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Anal. Chern. 1983, 55, 2026-2032
Speciation of Inorganic and Organometallic Compounds in Solid Biological Samples by Thermal Vaporization and Plasma Emission Spectrometry Shigeki Hanamura, B. W. Smith, and J. D. Winefordner* Department of Chemistry, University of Florida, Gainesville, Florida 32611
By means of thermal Vaporization, Inorganic, organlc, and metallorganic species are separated and elemental emission In a microwave plasma is detected as a functlon of vaporlzatlon temperature. Solid samples of 250 mg or more are used to avoid problems with sample heterogenelty. The preclslon of characteristic appearance temperatures Is f2 ' C . The slngle electrode atmosphere pressure mlcrowave plasma system Is extremely tolerant to the Introduction of water, organlc solvents, and air. The measurement system contained a repetltlon wavelength scan device to allow background correctlon. The plasma temperature was 5500 K. The system was used to measure C, H, N, 0, and Hg In orchard leaves and in tuna fish.
Speciation means the separation and determination of trace inorganic, organic, and organometallic species in real samples ( I ) . The separation processes, which have been most useful for molecular species in solid samples, include volatilization and/or gas chromatography, extraction and partition chromatography, fusion and dissolution, and direct methods. Extraction and partition chromatography are normally time-consuming and limited by the purity of the reagents used for sample dissolution and extraction. Fusion and dissolution are plagued even more with the difficulties listed for extraction and partition chromatography. Direct methods are normally impossible because the detection method will be subject to considerable interferences. Volatilization with gas chromatography is an excellent choice but the sample size must be small, e.g., 510 pg (2)which results in difficulties in sample homogeneity in some cases, and the column temperature may cause thermal decomposition for some species. Volatilization, as a separation method, is certainly more complex than extraction but is capable of separating molecular species in large (up to 1 g) samples. The most powerful detection approach has involved mass spectrometry, which has been used to detect molecular species generated by volatilization from minerals (3,4),oil shales (5), polymers (6),and lunar soils (7). Unfortunately, mass spectrometric detectors are more difficult to use, require very small (- 1pg) sample sizes, and are much too expensive for many laboratories. Methods, such as X-ray diffraction, infrared absorption spectrometry, and NMR, are much too insensitive to be used for trace analysis. Electrochemical detectors (8, 9) are useful for a number of metals and require the species of interest to be in solution. Gas chromatographic detectors (1&13) based upon flame ionization, electron capture, thermal conductivity, and photoionization are generally nonselective and rely totally upon the separation power of gas chromatography. Recently Fuwa et al. (24)has published a critical review of spectrochemical methods for chemical speciation using element specific detectors with various chromatographic techniques. Atomic spectrometric detectors are inexpensive, sensitive, precise, elemental selective, and simple to use. Atomic spectrometric devices (1, 15, 16) based upon atomic
absorption in flames (15,16)and furnaces, atomic fluorescence in flames (15) and furnaces (151,atomic emission in flames (15),arcs (15),and plasmas (15,17,18) have been used with great success to detect elements of separated molecular species. The approach which shows considerable promise for speciation of molecular compounds (inorganic and metalloorganic) in solid biological and environmental materials is evolved gas analysis/microwave induced emission detection. Evolved gas analysis involves volatilization of the molecular species as a function of temperature. This technique was first proposed by Hanamura (19)and later developed by Bauer and Natusch (17) and Mitchell et al. (18). Bauer and Natusch (17) developed an evolved gas analysis/microwave emission spectrometer for identifying trace inorganic compounds in solid samples. The solid samples were heated from 25 to 1000 OC at 140 bC/min, and the molecular components were vaporized into a low power atmospheric pressure, He microwave induced plasma. Both metals and nonmetals could be measured. These workers indicated some difficulties with the efficiency of mass transfer of the evolved vapors to the microwave plasma and the lower power (250 mg, to reduce problems due to sample heterogeneity as recommended by the NBS certification of standard reference materials (20). The present approach follows the one orginally described by Hanamura (19) and consisted of a thermal vaporization separation and a "high" power (-500 W) microwave emission plasma to measure elemental species in the envolved gases which are carried into the plasma by Ar, N2, or He. In the present method, solid samples (liquid samples could also be used) are used directly, and so no chemical pretreatment steps are necessary; the sample size (20) can be considerably larger (2250 mg) than in previous methods which minimizes heterogeneity problems in environmental and biological solid samples. Also in the present method, the single electrode microwave plasma emission allows very sensitive detection of both metals and nonmetals and the higher power minimizes sample matrix interferences. The need for high sensitivity is apparent when one considers the requirement of a low temperature ramp (- 15 "C/min) over the temperature range of 25 OC to 450 "C; the low rate of temperature rise is needed to achieve higher resolution in the vaporization separation process. The analytical results consist of the emission signal of the analyte species recorded as a function of the temperature of the sample, resulting in a plot similar to differential thermal analysis. Because analyte species in the solid sample vaporize a t different temperatures, each species produces a peak a t a temperature characteristic of the analyte species and the sample type.
