Anal. Chem. 1984, 56, 1545-1548
1545
Hydride Generation and Atomic Emission Spectrometry with Helium Glow Discharge Detection for Analysis of Biological Samples K a z u k o Matsumoto,* Toshio Ishiwatari, and Keiichiro F u w a Department of Chemistry, Faculty of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, J a p a n Hydride accumulation has been widely utilized for the atomic spectrometry of Ge, Sn, Pb, As, Sb, Bi, Se, and T e (1). For the detection of these elements, ICP (inductively coupled argon plasma) (2-4), CMP (capacitatively coupled microwave plasma) (5), APAN (atmospheric pressure active nitrogen after-glow) (6), and AFS (atomic fluorescence spectrometry) (7, 8) have been investigated, as well as AAS (atomic absorption spectrometry) (9-12). A helium glow discharge detector for atomic emission spectrometry has also been studied in connection with a hydride generation technique (13,14). Although the detector is highly sensitive to As, Ge, Sb, and Sn, no attempt has been reported concerning its application to the hydrides of other elements. In the present study, the detector is applied to the determination of Pb and Se, together with Ge, As, Sb, and Sn. A new hydride generation system is developed on the basis of consideration of the chemical properties of each hydride and also of the optimum conditions of stable and continuous operation of the He glow discharge detector. The effects of the electrode gap and the power on the sensitivity of each element are examined, and their relation is discussed. The method is successfully applied to the analysis of biological materials.
EXPERIMENTAL SECTION A Helium Glow Discharge Detector. A schematic diagram of the helium glow discharge detector used in the present study is shown in Figure 1. The cell is made of quartz and the electrode holders are brass. The design is basically the same as that reported in ref 15 and 16. However, the holders are considerably larger ( 3 cm in diameter and 5 cm long) compared to the previously reported one, and therefore the heat produced in the electrodes is transferred to the holders and is efficiently air-cooled owing to the large surface area of the holders. Although the holders are designed so that they can be water cooled, it has been shown that water cooling makes the electrodes too cool to maintain a stable discharge. The electrodes used are W-2% Tho2and are 1.6 mm in diameter. The electrode gap can be easily adjusted with a screw and the electrodes can be easily removed and cleaned when they are stained. The direct current power supply is Model 6522A from Hewlett-Packard (0-100 mA, 0-2 kV) and is used in the constant-current mode. A lO-kQresistance is inserted in series as a ballast resistor for stable discharge. The light detection system was based on a Nippon Jarrell-Ash 0.5-m Ebert monochromator. A lens with a focal length of 10 cm focused a 1:l image of the glow discharge on the entrance slit (40 wm) of the monochromator. A Hamamatsu RlO6UH photomultiplier was used for Se and a R456 photomultiplier was used for the other elements. The current was amplified with a direct current amplifier, Model 427, from Keithley Co., and was recorded on a strip chart recorder. A Hydride Generation System. As is already known for hydride generation-AAS, the reduction reaction with NaBH, produces much hydrogen gas, which extinguishes the He glow discharge. In the previously reported hydride generation systems (13,14,17,18), a hydride trap immersed in a liquid N2bath was directly connected to a He glow detector and, therefore, the discharge was off while the hydride generation reaction proceeded and much hydrogen gas was evolved. After the reaction was completed, the discharge was initiated and stabilized for 1 min. The hydride was then introduced into the discharge by allowing the trap stand at room temperature or by rapidly warming to room temperature by use of a fan. In the present study, an improved hydride generation system has been constructed, as shown in Figure 2. The main improvement is that the He flow path is divided into two, one of which leads to the glow discharge through 0003-2700/84/0356-1545$01.50/0
