50 ml of concentrated H2S04 with 300 ml of distilled-deionized water. T o this mixture is added 300 ml of concentrated HC1, followed by dilution to 1 1. with distilled-deionized water. Sample Preparation. The sample of food or biological tissue (1-3 g, dry weight) is weighed into a 100-ml Kjeldahl flask, and 30 ml of a (4:l:l) mixture of concentrated, reagent grade "03, HzS04, and HC104 is added t o the flask. At least one reagent blank consisting of 30 ml of the ternary acid mixture is prepared with each set of 10-12 samples. The sample oxidation is carried out using one or more 6-unit, micro-Kjeldahl digestion racks equipped with a glass manifold, water-cooled condenser, receiving flask, and dilute (10% w/v) NaOH scrubbing tower. The sample-acid mixture is initially heated very gently. After the sample foaming subsides, the temperature is raised to produce steady boiling. Sample charring is avoided by cautious heating during the He104 reaction. If slight charring does occur, analyte loss can be prevented by immediate addition of small (1 ml) increments of concentrated "03 and/or rapid cooling of the sample-acid mixture in an ice water ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976

121

8 20 SEC

Table I. Operating Parameters Element

As

Se

Sb

Te

Source Source power o r current Slit width, m m Spectral band width, n m ’ Wavelength, n m

EDL

EDL

EDL

HCL

8 watt 3

6 watt

7 watt

3

0.3

30 m A 1

2.0 197.20

2.0 196.0

0.2 217.6

0.7 214.3

a T h e 1 9 3 . 7 - n m resonance line of As is a b o u t twice as sensitive as t h e 1 9 7 . 2 - n m line a n d can b e employed with s o m e sacrifice of linear range.

bath. Excess HClO4 is boiled off and the residual H2S04 heated strongly to fumes of sulfur trioxide. G. F. Smith’s monograph on the use of perchloric acid may be consulted for a more thorough treatment of necessary precautions (16). These vigorous acid-oxidation conditions are required both to prevent volatilization loss of the analyte(s) and to oxidize the sample completely. Unoxidized organic moieties in the digest appear to alter the rate of release of the hydrides during the determinative step of the procedure. The digestion is normally complete in 1.5 to 2 hr. The digests, when cooled to room temperature, are transferred to 100-ml volumetric flasks. Concentrated HC1, 30 ml, is added to each flask and the solution diluted to 100 ml with distilled-deionized water. Instrumental Conditions. With the burner drain tube tightly stoppered, the hydrogen diffusion flame is established, using a hydrogen flow rate of approximately 2 l./min (10 psig) and a nitrogen diluent flow rate of about 6 l./min (30 psig). The nitrogen is introduced through the auxiliary oxidant port of the burner, and the normal nebulizer tube is removed from the chamber and closed off. The hydrides are introduced through the nebulizer port. Optimization of the absorption signal is normally obtained by adjusting the position of the burner head to a height just below the optical path of the line source. Typical instrument parameters are shown in Table I. Prior to the actual determinations, the duration and flow rates of the NaBH4 introduction and distilled water wash, and the duration of the rest period were adjusted to ensure complete reaction of the analyte(s) and to maximize the absorption signal. Typical NaBH4 solution and distilled water wash flow rates were 24-32 and 20-40 ml/min, respectively. The latter was not critical since it served only to rinse unspent NaBH4 from the capillary. The flow rates were achieved by regulating the pressure (3-5 psig) on the reagent reservoirs and adjustment of the Teflon needle valves in the reaction head. The 4-cam timer (90 sec/rev) which controlled the two normally closed solenoid valves by actuating microswitches, was adjusted to provide 15 sec open (on) time for the NaBH4 solenoid, 5 sec rest period, and 5-15 sec for the distilled water (wash cycle) solenoid. The resulting 6-8 ml NaBH4 addition resulted in well defined, reproducible absorption peaks for each analyte. Sufficient accuracy was obtained by peak height measurement. Signal integration was possible but was not used for routine analysis. Correction for non-specific background absorption proved entirely unnecessary. Determination of Arsenic or Antimony. An aliquot of the acidified sample digest (maximum of 20 ml, which is equivalent to 1 g of sample when 5 g (wet weight) is digested) is transferred to an 8-in test tube reaction cell and diluted to 20 ml with the acid diluent as necessary. The NaI prereductant (10% w/v) is added to the reaction cell: 0.5 ml is sufficient to reduce As5+ or Sb5+ to the trivalent state. A minimum of 1-min reaction time is required to prereduce the As and Sb. The test tube is affixed to the reaction head of the hydride generator, and the timer is actuated. The reaction gases are transported to the burner, and the transient absorbance signals are recorded. Standards, typically ranging from 0.05 to 0.70 pg As or Sb, are prepared by appropriate microliter additions of a 10 wg/ml mixed As-Sb stock solution (prepared daily in 30% v/v HCl) to 20-ml volumes of the acid diluent. Standards and reagent blanks are treated with the NaI prereductant prior to hydride generation. The prereductant is essential to the generation of stibine, SbH3, under the uniform conditions of acidity (5%H2S04,30% HC1) used throughout for As, Sb, Se, and Te. Arsine, ASH,?,is quantitatively produced either in the presence or absence of the NaI, i.e., quantitative reduction of As5+ or As3+ is achieved by the NaBH4. How122

ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976

T I M E , SEC.

Figure 3. Absorbance vs. time peaks for 0.5 M g A s (A) without

Nal prereductant. (B) with Nal prereductant. Peak areas are identical over 30-sec integration.Base lines are offset to avoid overlap 07

06

05

E

04

d 0

9

03

02

01

01

02

03

04

05

06

MICROGRAMS OF ELEMENT

Figure 4. Analytical calibration curves for As, Se, Sb, and Te ever, the appearance of the As absorption signal is delayed and the signal duration is somewhat longer without the NaI prereductant (Figure 3). The peak areas, integrated over 30 sec, are identical for As5+ compared with As3+ initial oxidation states. Determination of Selenium o r Tellurium. Sample, blank, and standard aliquots for Se or Te determination are treated just as those for As or Sb determination with the important exception that no prereductant is added. A prereductant would prevent generation of H2Se or H2Te by possible reduction to the elemental form.

RESULTS AND DISCUSSION Sensitivity and Linearity. Both t h e sensitivity a n d the linearity are illustrated i n Figure 4. The analytical calibration curves for As, S e , and Sb are all linear to a b o u t 0.5 Mg of the e l e m e n t w h e n using EDL’s as the p r i m a r y source, peak height m e a s u r e m e n t s , a n d m o d e r a t e i n s t r u m e n t d a m p i n g (T = 1 sec). Under the s a m e conditions, b u t using an HCL as p r i m a r y source, t h e Te calibration curve is line a r t o a b o u t 0.4 Mg.

Table 11. Analysis of NBS Reference Materials, ppm (dry weight) AAS (Hydride)a

NBS~

Independent NAA

Orchard Leaves (SRM 1571) As 11 *1 11 r 2 ... Se 0.055 t 0.009 0.08 t 0.01 ... Sb 2.99 t 0.05 ... 3.0C Bovine Liver (SRM 1577) As 0.049 f 0.006 0.055d ... Se 0.98 t 0.01 1.1 t 0.1 ... Sb Not Detected ... 0.034e Tunaf 4.6 t 0.3 3.6 + 0.2g As 3.3 t 0.2 *.. 3.3 t 0.3 Se 3.6 t 0.1. a Mean and standard deviation based on the analyses of three replicate 1-g samples. b Estimate of uncertainties in no case less than 95% confidence 1imits.c (19).d NBS Informational value (1 7). e ( 2 0 ) .f NBS proposed research material. NBS informational As and Se values from ref (18). g FDA NAA value.

Flgure 5. Typical precision of measurement for 10 replicate standard solutions

Table 111. Arsenic Confirmation Analyses, ppm (dry weight) Sample

Neutron activationa

Spectrophotometricb

AAS (hydride)c

40.1 37 t 3 Haddock 40 ?: 2 8.3 7.5 5 0.4 Perch 9.0 t 0.4 19.0 t 0.9 15.8 15.3 t 0.7 Flounder 12 5 1 11.3 11 t 2 Cod a NAA based on 1 - 2 analyses. b Single analysis. C Based on 5 replicate analyses over a 4-month period.