0003-2700/83/0355-2026$01.50/0@ 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983
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____ ---Table I. Equipment Used in Evolved Gas Analysis/Microwave Emission Spectrometer System
E-rl
,
I
CAdRlEa GAS
Figure 1. Block diagram of the evolved gasiplasma emission spectrometer system.
1 to
Plasma Torch
Heoter ( 2 5 0 ~ ) Oucirtz Crucible
Ceramic Insulator
Silica tube (0D 3 0 m m ) Silica tube (0 D 15mrnl
Thermocouple output
'!A -' 7 I
Rubber Stopper
Carrier gas
Figure 2. Schematic diagram of evolved gas furnace.
EXPERIMENTAL SECTION Apparatus. A block diagram of the evolved gas analysis/ plasma emission spectrometer system is given in Figure 1. The instrumental components, including models and manufacturers, are listed in Table I. 'The operating conditions for the vaporization system and for the microwave plasma are given in Table 11. The furnace vaporizer is shown schematically in Figure 2. The sample is held in a quartz crucible; as the sample is heated, the volatile constituents are swept by the carrier gas flowing past the indented top (of the quartz furnace tube. An alumel-chrome1 thermocouple (B+S gaLcge 28) is used to monitor the temperature of the quartz sample crucible. The temperature of thle furnace, which was increased at a rate of 15 'C/mlin for most samples, was determined by a 250-UT heating coil controlled by a linear temperature programming unit taken from a commercial gas chromatograph. The 2450-MHz microwave plasma torch, which was operated with Ar, N P ,or He at 500 W, is schematically shown in Figure 3. The plasma torch and power generator system was similar to the one described by Murayama (22,23),the modifications have been described by Hmamura (21). The plasma operating characteristics are given in Table 11. The monochromator was modified to allow for repetitive wavelength scans of each spectral line (the experimental characteristics are given in 'Table 11). A relay, which is coritrolled by the timer, reverses a two-phase capacitor motor, which in turn changes the direction of the grating scan. The repeateld scanning
Photomultiplier Tube (Hamamatsu Corp., Middlesex, N J ) R-955 for nonmetal analysis R-919 for As, Hg Monochromator (Jlarrell-Ash, Waltham, MA) No. 82.000 0.5 m scanning spectrometer with 1180 grooves/mm grating (a) reciprocal linear dispersion at exit slit, 1.6 nm/mm (b) resolution, 0.2 nm in the first order Magnetron (Hitachi, Tokyo) Magnetron H 30B2L (for microwave oven) permanent magnet, air cooling system oukput frequenc:y, 2450 MHz plate voltage (max), 4.5 kV plate current (rnax), 350 mA output power, 885 W Power Supply, Constant Current, for Magnetron (Universal Voltronics Co., Mt. Kisco, NY) output voltage, 11-2.5 kV current, 100-400 mA regulation, less than * 1%for t 10% load changes Lock.In Amplifier (Princeton Applied Research, Princeton, N J ) Model HR-8 Power Supply for Photomultiplier Tube (Keithley Instruments, Co., Cleveland, OH) Model No. 246 max. supply voltage, 2999 V Plasma Torch (Laboratory Constructed) originally designed by S. Muramama improved by author (21 ) Recorder (Honeyvvell, Inc., Ft. Washington, PA) Electronik 196, two pen recorder Sample Heating System (Laboratory Constructed) heater, 300 W column oven linear temperature programmer rate 0.5 "C/ min to 20 "C/min (Varian, Palo Alto, CA) I
Table 11. Operating Characteristics of Experimental System Helium Plasma He flow rate, 3.6 L/min sample vapor carrier flow, 0.4 L/min Argon Plasma Ar flow rate, 3.6 L/min sample vapor carrier flow, 0.4 L/min (argon passed through the water gas wash bottle to become saturated in water vapor producing a more stable plasma) Nitrogen Plasma N , flow rate, 3.6 L/min sample vapor carrier flow, 0.4 L/min Monochromator slit width, 0.03 mm (spectral band-pass = 0.05 nm) slit height, 2 mm scanning speed (repeated scan modes), 2.0 nm/min Observation Area center of plasma-just above the Pt electrode (- 1-2 nim) Magnetron current, 200 mA supply voltage, 2 kV
