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Anal. Chem. 1988, 58, 2534-2541
(5) McCloskey, J. A. I n Mass Spectrometry in the Health and Life Sciences; Burlingame, A. L., Castagnoll, N., Jr., Eds.; Elsevier: Amsterdam, 1985: p 521 and references thereln. (6) Slowikowski, D. L.: Schram, K. H. Nucleosides Nucleotides 1985,4 , 309-345. (7) Budzikiewicz, H. Mass Spectrom. Rev. 1985,4 . 145. (8) McLafferty. F. W. Acc. Chem. Res. 1980, 13,33. (9) Wickramanayake, P. P.; Arbogast. B. L.; Buhler, D. R.; Deinzer, M. L.; Burlingame, A. L. J. A m . Chem. SOC.1995, 107,2485-2468. (IO) MRchum, R. K.; Freeman, J. P.; Beland. F. A.; Kadlubar, F. F.; in Mass Spectrometry in the Heanh and Life Sciences: Burlingame, A. L.. Castagnoli, N., Jr., Eds.; Elsevier: Amsterdam, 1985; p 547. (11) Kingston, E. E.; Beynon, J. H.; Newton, R . P.; Liehr, J. G. Biomed. Mass Spectrom. 1985, 12,525. (12) Sindona, G.; Uccella, N.; Weciawek, K. J. Chem. Res. Synop. 1982, 1184- 1 185. (13) Panico, M.; Sindona, G.; Uccella, N. J. Am. Chem. SOC. 1983, 705, 5607-5610. (14) Neri. N.; Sindona, G.; Uccella, N. Gass. Chim. I t a / , 773, 197-202. (15) Crow, F. W.; Tomer, K. 6.; Gross, M. L.; McCloskey, J. A.; Bergstrom, D. E. Anal. Biochem. 1984. 139,243-262. (16) Cerny, R. L.; Gross, M. L.: Grotjahn, L. Anal. Biochem., in press. (17) Bodley, J. W.: Upham, R.; Crow, F. W.; Tomer, K. 6.: Gross, M. L. Arch. Biochem. Biophys 1984,230, 590-593. (18) Soman, G.; Tomer, K. 6.; Graves, D. J. Anal. Biochem. 1983, 734, 101-110. (19) Bull, L. 6.; Culvenor, C. C. J.; Dick, A. T. The fyrroi North-Holland: Amsterdam, 1968. (20) McLean, E. K. Pharmacol. Rev. 1970,22, 429. (21) Leonard The Alkaloids; Manske and Holmes, Eds.; Academic Press: New York, 1960; Vol. 6, pp 35-121. (22) Mattocks. A. R. I n @tochemical Ecology; Harborne, J. B., Ed.; Academic Press: London, 1972; pp 179-200. (23) Deinzer, M.; Thomson, P.; Burgett, M.; Isaacson, D. Science 1977, 195,497. (24) Culvenor, C. C. J.; Edgar, J. A.; Smith, L. W. J. Agric. Food Chem. l981,29,958. Cooke, M. P.; King, R. R.; Mohamad, P. A. J. Am. (25) Dickinson, J. 0.; Vet. Med. Assoc. 1978, 169, 1192. (26) Dickinson, J. 0.; King, R. R . I n Effects of Poisonous Plants on Livestock; Keeler, R. F., Van Kampen, K. R., James, L. F., Eds.; Academic Press: New York, 1978; pp 201-208.
.
(27) Deinzer, M. L.; Arbogast, B. L.; Buhler, D. R.: Cheeke, P. R. Anal. Chem. 1982,5 4 , 1811. (28) Allen, J. R.; Hsu, I. C.; Carstens, L. A. Cancer Res. 1975,35, 997. (29) Svoboda, D. J.: Reddy, J. K. Cancer Res. 1972,32,908. (30) Ames, B. N.: Durston, W. E.; Yamasaki, E.; Lee, F. D. R o c . Natl. Acad. Sci. U . S . A . 1973,70,2281. (31) Black, D. N.; Jago, M. V. Biochem. J. 1970, 118,347. (32) Hsu, I. C.; Robertson, K. A.; Allen, J. R. Chem.-Biol. Interact. 1978, 12,19. (33) Eastman, D. F.; Dimenna, G. P.; Sgall, H. J. Dmg. Met. Disp. 1982, 70. 696. (34) Segall, H. J.; Wilson, D. W.; Dallas, J. L.; Haddon, W. F. Science 1986, 229,472. (35) Gross, M. L.; Chess, E. K.; Lyon, P. A.; Crow, F. W.; Evans, S.; Tudge. H. Int. J. Mass Spectrom Ion Fhys. 1982,42, 243-254. (36) Hoskins, W. M.; Crout. D. H. G. J. Chem. Soc., Perkin Trans. 7 1977, 538. (37) Culvenor, C. C. J.; Edgar, J. A.: Smith, L. W.; Tweeddale, H. J. Aust. J . Chem. 1970,23, 1853. (38) Blume, R . C.; Llndwall, H. G. J. Org. Chem. 1945, 70, 255-258. (39) Josey, A. D.; Jenner, E. L. J. Org. Chem. 1962,27, 2466-2470. (40) Adams, R.; Miyano, S.;Fles, D. J. Am. Chem. SOC. 1980,82,1466. (41) Jensen, N. J.; Tomer, K. 6.; Gross, M. L. J. Am. Chem. SOC.1985, 107, 1863-1868. (42) Organic Chemistry of Nucleic Acids, Part A , Kochetkov, N. K., Budovski, E. I., Eds.; Plenum Press: New York, 1971. (43) Lewin, S.:Humphreys, D. A. J . Chem. SOC.B 1966,210. (44) Rawitscher, M.; Strtevant, J. M. J. Am. Chem. SOC. 1960,82,3739. (45) Lavery, R.; Pullman, A,; Pullman, B. Theor. Chim. Acta 1978,50, 67-73. (46) Wilson, M. S.;McCloskey. J. A. J. Am. Chem. SOC. 1975, 97, 3436-3444.