the sample solution and the hydride trap. The hydrogen evolved in the reaction chamber is vented before it enters the discharge. The other path is for the maintenance of the discharge; the discharge is possible even while the reduction reaction proceeds and hydrogen gas is evolved owing to this extra He path. These two He paths enable continuous operation of the glow discharge throughout the whole analytical procedure. H,O and CO, are also produced as byproducts of the reaction. These gases cause molecular emissions such as OH, CO, or CO' bands, which considerably destabilize the background emission and also decrease the analytical sensitivity. These byproducts must be removed as completely as possible from the hydride gas. The water trap immersed in a dry ice-2-propanol bath and the COz trap (a column packed with NaOH) in Figure 2 are used to remove these undesired byproducts. Since hydrides of As, Ge, and Sn are not reactive toward NaOH, a NaOH column was inserted in the He path as shown in Figure 2. However, as hydrides of Pb, Sb, and Se react with NaOH, the COz trap was not used for these elements. Since no neighboring emission lines due to CO, are observed for the analytical lines of Pb and Sb, no serious interference problem was created for these elements, even though no CO, trap was used. However, for Se, COz causes a considerable blank signal and must therefore be removed by some other method. For the determination of Se, a column, designated as SC in Figure 2 and packed with Chromosorb 102 (60-80 mesh), was employed for the separation of H2Sefrom CO,. After both HzSe and C 0 2 were trapped in the column immersed in a liquid Nz bath, it was removed from the bath and allowed to stand at room temperature. Since H2Se vaporizes about 1.5 min later than COz does, only HzSe can be once again trapped in a U-shaped column (H),packed with quartz wool and immersed in liquid N,. In the final step, the H,Se was vaporized and introduced into the He glow discharge. Reagents. Standard solutions were prepared from the following reagents: As(II1) from As2O3,dissolved first with 1 M NaOH and then acidified with HC1; As(V) from Na2HAs04.7H20, dissolved with HC1; Ge(1V) from GeO,, dissolved with KOH; Pb(I1) from the metal dissolved with HNO,; Sb(II1) from potassium antimonyltartarate, K(SbO)C4H406.0.5H,0dissolved in H20, Se(1V) from the metal dissolved with HNO,; Sn(1V) from the metal dissolved with HNO, and HC1. Analytical Procedure. For the determination of As, Ge, Pb, Sb, and Sn, the procedure is as follows: 10 mL of sample solution, acidified to 0.2 M HC1, was placed in a 50-mL reaction vessel (G, Figure 2), while He flowed through the bypass to maintain the discharge. In the case of Pb determination, 1mL of 2 M NazS20B was added to the sample solution for the enhancement of the sensitivity (19). After the reaction vessel was filled with He gas (flow rate 0.3 L/min) and the hydride trap H was immersed in liquid Nz, 2.3 mL of 4% NaBH, solution was added with a peristaltic pump (PP) for 40 s. For the determination of Pb, 2.8 mL of 4% NaBH, solution was added for 50 s. The evolved hydride was trapped for 3 min. After the four-way valve was turned so that He gas flowed into the discharge cell D, the liquid N2 Dewar was removed from the trap H and the hydride was introduced into the discharge. In the measurement of As, Ge, and Sn, a nichrome wire was used to help the rapid vaporization of the hydride gas. The peak height of the signal was used for the calibration. For the determination of Se, sample solution was acidified to 4 M HCl and the separation column (SC) was immersed in liquid Nz. A 1.7-mL portion of 4% NaBH4 solution was added for 30 s and the evolved H2Se and COP were trapped for 2 min. After three-way valves were turned so that He flowed in the bypass B,, the SC was left standing in the air for 2.5 min, while COz was vented. After all the C02 was vented, the hydride trap H was immersed in liquid Nz and the H,Se evolved from the column 0 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
n
Table I. Wavelengths and Relative Sensitivities of Emission Lines for As,Ge, Se, and Sn ele- wavelength, ment nm RS' As
Ge
a
Flgure 1. Schematic diagram of the helium glow discharge detector: (A) screw (brass) for adjustment of the electrode gap; (S) electrode holder (brass); (E) tungsten-2 % Tho2 electrode; (D) quartz discharge cell; (W) quartz window; (I) helium gas inlet (perpendicuhr to the sheet); (0) helium gas outlet capillary (perpendicular to the sheet).