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-
method is necessa.ry to discriminate the emission signal of the analyte from the plasma emission background. In Figure 4, a comparison is given of the importance of the present repetitive scan method as compared to the conventional fixed wavelength approach. In Figure 4a, the emission signal as a function of time
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 13,NOVEMBER 1983
Table 111. Recommended Minimum Sample Weight of NBS Certified Standard Reference Materials (20) 1
1
.
1 Observation Window Electrode Cooling Woter Tube
SRM no.
name of material
min amt of sample, mg
1566 1569 1577 1567 1568 1570 1571 1573 1575
Oyster Brewers Yeast Eovine Liver Wheat Flour Rice Flour Spinach Orchard Leaves Tomato Leaves Pine Needles
250 150 250 400
400 300 250 500 500
_ _ _ I _
_ I _ _ I _
Tube
-Sheath
Table IV. Characteristic Temperatures (Peak Values) for Hg and As Compounds
gos
molecular species 'Sornple V o w with Carrier gas
Figure 3. Schematic diagram of microwave single electrode plasma torch assembly.
sample
freeze dried fish pure pure pure pure pure pure pure As203 pure As82 pure pure NaAsO, pure C,H ,As0 pure OAsCH30,Na~5H,0 pure C,H,AsNO, pure HgC1, HgC4 HgCl HgO HgBr, HgI 2 CH,HgCl AsBr,
characteris tic temp,a "C 72c (104) 75 97
89,133 69 74 33 22 67 152 180 223 144 124 287
a Characteristic temperatures determined by extrapolation of the linear portion of the low temperature wing of the peak to the base line. Values have a precision of i.2 "C. Direct. Cold trapped.
__-
Flgure 4. Hypothetlcal temporal outpout of evolved gas/plasma emission system for separation of three molecular specles (A, B, C). Plots are plasma emission intensity vs. time or temperature of sample: (a) Wavelength of spectrometer is constant at A,. (b) Wavelength of spectrometer is repetitively scanned over region AA around A,. The short fluctuating segments represent background emisslon. A spike on the fluctuation represents analyte emlssion.
(for the fixed wavelength approach) is given for a hypothetical sample containing three molecular species A, B, and C. I t is difficult to separate the emission signals into those due to the analyte species and those due to the background emission. In Figure 4b, the same hypothetical sample is vaporized as a function of time but now the repetitive wavelength scanning mode is being used. In the present work, the wavelength scanning (2 nm/min) occurs over the region *LO nm (spectral band-pass is 0.05 nm) of A, (the wavelength setting of the spectrometer). Because of the rather low rate of temperature rise, it is possible to obtain a sufficient number of repetitive scans to reproduce the temporal characteristics of the vaporization separation process. The background emission is due to a combination of the following processes: carrier gas emission (Ar, N2,or He); and emission of sample concomitants being evolved simultaneously with the analytes. Procedure. In Figure 5, actual vaporization separations for As and Hg species in orchard leaves, As species in oyster tissue, and Hg in tuna fih tissue are shown; the temperature rise is shown along with the repetitive wavelength scans. The peaks due to the As and Hg species are clearly shown and the ease of background correction is evident. For each of the cases in Figure 5, the dry sample of 1g (60 mesh) was placed in a quartz crucible (2 mm thick, 25 mm i.d., and 25 mm high); the sample thickness should be less than 3 mm in the bottom of the crucible to minimize tailing of the peaks of the separated species.