RECEIVED for review January 21, 1986. Accepted June 17, 1986. This work was partially supported by the Midwest Center for Mass Spectrometry, a National Science Foundation Regional Instrumentation Facility (Grant No. CHE-8211164), and grants from the National Institutes of Health (No. ES 00210, CA 22524, and ES 00040).
Microwave Energy for Acid Decomposition at Elevated Temperatures and Pressures Using Biological and Botanical Samples H.M. Kingston* and L. B. Jassie' Center for Analytical Chemistry, National Measurement Laboratory, National Bureau of Standards, Gaithersburg, Maryland 20899
A closed v d mkrowave digestion system Is described. I n situ measurement of elevated temperatures and pressures in closed Teflon PFA vessels during acid decompodtlon of organic samples Is demonstrated. Temperature pmfWes for the acid decomposition of biological and botanical SRMs are modeled by the diesolvkrg add. Microwave power absorption of nitric, hydrofluoric, sulfuric, and hydrochloric aclds Is compared. An equation Is applied to acid microwave interactions to predict the time needed to reach target temperatures during sample dissolution. Reaction control techniques and safety precautions are recommended.
Sample preparation is a critical step in chemical analysis. It frequently establishes the lower limit in an elemental IResearch Associate, CEM Corp., I n d i a n T r a i l , N C 28079.
analysis through its influence on the analytical blank. Acid decompositions that are necessary prior to instrumental trace element analysis are time-consuming and are usually the slowest step in the analysis. Whereas phenomenal advances have been made in analytical instrumentation, sample preparation methods have not changed significantly in recent years. Indeed, the impetus to prepare large numbers of samples in less time and with greater efficiency is fostered by multielemental instruments that analyze samples in a fraction of the time needed to prepare them. The use of microwave energy as the heat source in acid digestion was first demonstrated a decade ago ( 1 , Z ) . Since that time there have been several papers describing specific applications to open beaker acid digestions where significant time savings have been demonstrated (3-7). Several studies have compared the technique with different digestion procedures and have applied it successfully to a variety of different samples (8-10). Open vessel work leads to corrosion of equipment and risks environmental contamination of the
0003-2700/86/0358-2534$01.50/00 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
sample, as well as mechanical or volatile loss of the analyte. These conditions also limit the maximum sample temperature to the boiling point of the acids. In an attempt to deal with these problems we and other researchers are using closed Teflon PFA digestion vessels to obtain elevated temperatures and pressures (11-14). Microwave digestions depend on the direct coupling of electromagnetic radiation with the mineral acid solvents used in decomposition; however, the relationship between acid and microwave interaction resulting in sample digestions has remained purely empirical. The rate of reaction and efficiency of acid decomposition increase dramatically with temperature. Reactions carried out at elevated pressures and temperatures require considerably less time to reach completion than do decompositions limited by the boiling points of the acids. In addition, substances that ordinarily would not be decomposed by these acids a t their normal boiling points react at elevated temperatures and pressures (15,16). Elevated pressure and temperature systems have been used routinely since the development of the Carius tube (16)and the implementation of steel-jacketed Teflon bombs (15,16).The use of unjacketed Teflon containers as pressure vessels coupled with a microwave heat source provides a unique intermediate pressure system. Because high temperatures and pressures are obtained in 1-2 min tremendous reductions in sample preparation time can be achieved (11-14). Control of the temperature and pressure during these closed vessel digestions is critical both to the efficiency and reproducibility of the digestion as well as to the safety of the operation. In the past the temperatures and pressures in these closed vessel acid dissolutions were not determined. An understanding of the acid coupling with the 2450-MHz microwave radiation and the temperatures and pressures produced in the sample digestions is necessary if this technique is to be generally applicable. This work describes the basic parameters governing closed vessel microwave digestions at elevated temperatures and pressures and identifies basic interactions between the acids and the 2450-MHz microwave. Real time measurements of temperature and pressure during acid digestion of biological and botanical SRMs are used to demonstrate the fundamental parameters that control the reactions. Safe operation procedures are presented that were achieved through equipment configuration and reaction control. The feasibility