1193.7 1197.0 1228.8 1235.0 1278.0 1265.2 1271.0 1303.9 1327.0 1422.7
0.14 0.33 1 0.19 0.30 1 0.38 0.33 0.10 0.02
ele- wavelength, ment nm RS ' Se Sn
1196.0 1204.0 1206.3 1224.6 1284.0 1286.3 1303.4 1317.5 1326.2 1452.5
0.36 1 0.37 0.12 1 0.49 0.31 0.17 0.26 0.04
Relative sensitivity.
Se. Six dolphin livers were kindly supplied by R. Tasukawa of the university of Ehime and were analyzed for Se and As after being homogenized. Two ginseng roots were analyzed for Ge after being dried and pulverized. The calculation of each analyte concentration was performed on the basis of the conventional calibration method, except for Se in bovine liver, for which the standard addition method was employed, since recovery tests by the conventional calibration method gave unsatisfactory results. The matrix of the standard solution for calibration was matched with that of acid-decomposed sample solution.
RESULTS AND DISCUSSION Selection of Analytical Lines. The analytical line for each element was determined on the basis of the relative sensitivity S, which is calculated from the analyte emission
s = (10 - I b ) / " Figure 2. Schematic diagram of hydride generation-helium flow discharge detector for determination of selenium: (C) helium gas cylinder; (F) flowmeter; (S) NaBH, solution: (PP) peristaltic pump; (G) reaction vessel; (B,, B2) bypass; (CT) CO, trap (NaOH); (W) water trap (dry ice-2-PrOH); (SC) separation column (Chromosorb 102, 60-80 mesh): (H) hydride trap; (LN) liquid N2 bath; (DC) dc power supply; (D) quartz discharge cell; (L) lens; (M) monochromator; (P) photomultiplier tube; (V) high voltage power supply; (A) amplifier: (R)recorder.
SC was trapped for 3 min. The procedure after this step was the same as that for the other hydrides. The nichrome wire was also used for the rapid vaporization of H2Se. Analysis of Biological Samples. In order to test the applicability of the present method to real samples, analyses of NBS SRMs, NIES CRM, dolphin livers, and ginseng roots were carried out. The decomposition procedures were as follows: Since trace amounts of As are leached out of Pyrex glass and give a blank signal, decomposition of samples for the determination of As was carried out in a Teflon beaker (100 mL). Ten milliliters of HN03 was added to 1-1.5 g of a sample, which was left standing overnight. Five milliliters of HNOBwere then added and the sample was heated at 150 OC for several hours. Five milliliters of HClO, and 3 mL of H2SO4 were added and the sample was heated at 250-300 OC until white fumes of HClO, appeared. Additional HC104 (3 mL) was added and the sample was further heated until the white fumes disappeared, which is the completion of the decomposition. The decomposed solution was filtered and diluted to 100 mL. For the determination of Se, no blank signal was observed. Therefore, a Pyrex glass vessel was used for decomposition. The decomposition procedure was the same as that for As. However, since a part of Se is Se(VI),which cannot be reduced to HzSe by NaBH,, Se(V1) has to be reduced to Se(1V) before measurement. The procedure is as follows: 50 mL of H20 and 25 mL of HCl were added to the decomposed solution, which was then boiled for 10 min to reduce Se(V1) to Se(1V). For the determination of Ge, samples were decomposed after the procedure for As. NBS SRM Orchard Leaves and NIES CRM Pepperbush were analyzed for As and NBS SRM Bovine Liver was analyzed for
(1)
intensity Io, the blank emission intensity I b and the relative standard deviation of the background emission intensity 0. For As, Ge, Se, and Sn, the relative sensitivity of each analytical line is tabulated in Table I. The line of each element used for analysis was determined as that which gives the best sensitivity. Effect of Acid Concentration, Since the optimum acid concentration must differ for each element, 0.2 M and 1 M HC1, H2S04,and HN03 were examined for each element. The result did not depend on the acid used. Since for As(III), Ge, and Sn the sensitivity did not differ from 0.2 M to 1 M, 0.2 M HC1 was used in the following