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The recommended minimum sample weight for NBS certified standard reference materials (20) is given in Table 111. Such sample weights will result in samples representative of total sample, and analytical results should be near the certified value. In general, the sample weight must be greater than 250 mg for animal tissue samples and greater than 500 mg for botanical tissue samples assuming a -60 mesh sample size. R E S U L T S A N D DISCUSSION Characteristic Temperatures. The vaporized molecular species, introduced into the microwave induced plasma, appear as peaks a t characteristic temperatures dependent primarily upon the molecular form and secondarily upon the sample composition. The characteristic temperatures of mercury and arsenic molecular species are listed in Table IV. The identification power of the thermal vaporization (evolved gas) / microwave emission detection is evident by the results in Table IV. The identification power would be considerably augmented by simultaneous detection of all elemental species since this would enable one to obtain empirical formulas of each compound, assuming the use of a calibrated detector. Detection Power. In Table V, the detection limits (amount of analyte producing a signal-to-noise ratio of 3) for several nonmetals (H, 0, N) and several metals (As, Hg) are given for three plasma types (He, Ar, and N2)with the evolved gas analysis/microwave emission spectrometric system. Estes et al. (24) have recently published excellent detection limits for a large number of elements in a microwave-excited atmospheric pressure helium plasma emission detector used in fused silica capillary gas chromatography; these workers obtained detection limits of 0.2 ng for As and 0.06 ng for Hg. Estes et al. (24)also have reported detection limits for H and C as 0.05 ng and 0.01 ng, respectively. Fry has obtained
ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983
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300
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15
20
25
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lminl
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Flgure 5. (a) A s in orchard leaves (NBS-SRkl-1571): total As, 5.2 pg; Ar plasma; Ar flow rate, 4 L/min; A, 234.9 nm; microwave power, 560 W. (b) Hg in orchard leaves (NBS-SRM-1571): total Hg, 57 ng; condlitions same as in 5a except 1,253.6 nm. As in oyster tissue (NBS-SRM-1566): total As, 11 yg; conditions, same as in 5a. (d) Hg in tuna fish (NBS-RM-50): total Hg, 1 yg; conditions, same as 5a except microwave power was 500 W. NOT€: The circles indicate tho analyte peak wavelengths.
Table V. Detection Limits (in p g ) for Several Nonmletals and Metals with the Evolved Gas Analysis/Microwave Emission Spectrometric System
hydrogen oxygen nitrogen carbon mercury arsenic
wavelength, nm
hlelium
656.28 777.19 746.53 247.86 253.65 234.98 193.70
0.4 (0.01)" 0.8 (0.004) 5 (0.03) 0.7 (0.00'1) 0.1 (0.001) 0.08 (0.001)
plasma gas argon 5 (0.08) 4 (0.07) 240 (4) 4 (0.07) 0.006 (0.0001) 0.05 (0.0008)
nitrogen 20 (0.3) 4 (0.07) 10 (0.2) 0.007 (0,001) 0.3 (0.004)
" Values in parentheses are in ng/s. detection limits of 0.5 yg for 0 (25),0.3 pg for N (26) and 01.1 pg for C (27)with inductively coupled plasma atomic emission by observing the emission in the far-red region from the area within the induction coills. The detection limits by our single electrode microwave plasma are generally in the range of 0.4 to 5 pg or 0.001 to 0.03 ng/s for the nonmetals (H, 0,N, C) in a He plasma, 4 to 240 yg or 0.07 to 4 ng/s in an Ar plasma, and 4 to 20 yg or 0.07 to 0.3 ng/s in a lrJ2 plasma. The detection limits which we obtained for Hg vary from 0.006 to 0.1 pg or 0.0001 to 0.001 ng/s in the three plasmas and for As varies from 0.08 to 0.3 pg or 0.0008 to 0.004 ng/s in the three plasmas. These values are superior to those obtained