of using thermodynamic parameters to predict the time and conditions necessary to reach specific temperatures is discussed. EXPERIMENTAL SECTION Reagents and Standards. The specific NBS Standard Reference Materials (SRMs) used were Oyster Tissue (SRM 1566), Rice Flour (SRM 1568), Wheat Flour (SRM 1567),Bovine Liver (SRM 1577a), and Human Urine (SRM 2670). Ultrapure acids were prepared by subboiling distillation ( I 7). Equipment. Digestions were accomplished by use of a microwave digestion system (CEM Corp. MDS-81) equipped with a 600-W magnetron operating on a 1-s duty cycle. Modifications to the unit include a 360' reversing turntable operating at 3 revolutions/min and a 12-position carousel. In addition, a variable-speed exhaust fan removed corrosive fumes through a 10 cm diameter X 2 m long PVC hose mounted at the back wall of the microwave cavity and vented to a fume hood. Further modifications included a flexible and a rigid waveguide attenuator cutoff mounted at the cavity wall to provide access for temperature and pressure lines. After any modification to the equipment, the unit was checked for radition leakage with a microwave survey meter (Holaday Industries, Model HI 1501) capable of measuring 0.01 mW. The magnetron in this system is protected from reflected microwave radiation by a nonlinear microwave device. Two types of wavelength attenuators were used (rigid and flexible) to transport the temperature and pressure probes through the wall of the microwave cavity. These stainless steel attenuators (0.8 cm i.d.) were affixed and grounded over small holes in the
PRESSURE TRANSDUCER7
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Flgure 1. Microwave digestion system and computer monitoring
system. cavity wall. A 35-cm extension (illustrated in Figure 1) proved to be effective for transporting the temperature and pressure lines through the microwave cavity wall without allowing any detectable radiation leakage (CO.01 mW). Temperature measurements were made with a copper/ constantan thermocouple with the tip shielded in a 0.16 cm diameter 316 stainless steel tube. The thermocouple leads were electromagnetically shielded in the cavity using tinned capper overbraid. Both the braid and the sheath were electroplatedwith a 10-14 bm coating of gold and were joined by a gold-plated collar. The thermocouple shielding was grounded to a copper plate on the outside wall of the cavity by a modified gold-plakd subminax connector. Thermocouple extension wires were fed through the wavelength attenuator cutoff on the outside of the cavity, to an HP-3054 data-logger computer system. A temperature compensation block provided a zero reference junction voltage. The measurement system is shown in Figure 1. Pressure measurements were made with a pressure transducer (Sentra, Model D-12-100) connected to the digestion vessel by l/s-in. (0.d.) Teflon PFA tubing. The pressure line extends from the digestion vessel through the wavelength attenuator cutoff through a pressure relief valve to the transducer. The 65 cm of tubing ahead of the sensor, including the valve, was filled with distilled water to isolate the valve and pressure apparatus to prevent corrosion. Digestions were carried out in closed W-mL Teflon PFA vessels (Savillex Corp.), which had been conditioned by annealing for a minimum of 90 h at 200 'C. All Teflon materials referred to are Teflon PFA, a tetrafluoroethylene with a fully fluorinated alkoxy side chain. These vessels were then cleaned by leaching in hot hydrochloric acid (1:l)followed by hot nitric acid (1:l). Prior to the actual digestion in the microwave unit, each vessel cap was closed to a torque of 24 N m. This was accomplished by adapting a set of vessel wrenches and a torque wrench (0-36 f 1% N m). Temperature and pressure conditions were monitored in vessels fitted with two '18 in. diameter transfer ports. Both the pressure and temperature lines were secured with '/a in. ferruled Teflon nuts. During temperature measurements the thermocouple was inserted into a protective Teflon or glass sleeve and secured. The digestion vessel temperature and pressure measurement connections are shown in Figure 2. Temperature and Pressure Calibration. Copper/ constantan thermocouples were calibrated against an NBS calibrated thermometer from 0 to 200 "C with an accuracy of 0.5 "C. Calibration of the pressure transducer was accomplished with an NBS certified air pressure gauge at ambient temperature. The calibration was linear over the range of C-17 atm to f0.007 atm. Power Measurements. The available power in the microwave cavity was determined by measuring the absolute change in temperature of 1 kg of distilled water, in a Teflon container, for 2 min. Power absorption profiles for water and several mineral acids were complied by measuring the temperature rise in weighed quantities of 200, 100, and 50 mL of material using Teflon containers such that the geometric configuration was kept relatively constant. Microwave power was controlled to produce a total rise in temperature of between 15 and 50 "C.