experiments. However, for P b and Sb, much COz is produced when 1 M acid is used and the He glow discharge flickers. Therefore, 0.2 M acid was employed. Since As(II1) and As(V) showed the same sensitivity at 0.2 M acid, As(II1) was used as standard solution for all of the following experiments. Since the solubility of HzSe in water (270 cm3/100 mL of H 2 0 at 25 "C, 1 atm) is considerably greater than that of AsH3 or GeH4, the efficiency of the hydride evolution from the sample solution is low. Although the sensitivity was compared for 3 M, 4 M, and 6 M HC1 solution, the sensitivity did not vary in this concentration range. Therefore, the following experiments were carried out with 4 M HC1. Effect of Electrode Gap. The sensitivities of As and Ge were examined with the electrode gap varied. For both elements, the sensitivity increases with increasing gap, reaching the greatest sensitivity when the gap is 1.5 mm. After that the sensitivity decreases, being constant a t gaps larger than 3-3.75 mm. Although the sensitivity is high a t an electrode gap of 1.5 mm, the analytical reproducibility is worse and the sensitivity rapidly decreases after repeated measurement, since the electrode surface is eroded and deteriorates. Therefore, the gap was set a t 4.5 mm for a stable continuous discharge. Effect of the Power Supplied to the Discharge. Since the discharge is stable between 10 and 30 W, the emission intensities of He I (587.6 nm) and the analytical lines of As,
ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
Table 11. Analytical Figures of Merit for Hydride Generation-Helium Glow Discharge Technique detection limit/ng' wavelength/ present hydrideupper limit of element nm method AAS linearitylng' 10000 0.2 8 As 228.8 5 00 0.1 40 Ge 265.2 2000 6 150 Pb 405.8 20000 0.8 5 Sb 252.9 5000 2 18 Se 204.0 500 0.2 5 Sn 284.0
1547
~~
' Sample volume 1 0 mL. technical report.
I&
RSD % a (amting) 5.0 (25) 7 . 1 (10) 5.1 (100) 8.5 (25) 6.4 (100) 7.2 (15)
The values are cited from ref 1except for Pb, whose detection limit is taken from Varian
Table 111. Analytical Results of Arsenic, Selenium, and Germanium in Various Biological Materials content, pg/g As Se sample orchard leaves pepperbushe bovine liverd dolphin liver A B C D E F
found 12.2 f 0.3 2.4 f 0.2 0.017 i 0.018 t 0.018 i: 0,019 t 0.016 i. 0.015 t
cert'
found
14i2 2.3 f 0.3
certa 0.08 t 0.01
1.08 r 0.08 86i 6 45f 3 2.14 t 0.07 8.3 t 0.4 48f 3 76t 7
0.003 0.004 0.001 0.003 0.002 0.004
recovery, % As
Se
102f
1.1i 0.1
1oog llOh
99h
sample
Ge found, ngig
Ge recovery, %
ginseng root A B
16i 3 15 2 4
101' 9oj
' Certified value. NBS SRM 1571. NIES CRM No. 1. NBS SRM 1577. e Content is expressed on the basis of wet weight. As, 10 pg, was added per 1 g of sample. Se, l,pg, was added per 1g of sample. Se, 50 pg, was added per 1 g of sample. Ge, 50 ng, was added per 1 g of sample. Ge, 25 ng, was added per 1 g of sample. Ge, Se, and Sn were examined in this power range. The emission intensity of He I 587.6 nm increases exponentially with increasing power. The intensity dependences of As, Se, and Ge on the power are shown in Figure 3. The emission intensity of As decreases slightly, while that of Se increases slightly with increasing power. There seems to be two possible reasons for the decrease in emission intensity: One is that the increased power level causes more material to enter the discharge from electrodes, which can cause a change in the effective ionization potential of the discharge and would cause a temperature increase (20,21). The low-temperature hydride gas generally moves along the outer relatively low-temperature region of the discharge, as was observed in primitive ICP about 15 years ago (22). Therefore, the efficiency of the hydride introduction into the discharge is decreased with increasing temperature and also the hydride density in the discharge is lowered. The other possible reason is that the ionization ratio of the analyte element is increased as the power is increased. In order to examine the effect of the ionization, the intensity