Table IV. Selenium Confirmation Analyses, ppm (dry weight) Sample

Neutron act ivat ion0

Fluorometryb

Haddock 1.6 i: 0.4 1.6 Perch 2.2 !: 0.2 2.3 Flounder 0.9 t 0.3 0.9 Cod 1.2 c 0.2 1.2 a NAA based on 3-5analyses. b Single analysis. C 5 replicate analyses over a 4-month period.

AAS (hydride)c

1.6 t 0.4 2.6 i 0.3 1.2 t 0.2 1.4 t 0.4 Based on

Rather than use the definition of sensitivity common to practicing atomic absorption spectroscopists, i.e., the concentration of analyte, in wg/ml, which will produce 1%absorption (0.0044 A), the authors prefer the use of the "slope" of the linear portion of the analytical calibration curve. For Se, As, Sb, and Te, the sensitivities are 1.10, 1.00,0.70, and 0.40A/pg, respectively. Accuracy. The accuracy of the analytical method was established by: (1) successful determination of As, Se, and Sb in NBS Standard Reference Materials (17); (2) agreement of results obtained by hydride generation-atomic absorption spectrometry with totally independent techniques; and (3) quantitative recovery of analytes added to a variety of foods. The third approach was especially important for S b and T e where native levels of the elements in foods or biological tissues either were not detected or were too low to be quantitated. Results of the analysis of NBS SRM 1571 (Orchard Leaves) and SRM 1577 (Bovine Liver) for As, Sb, and Se are given in Table 11. Results are also given for a Tuna which will be an NBS Research Material. Since the Tuna has not been certified, all of the tentative NBS values must be considered as informational values only (18).With the possible exception of Se in Orchard Leaves, and As in

s J d J d - d d c - - / i i N c - . / r l l - N c ^ SELEhlUM

Figure 6. Reproducibility of the determination of As and Se in oyster

meats: duplicate measurements Tuna, the results obtained by the hydride evolution method agree quite well with NBS certified or informational values. FDA independent NAA analysis gave an As value for the Tuna of 3.6 f 0.2 ppm. The Orchard Leaves, which contain rather large amounts of As and Sb (NAA value) (19) and relatively little Se, and the Bovine Liver, which contains a substantial amount of Se but little As and Sb (NAA value) (20), provide excellent examples of the accuracy of the method over a large range of concentrations for two very different natural materials. Further confirmation of accuracy is given by the agreement among values obtained by independent analytical methods shown in Tables I11 and IV. Frozen fillets of fish were purchased, coarsely ground with a meat grinder, blended and lyophilized. The dried products were further ground and blended prior to analysis by y ray spectrometry (NAA), fluorometry (for Se), absorption spectrophotometry (for As), and atomic absorption spectrometry (hydride volatilization). The results agree well within experimental error. During the course of hundreds of analyses, a large volume of recovery data was collected. The recovery data were obtained by a single addition of the analytes t o each commodity. In general, enough analyte was added to fortify the samples by amounts ranging from approximately the native level to as much as fivefold greater than the native level. Since Sb and Te either were not detected or could not be quantitated in any food, additions were made a t the 1-pg level. For a wide variety of foods and spiking levels, the relative recoveries were 98 f 5% for As, 104 f 7% for Sb, 106 f 6% for Se, and 100 f 7% for Te. Precision a n d Detection Limits. Ten replicate measurements each of the As peak for a standard solution containing 0.025 pg of the analyte and the reagent blank solution were made (Figure 5). The shoulder on the trailing (left) side of the analyte peak corresponded to the NaBH4 solenoid shut-down whereas the small peak further to the left of each analyte peak resulted from the distilled water wash cycle. The average absorbance of the standard was ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976

123

Table V. Interlaboratory Study: Arsenic? ppm (dry weight) Laboratory

1 2

Haddock

36.0 35.1

Flounder

Cod

Tuna

Eggs

Chicken livers

16.1 14.8 14.2 16.3 15.4 ( 1 . 0 )

11.1 10.2 10.1 10.9 10.6 ( 0 . 5 )

3.3 3.2 3.4 3.4 3.3 (0.1)