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SUBMINAX CONNECTOR COLLAR-
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Dissolution Procedures. Samples of Oyster Tissue, Bovine Liver, Rice, and Wheat Flour SRMs were weighed directly into digestion vessels to which 5-mL portions of concentrated nitric acid were then added and set to predigest on a hot plate until nearly dry. An additional 5 mL quantity of concentrated nitric acid wm added to each sample and the vessels were sealed under class 100 air before transfer to the microwave system for digestion. The sealed (torqued) vessels were then positioned in the microwave carousel. Temperature and pressure sensors were mounted in the transfer ports of the vessel. Each of these materials was then digested individually. Two different dissolution procedures were followed. The first program consisted of a two-stage power and time setting. The system was operated at full power for 1.5 min and then power was reduced. The second dissolution program used a power level of either 25 or 35% for 8 min. The samples of SRM Human Urine were reconstituted with a weighed aliquot of water, spiked with a known quantity of vanadium-50 separated isotope, and evaporated to dryness. Five milliliters of concentrated nitric acid was added and the vessels were torqued closed. Eight samples were digested simultaneously using 100% power for 2.5 min, followed by 80% power for 3.5 min. The temperature and pressure were measured on only one vessel in each of two groups. Amino Acid Analysis. To assess the level of acid decomposition in selected samples, the residual amino acids were analyzed by using a modified o-phthalaldehdye fluorometric procedure (18). A single reference standard of 100 mg/L norleucine was prepared in 1 M nitric acid and sample fluorescence was measured with a Foci Mark I spectrofluorometer using 351 nm as the excitation wavelength and 450 nm as the emission maximum. For additional sensitivity, the standard was diluted 1 : l O and 1:lOO. RESULTS AND DISCUSSION Measurement of Temperature and Pressure in the Microwave System. Accurate temperature measurement
in the microwave environment requires a specific configuration of the thermocouple shielding. Shielding similar to that found in conventional home microwave temperature probes proved unsatisfactory. Commercial probes are generally thermistors inside stainless steel tubes and copper braid and cannot be operated at temperatures above 100 OC. These probes require placement inside a microwave absorber while in the microwave field and are isolated from the field when surrounded by more than 2.5 cm of an aqueous medium. Thermocouples with stainless steel shielding (l/g in. o.d.), as well as the tin-plated copper connecting braid, were tested but exhibited heat buildup and transient temperature spikes induced by the
microwave field. These thermocouple shieldings welded to the Teflon-coated stainless steel cavity walls, or to themselves, when they came in contact during magnetron operation. Although very small diameter shielding (1/16 in.) reduced self-heating, continual failures of the probe sheath were finally traced to spark discharges coming from the probe during use (usually at the tip, which also exhibited accelerated surface oxidation). These sparks were sufficiently powerful to penetrate Teflon or glass tubes used to isolate the thermocouples from the samples during digestion. Problems caused by metallic temperature sensors in the microwave field involve surface effects associated with the shielding and connecting braid. It is essential to provide an efficient path to the cavity wall where the energy can be dissipated by grounding. The analytical probe developed here functions while exposed to full microwave power without exhibiting the traditional arcing and self-heating of commercial probes that require insertion into a microwave absorber. Gold plating proved effective in preventing all problems associated with the electromagnetic interactions of the temperature probes. Approximately 50% of the probes fail when first tested and no reliable way has been found to evaluate them outside the microwave environment. Failures usually are associated with the loss of integrity of the gold plating. The thermocouple shielding (or unshielded thermocouple wires) must not pass through the wavelength attenuators without being grounded at the cavity wall, otherwise a large quantity of microwave energy will be transported out of the cavity. Under these conditions the wires act as antennas and transport the energy through the wavelength attenuators. The system should be checked for microwave energy leaks after any structural modifications. To avoid problems of microwave interactions with metal and electronics, the pressure measurements were made using pressure transducers located outside the microwave cavity. They were connected to the digestion vessel through the wavelength attenuator by l/g in. 0.d. thick-walled Teflon tubing and required installation of a reversing turntable to prevent tangling of the pressure tubing and thermocouple probe. A relief valve inserted in the pressure line prevented over-pressurization of the digestion vessels and proved a viable method of controlling the reaction. This control mechanism will be shown in more detail in later examples. Influence of Equipment Configuration on Results. Configurations and modifications of the equipment significantly influenced the experimental results. The microwave unit was equipped with a nonreciprocal device, employing ferrites and static magnetic fields, that prevents reflected radiation from returning to the magnetron which could change its temperature and therefore its power output (19,20). A single 5-mL sample of nitric acid consumed as little as 40 W (at 100% power), reflecting approximately 93% of the power back to the waveguide and magnetron. Small sample loads are often the cause of magnetron failure in conventional equipment. A homogeneous microwave field is essential in order to obtain reproducible results. Placement of samples in stationary positions in the cavity led to temperature differences of as much as 50%. To achieve field uniformity the turntable was modified to rotate 360° in 20 s, and then reverse direction by magnetically controlled microswitches installed under the turning mechanism. Moving the vessels through the field at a high rate of speed assured uniform power consumption so that identical temperature measurements were achieved in multiple samples (within measurement error). Fractional power settings, using the unique 1-s duty cycle, produced a smooth continuous temperature rise that was indistinguishable from the use of continuous power.