by using the ICP-AES method but inferior to those obtained by Estes et al. (25). However, it must be stressed that the atmospheric pressure microwave He plasma system used by Estes et al. (24)was operated at 45-75 W and would very likely have little tolerance for solvents or vaporized molecular constituents of the sample matrices, especially when samples of 250 mg or larger are thermally volatilized. Effect of Plasma Gas. The effect of plasma gas type upon the detection limits has already been discussed (see Table V). In Figure 6, the microwave background emission spectra of the three plasma types (He, Ar, N,) are given, and in Table VI, the intensity ratios (normalized for each case to the most
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983 CH 3064
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0 777.2 M
( a ) He Plasma H656.3~
0 844.7 M
0 615 8 pII
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( c ) N, Plasma
I
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200
300
400
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i
600
700
I 800
1 850
wavelength (nm)
Flgure 6. Background emission spectra for the He (a), Ar (b), and N, (c) microwave plasmas. The flow rate of the plasma gas in each case is 3.5 L/min. The magnetron output power In each case in 400 W. The observation point for each case is in the center of the plasma, just above
the electrode. intense line) are given for several intense lines of H, 0,N, and As. In Figure 7, the microwave emission spectra of As in the three plasma types are given. Based upon signal levels for H, 0, N, and As and based upon detection limits for H, 0, and N, it is clear that the He plasma is the best. On the other hand, for Hg and As and for other heavy metals (preliminary work on Pb, Cd, etc.), based upon detection limits (see Table IV),the Ar plasma is the best. These results agree in principle with the results obtained by Mermet (28)for both microwave and rf plasmas. Temperature of Plasma. The plasma temperature was estimated by the two-line emission ratio method (29) using two Cu lines (521.820 and 510.554 nm). The plasma temperatures for Ar given as a function of distance from the plasma center varied from 5500 K in the center to 5000 K, at a radius of 1.5 mm. These temperatures represent averaged
lateral (not Abel inverted radial) values and have an estimated error of f500 K. The luminous plasma has a radius of 3 mm. Preliminary temperature measurements for the N2 and He plasmas indicate values similar to those obtained for the Ar plasma. Temperatures obtained by using a Pt-coated plasma electrode were generally lower (by -200 K) than those obtained by using a thoriated-W electrode. All of these temperatures were obtained by using a poorly optimized instrumental system producing poor signal-to-noise ratios for the two copper lines, and so no values will be given here; further work is in progress to measure both lateral electronic excitation temperatures and electron number density. Applications of Method. In Figures 8 and 9, the evolved gas analysis/microwave plasma emission spectrometer is used for the measurement of C, H, N, 0, and Hg (Figure 9 only) in orchard leaves (NBS-SRM-1571) and in tuna fish (NBS-
ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983 --__^_._l_l
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Table VI. Intensity Ratios of the Most Intense Lines of H, 0, N, and As in the Three Plasma Types plasma gas element hydrogen oxygen
nitrogen arsenic
h,nm
He
486.:13 656.28 4 36.83 777.119 77 7.42 7 7 7.54 844.63 844.64 84 4 . 68 742.96 744.23 746.53 193.'70 197.20 198.!37 200.:33 228.131 234.98
11 100 10 100 66 24
Ar 95 100 W.D. 100 87 36
12
9
N.D.
33 64 100 65 60 28 46 100 81
34 64 100 31 28 10 15 100 93
-
-___--~-
N, 110 100 N.D. 1010 816 45
19 21
6 9 100 87 "
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0
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io
,,me
400
y10
SOmDle T l m p l r o l " , ~ 1-c
IL
500
1
I5
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35
40
1rnl"l
Figure 8. Evolved gas, analysis/plasma emission spectrometer application to the measurement of C, H, N, and 0 in orchard leavies (NBS-SRM-1571). Conditions: He plasma, He plasma gas flow rate, 3.6 L/min, He carrier gas flow rate, 0.4 L/min; microwave power, 500 W; sample weight, 330 mg for 0, 305 mg for H, 312 mg for N, 310 mg for C. Emission wavelengths: 0, 777.2 nm; H, 656.3 nm; C, 247.8 nm; and N, 746.5 nm.
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