dependences of Sn I (284.0 nm) and Sn I1 (189.9 nm) were measured. The result is that both lines almost linearly decrease with increasing power, therfore the decreasing trend for Sn I cannot be ascribed to analyte ionization. It seems rather to be due to the decrease of the hydride introduction ratio into the discharge. As for the analytical sensitivity, since the background emission, as well as the blank emission due to COz, decreased as the power decreased, all elements gave their best sensitivities a t 10 W, which is unexpected, since Feldman set the current a t the highest possible value (13). Effect of Helium Flow Rate. The effect of the helium flow rate was examined for the range from 0.2 to 1.0 L/min. In order not to change the hydride evolution efficiency from
0 -.--I10
7n
30
Power I W I
"
1-1
10
jti
do Powpr
1w1
?owe
I W
Flgure 3. Effect of power on the emission intensities of As, Se, and Ge. As, 25 ng, Se, 100 ng, and Ge, 10 ng, were used with the He
flow rate of 0.6 L/min. the solution, the He flow rate was fixed a t 0.3 L/min while the hydride is trapped and was varied when the hydride was vaporized from the trap and introduced into the discharge. The change of He I (587.6 nm) intensity is shown in Figure 4 as the function of He flow rate and also of the power. Figure 5 shows the intensity dependence of As on He flow rate. The He and As emission lines show a similar dependence on He flow rate. Although the sensitikity of As is the same both at He 0.5-0.7 L/min (power 60 W) and at 1.0 L/min (power 30 W), the He flow rate of 1.0 L/min is so high that the inner pressure of the discharge cell is high and sometimes gas leaks occur. The discharge was usually operated at 10 W and a He flow rate of 0.5-0.7 L/min. The effect of He flow rate was also examined for Se, Ge, and Sn while the power was fixed at 10 W. The result is shown in Figure 6. As a result of these observations, the He flow rate was fixed a t 0.6 L/min for all of the elements. Detection Limits a n d Dynamic Ranges. The detection limit and dynamic range for each element are shown in Table 11, together with those for AAS. These detection limits are 1 to 2 orders of magnitude improved compared with those
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
On the other hand, chemical interferences are observed as previously reported in the hydride generation-AAS (23). Therefore the interferences of the elements comprising biological materials are investigated in the present system. The most severely interfering elements are Co(II), Cu(II), Ni(II), and Pt(1V). The recoveries of these elements when present 1pg/mL in 2.5 ng/mL As solution are 82,54, 3, and 36%, at L LI 05 ID 0 05 ID respectively, and those for 1.0 ng/mL Ge solution are 69, 53, He Gar Rule IL,nilnl Hc Gas Flow Rata I L 01 H e Gas Flow R a t e I 0, and 33%. These negative interferences can be removed by Figure 4. Effect of helium gas flow rate on the emission intensity of the addition of 2% KI and 0.1 M malic acid as reported He I 587.6 nm at various power levels: (left) power 10 W; (middle) previously (24). For the analysis of real samples, the standard power 20 W; (right) power 30 W. addition method must be employed or these masking reagents have to be added. 15 Analytical Results of Biological Samples. The anaI A lytical results for As and Se in NBS SRMs, NIES CRM, and six dolphin livers and also Ge in ginseng roots are summarized in Table 111. Some of the dolphin livers contain unexpectedly high Se, which is interesting with respect to the recently reported possible detoxification effect of Se against Hg. As a summary, He glow discharge is a highly sensitive detector for hydride-forming elements. The detection limits are 1 to 2 orders of magnitude improved compared with those obtained by usual hydride generation-AAS. The discharge , -.- 20:; is stable for a long period. It flickers only slightly with hydride sow introduction. 05
Flow
10
L n> n !
11,
t-
I r=’‘ --.--
0
r’
0.1
0.5
Registry No. As, 7440-38-2;Ge, 7440-56-4;Pb, 7439-92-1;Sb, 7440-36-0;Se, 7782-49-2;Sn, 7440-31-5.