ANALYTICAL CHEMISTRY, VOL. 58,NO. 12, OCTOBER 1986
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Flgure 3. Temperature and pressure conditions for 5 mL of nitric acid and 0.25 g of Bovine Liver (SRM 1577a)in 5 mL of nitric acid exposed to 25% power for 8 min. The working temperature and pressure range for Teflon vessels used is determined by the physical properties of the vessel material, engineering, and conditioning. Conditioning was found necessary to reliably maintain pressure at elevated temperatures. Annealing the Teflon PFA vessel for extended periods (in this instance 96 h at 200 "C) has been shown to improve its tensile properties and increase the melt viscosity (21). After annealing, the vessels exhibited a weight loss and 170reduction in overall dimensions. Teflon PFA has a melting point between 302 and 306 "C, although its useful limit may be somewhat lower (21). Because Teflon is transparent to microwave radiation, it does not absorb energy directly from the microwave but does absorb heat from the sample through conduction. When internal temperature readings of 150 "C were observed, the outside of the vessel was only warm to the touch. If the internal temperature was sustained for more than 2 min, then the outer surface temperature increased markedly. Pressure rather than temperature was more often the limiting parameter for nitric acid and organic sample digestions. The upper working range for pressure was determined by considering the change in tensile strength, from 31 MPa (4500 psi) at 23 "C to 14 MPa (2000 psi) at 250 "C (21). At approximately 1.1MPa (155-160 psi), usually at temperatures between 200 and 260 "C, the vessel caps developed external radial cracks. This practical observation of the strength of the material and the vessel design prompted the use of 150 psi as the safe upper pressure limit. After this pressure limit was established, the external relief valve was calibrated and set to release at this pressure. Acid Digestion of Biological Samples. Biological and botanical materials are excellent samples for demonstrating the feasibility of the closed vessel microwave procedure and evaluating fundamental relationships. When decomposed in a closed system, organic samples contribute additional stresses which must be overcome. First, they produce gaseous degradation products that increase the pressure of the system. Second, organic samples often undergo exothermic reactions that increase the temperature of the entire system. To limit the complexity of the observations to a single power-absorbing component, nitric acid was chosen as the digesting acid. Figure 3 shows the temperature and pressure profile of 7.2 g (5 mL) of concentrated nitric acid in a closed Teflon vessel exposed to 25% power for 8 min. Measuring the temperature and pressure in situ yielded data that were reproducible to within 5%. The pressure values agreed with the literature values for the partial pressures of nitric acid to within 15% over the range of 21-150 "C (22). If the integrity of the vessel
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Flgure 5. Temperature and pressure conditions for 0.25-9 samples of Bovine Liver (SRM 1577a)and Wheat Flour (SRM 1567) in 5 mL of nitric acid exposed to 35% and 30% power, respectively, for 8 min. and associated measurement apparatus was maintained, then a plot of temperature vs. pressure should show overlap of the heating and cooling curves. This is indeed the case as shown in Figure 4. Using a similar procedure for water, literature values (22) for the vapor pressure at its corresponding temperatures were also obtained within measurement error. When 7.2 g of nitric acid is added to a 0.25-g sample of bovine liver, the same microwave program (25% power for 8 min) results in the profile shown in Figure 3. It is apparent that the temperature and pressure curves are similar to those generated for pure nitric acid. This demonstrates that through its coupling with the 2450-MHz microwave, the nitric acid is the fundamental producer of heat in the reaction. A plot of temperature vs. pressure reveals that additional pressure above the partial pressure of nitric acid exists as seen in Figure 4. This is the additional residual pressure of 1.8 atm at 100 "C contributed from the undissolved COB and NO digestion products of the organic material and nitric acid [e.g., (CH,), + 2HN03 = C02 2N0 + 2H20] (23). Three different procedures were used in controlling the reaction in the digestion vessel. The simplest utilized low proportional power delivery that resulted in a gradual temperature rise as demonstrated by the nitric acid and bovine liver (Figure 3). Excess pressure from gas buildup is not
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2538
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
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purged from the vessel by this method. A second method is demonstrated in Figure 5 where the pressure is controlled by using the external valve to limit the pressure in the vessel. Because the valve reseals as soon as the pressure drops below a predetermined level, large pressure variations are prevented. This was also found to be an effective and practical method of controlling conditions in the digestion vessel, allowing the temperature to continue to rise without over-pressurizing the vessel. This controlling technique would not be appropriate for samples containing volatile analytes. If uncontrolled sample boiling occurred due to a large pressure drop, the sample could be compromised by aerosol particles escaping through the pressure line. Sample boiling was'never observed during or after valve release. Oyster tissue and rice flour samples demonstrate a third programming technique where the temperature and pressure increase rapidly in a two-stage program (Figure 6). These graphs illustrate an exothermic reaction that is essentially out of the control of the microwave power programming. The large temperature and pressure spikes after the initial power input in the oyster tissue and after complete shutdown of all power in the rice flour are attributed to the interaction of the sample and the nitric acid, which yielded an exothermic reaction. These exothermic reactions were common in the organic samples investigated when full power was used to increase the temperature as fast as possible in single samples. Using a fractional power setting to slowly ramp the temperature and reaction conditions reduced this tendency. Another technique found to be effective was to react samples for a short time on a hot plate prior to placement in the microwave. Predigestion combined with low power settings permits controlled decompositions, as demonstrated in Figures 3 and 7. Rice flour gave results identical with those shown for wheat flour in Figure 7 . Multiple sample behavior is essentially identical with that of single samples. Because small samples absorb only a fraction of the available energy, power consumption is not a limiting factor when increasing the number of simultaneous digestions. A two-stage program for digesting eight human urine samples simultaneously is shown in Figure 8. Prior to the addition of 7.2 g of nitric acid, the samples were evaporated to dryness in the digestion vessels. Several sets of these samples, which behaved identically, were digested. This two-stage power program provided extended time at sustained higher temperatures and pressures than would a more con-
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servative single-stage program. It also demonstrates how rapidly the temperature can be increased in the microwave system at full power. Other researchers have compared the results of elemental determinations using classical dissolution techniques with open vessel microwave digested samples and found good agreement between the digestion methods for these biological SRMs using ICP analysis (7, 8). It is generally accepted that organic materials are not totally decomposed to COz and water by nitric acid; rather, they have incomplete digestion products remaining in solution with the inorganic ionic species of interest (16). Because the materials in these studies were biological and botanical, the amino acids remaining after dissolution were used as a comparative method for evaluating the completeness of the decompositions. Amino acids in human urine were typically reduced by a factor of IO5 after digestion. Amino acid concentrations in the wheat flour samples were reduced to below 5 X Mg/g, based on the lower limit of detection. These human urine samples were compared with samples digested on hot plates for 4 days (including HN03 and HC104 as part of the standard hot plate procedure). The vanadium concentrations of both sets were subsequently analyzed by isotope dilution mass spectrometry (IDMS) (24). Neither sample digestion exhibited any interference from organic decomposition products remaining in the samples, and no equilibration differences were observed for isotopic spike equilibration, essential in IDMS measurements. The acid blank is significantly improved when using these vessels as closed digestion containers because evaporation during dissolution is prevented, thus permitting the use of less reagent. By use of closed vessels, the vanadium blank associated with the analysis of Human Serum (SRM 909) was
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
reduced by a fador of almost 3 from that normally found with open beaker digestions (24). Because the containers are closed to the atmosphere during digestion, they are not vulnerable to contamination from the air. Sealing and unsealing samples under class 100 clean laboratory conditions prevent external contamination during the entire decomposition procedure. Caution. Because of direct power transfer to the acid in a closed system, large pressures develop rapidly. A pressure relief valve effectively prevents the uncontrolled release of superheated acid from the digestion system. This precaution is recommended to prevent injury to individuals and damage to the equipment during normal use and should be required when developmental work is being done. Interaction of t h e Mineral Acids with t h e 2450-MHz Microwave Radiation. The amount of power transmitted to the sample chamber depends not only on the magnetron but also to a large extent on the waveguide and the ratio of cavity dimensions to the waveguide. Ordinarily, the available microwave power in the sample cavity is determined by measuring the temperature rise of a quantity of water large enough to absorb all the microwave energy delivered (25-27). The evaluation of the available power is derived from fundamental thermodynamic relationships used to calculate the amount of energy transferred by microwave radiation to a tissue during therapeutic diathermy (28)and to evaluate total body heat (29-31). The relationship can be expressed as Wa =
Kc AT m t
where Wa is the apparent power absorbed by the sample (in watts), K is the conversion factor (from thermochemical calories to watts 4.184), c is the heat capacity (or thermal capacity) in cal g-' deg-', AT = T f-Ti (final temperature initial temperature, in deg), m is the mass of sample (in grams), and t is time (in seconds). In 2 min at full power, 1 L of distilled water increases 16.6 f 0.6 "C. A simplified form of the equation is constructed by combining the heat capacity, conversion factor, time, and mass into a single constant for the evaluation of microwave power in commercial equipment (25, 26). Potential sources of error in using this equation are the changes in heat capacity for water with increasing temperature. At 21 "C, c is 0.99869 and at 65 "C it is 1.00000 cal/g, which represents a range of 0.13% in 44 "C. This error will increase to 1% if the AT is 160 "C. Another is the heat loss during microwave absorption, which decreases the apparent power. A third source of error in these calculations results from the change in the dielectric behavior of water causing a decrease in absorption of microwave energy with increasing temperature. Changes in the dielectric