1 .o
h e Gas Flow R a t e ( L / m i n )
Figure 5. Effect of helium various power levels.
05 10 He Gas Flow Rate I L min!
gas
flow rate on the sensitivity of As at
0
He
083
Flow Rate ( L l r n l n !
He Gar Flow Rate i L
nit”
1
Figure 8. Effect of helium gas flow rate on the emission intensltles of Se, Ge, and Sn. Se, 100 ng, Ge, 10 ng, and Sn, 15 ng, were used and the power was 10 W for all three elements.
obtained by usual hydride generation-AAS. Interferences of Coexisting Elements. Since hydrides of analyte elements are isolated as gases from the matrix elements in the sample solution, little optical interference due to neighboring emission lines exists in the present method. The possible interfering elements are only other hydrideforming elements and Hg, which is also produced by the reaction with Na13H4. Although Ge (265.158nm), Se (265.148 nm), and Sb (265.261 nm) seem to interfere, hydrides of the latter two elements are decomposed by the NaOH used as a COz absorber and therefore cause no interference. Likewise, Sn 203.95 and 204.12 nm) and Te (203.979 nm) cause no interference on Se (203.985 nm), since these are separated by a Chromosorb 102 column.
LITERATURE CITED Robbins, W. B.; Caruso, J. A. Anal, Chem. 1979, 5 1 , 889A-899A. Thompson, M.; Phalavanpour, B.; Walton, S. J.; Kirkbright, G. F. Analyst (London) 1978, 103, 568-579. Thompson, M . ; Phalavanpour, B. Anal. Chim. Acta 1979, 109, 251-256. Ikeda, M.; Nishibe, J.; Hamada, S.; Tsujino, R . Anal. Chim. Acta 1981, 125, 109-115. Nakajima, R. Bunseki Kagaku 1976, 25, 889-871. D’silva, A. D.; Rice, G. W.; Fassel, V. A. Appi. Spectrosc. 1980, 3 4 , 578-584. Thompson, K. C. Analyst (London) 1975, 100, 307-310. Kobayashi, S.; Nakahara, T.; Musha, S. Talanta 1979, 2 6 , 951-957. Smith, A. E. Analysf (London) 1975, 100, 300-306. Thompson, K. C.; Thomerson, D. R. Analyst (London) 1974, 9 9 , 595-601. Slemer, D. D.; Koteel, P. Anal. Chem. 1977, 4 9 , 1096-1099. Asmode, J.-F.; Foster, P.; Van’t Dack, L. Anal. Andreae, M. 0.; Chem. 1981, 53, 1766-1771. Feldman, C. Anal. Chem. 1979, 5 1 , 664-669. Braman, R. S.;Tompkins, M. A. Anal. Chem. 1978, 5 0 , 1088-1093. Braman, R. S.;Dynako, A. Anal. Chem. 1968, 4 0 , 95-106. Feldman, C.;Batlstoni, D. A. Anal. Chem. 1977, 4 9 , 2215-2221. Braman, R. S.; Johnson, D. L.; Foreback, C. C.; Ammons, J. M.; Bricker, J. L. Anal. Chem. 1977, 4 9 , 621-625. Braman, R. S.;Johnson, D. L.; Foreback, C. C. Anal. Chem. 1972, 4 4 , 2195-2199. Gin, K.; Taga, T. Bunseki Kagaku 1980, 2 9 , 522-526. Semenova, 0. P. Izv. Akad. Nauk SSSR, Ser. Fiz. 1945, 9 , 7. I.5-7. I . a. -. Saha, M. N. Philos. Mag. 1920, 4 0 , 472-488. Dickinson. (22) ~. .. , G. W.: Fassel. V. A. Anal. Chem. 1969, 4 1 . 1021-1024. (23) Smith, A. E. Analyst (London) 1975, 100, 300-306. (24) Jin, K.; Terada, M.; Taga, M. Bull. Chem. SOC. Jpn. 1981, 5 4 , 2934-2936.
RECEIVED for review October 19, 1983. Accepted February 24, 1984.