constant caused by increases in temperature are not addressed in this equation. In making power absorption measurements for different quantities of the mineral acids, the time of exposure was reduced for smaller samples so that the influence of the AT on the fundamental thermodynamic parameters would be minimized. The heat capacity for the acids is also affected by their concentration because they are not pure materials, but instead are mixtures of acid and water even in their normal "concentrated" form. The heat capacity values used for these and other calculations were converted from compiled tables of molal heat capacities in the literature (32, 33) and from individual physical chemistry data (where it existed) for the acid at atmospheric pressure (32-35). Several observations were made about the interactions of various sample sizes of the pure acids with the available microwave power. Samples smaller than 500 g absorb energy proportional to their total mass. Very small samples, such as 5 mL (7.2 g) of nitric acid, abosorb as little as 40 W of the 574 W delivered to the cavity. Both the acids and water
2539
Table I. Power Absorbed by Small Volumes of Concentrated Mineral Acids Compared to Distilled Water (s = 1 standard deviation, n = 4-12) Dower absorbed, W reagent water "03 HF H2S04
HCl (6 M)
50 mL 286 228 162 130
f f f f
42 55 34 47
100 mL
368 318 246 185 175
f f f f f
34 30 31 27 53
200 mL 434 465 380 370 287
f f f f f
23 24 61 44 43
consume nearly all the available power when they exceed 500 g. Above 500 g the size and number of vessels containing material had essentially no influence on the final temperature. On a mass basis, the acids tested absorb 2 4 5 0 - m microwave ~ energy with an efficiency similar to, but slightly less than, water. The amount of energy absorbed depends on the specific acid, its concentration, and the total quantity. Below 500 g the microwave absorption differed by as much at 50% from that of water. Nitric acid is nearly as efficient an absorber of power as water, followed by hydrofluoric sulfuric, and hydrochloric acids (Table I). The heat capacity of each acid increases as the concentration decreases and approaches that of pure water. Because of this property, power absorption increases as the acid becomes more dilute. This is demonstrated by the measured apparent power absorption of 200 mL of 12,6, and 1M hydrochloric acid which was 256 f 5.5, 250 f 3.7, and 341 f 3.4 W (n = 5)) respectively, compared with the same volume of water at 434 f 23 W. Prediction of Time Required To Reach a Given Temperature. Utilizing the technique and generalizing its application to other sample types depends on the ability to predict, or at least approximate, the time necessary to reach a target temperature. This requires knowledge of the power absorption of the sample and the final conditions required by the digestion. Equation 1 (rearranged for time) was used to predict the time required to reach 71 "C for 280 g of nitric acid from an original temperature of 21.4 "C. By use of eq 1 to calculate the power absorption Wa (465 f 24 W,n = 6)) and using the heat capacity for concentrated nitric acid (at 25 "C, 0.579 cal/g), the predicted time was 72 f 4 s. The actual time required to reach this temperature was 70 s. Large acid samples (>200 g) provide the ideal case where predictions agree well with actual results because AT is small, minimizing the change in the heat capacity and heat loss. Nonideal conditions that exist when using small samples (including significant heat loss from the vessels and large temperature ranges) make it difficult to predict the time necessary to reach a specific temperature for actual digestions. As a result of heat losses from the container, the power program needed to raise the temperature of the sample must be distinguished from the power program necessary to maintain that elevated temperature. The equation only addresses the increasing temperature portion of the programming and does not address the loss of heat, which increases as the temperature increases. Therefore, the equation can only be used to estimate the initial temperature rise and is not applicable to maintaining the temperature of the sample. Predicting the time necessary to reach a given temperature for each of eight individual 7.2-g samples required measuring the power absorbed by 57.6 g of nitric acid at 100% power, which yielded 192 W. These samples were each 10 g of human urine taken to dryness prior to the addition of the 7.2 g of nitric acid (Figure 8)) thus leaving the concentrated nitric acid to abqorb the microwave energy. Table I1 shows the calculated times necessary to reach various temperatures compared with the actual time needed to achieve them. These calculations
2540
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
Table 11. Time Required to Reach Elevated Temperatures for Human Urine (Using 7.2 g of Nitric Acid in Each of Eight Vessels) time, s temp reached, "C
predicted
actual
110 130 150 160 170 180
63 f 13 78 f 16 92 f 19 100 f 21 107 f 23 114 f 24
54 72 81 102 113 144
CONCLUSION
Table 111. Apparent Watts of Absorbed Power Calculated for One 7.2-g Sample of Concentrated Nitric Acid (25% Power for 8 min) temp, "C
power, W
temp, "C
power, W
100 114 130 140
10.2 10.2 8.20 7.66
150 160 170
6.55 5.87 5.47
Table IV. Actual Time Required for a Single (0.25 g) Sample of Biological Material in 7.2 g of Concentrated Nitric Acid to Reach a Particular Temperature (at 25% Power) sample
140 "C
nitric acid bovine liver oyster tissue wheat flour rice flour
267 239 273 225 237
time, s 150 "C 160 "C 339 319 338 306 298
408 400 417 369 330
as a model for the decomposition of the biologicals, the profiles were nearly identical (Figures 3 and 7). For samples less than 0.5 g, the time prediction for oyster tissue was consistently within 2% of this model, whereas the wheat and rice flours deviated significantly from the model at the higher temperature. With this model the analyst can set the decomposition conditions for a variety of similar materials.
170 "C 471 468 468 396 363
appear to give a good approximation of the time required to reach target temperatures. At higher temperatures it would be expected that the predictions would underestimate the actual due to the loss of heat. As can be seen, this in fact occurs a t temperatures over 150 "C. A program of 25% power was used for pure concentrated nitric acid; this duplicated the conditions used in the digestion scheme of the four biological SRMs. Equation 1 was used to calculate the apparent power absorption (Wa) at various temperatures. Table I11 is a comparison of Wa calculated at different temperatures and shows the effect of the heat loss with increasing temperature and time. The reduction in power from 10 W at 114 "C to 5.5 W at 170 "C reflects the heat loss from the container over the last 5.5 min of the heating cycle. The initial heating from the start to 2.5 min is essentially linear until the heat loss becomes significant. Table I11 shows that a single apparent power estimate cannot be used to predict accurately over a wide range of temperatures and heat loss conditions. In Table IV the time necessary to reach various elevated temperatures for nitric acid and for each of the biological digestions is shown. The absorption of power from the microwave field by nitric acid is the major influence on the rate of temperature increase. The presence of the sample does not change the absorption characteristics of the acid enough to cause a significant difference in its performance. Because the acids appear to absorb microwave radiation independent of the material being dissolved, realistic time estimates for sample heating can be modeled by the acid temperature profile. If they are digested using the same program, power consumption and heat loss can be predicted for a variety of samples digested under these same conditions. When the nitric acid temperature vs. time profile was used
The amount of power absorbed by each acid depends on the particular acid, the concentration of that acid, and the total amount of acid present. Once the absorbed power for a quantity of acid has been determined, the thermodynamic relationship can be used to predict several parameters in the digestion. While time predictions were demonstrated, temperature can also be calculated by using a simple rearrangement of the same equation. These temperature predictions have the same accuracy and limitations that are described for time. Previously, trial and error adjustments were necessary to develop sample digestion procedures for the microwave system because the basic relationships described were not applied to acid digestions. Power absorption is predictable for all the acids tested and should be used to aid in designing the dissolution procedure. Measurement of the acid temperature can be used to predict the behavior of the sample and will be applicable for a variety of different samples. These calculations were made for single acid systems because the relationship has not been expanded for mixed acid systems. The mineral acids are actually multiple component systems with some water present, even in their concentrated form. However, an extension of this relationship to encompass additional parameters may permit the prediction of time and temperature for mixed acids. These investigations will be addressed separately in future work. Closed Teflon PFA vessels enable higher temperatures to be reached while lowering the blank by reducing the amount of acid necessary and isolating the sample from the atmosphere. Teflon PFA, because of its low surface contamination and nonwetting characteristics is a superior material for acid dissolution of trace elemental samples. However, the effects of pressure during digestion and the reuse of the containers under these conditions remain to be evaluated. These closed vessel microwave digestion techniques possess the potential to bridge a gap between the new generation of analytical instrumentation and traditional sample preparation methodologies. A previously variable process may now be sufficiently structured to permit future automation. ACKNOWLEDGMENT
The authors wish to express their appreciation to Vivian B. Parker and David Smith-Magowan for their help in obtaining and transforming necessary physical chemistry data. For equipment modifications and technical assistance we express our appreciation to Michael Collins, Ron Goetchius, B. F. Armstrong, Dennis Manchester, Bill Bowman, Bill King, and Lori Briggs. Registry No. "03, 7697-37-2; HF, 7664-39-3; H2S04, 7664-93-9; HC1, 7647-01-0. LITERATURE CITED Abu-Samra, A.; Morris, J. S.; Koirtyohann, S. R. Anal. Chern. 1975, 4 7 , 1475-1477. Abu-Samra, A.; Morris, J. S.;Koirtyohann, S . R. Trace Subst. Environ. Health 1975, 9 , 297-301. Brown, A. 8.; Keyzer, H. Contrib. Geol. 1978, 16, 85-87. Barren, P.; DavMowski, L. J., Jr.; Penaro. K. W.; Copeland, T. R. Anal. Chem. 1978, 7 , 1021-1023. Cooley, T. N.; Martin, D. F.; Quincei, R. H. J . Environ. Sci. Health. Part A 1977, A 12 (1&2), 15-19. Andoh, K.; Saitoh, Y.; Takatani, A,; Takahashi, F.; Tazuya, Y.; Tsunajima, K.; Motoki, C.; Yasuoka, K.; Yamaji. Y.; Natsuoka, C. Kenkyu Kiyo-Tokushima Bunri Daigaku 1982, 25, 113-125. Chern. Abstr. 1982, 9 7 , 125965~.
Anal. Chem. 1986, 58,2541-2548 (7) White, R. 1.; Douthit, G. E. J . Assoc. Off. Anal. Chem. 1985, 68(4), 766-769. (8) Nadkarnl, R. A. Anal. Chem. 1984, 56, 2233-2237. (9) De Boer. J. L. M.; Maessen, F. J. M. J. Spectrochlm. Acta, Part B 1983, 38,739-746. (IO) Matsumura, S.; Karal, I.; Takise, S.; Kiyota, I.; Shlnagawa, K.; Horiguchi, S. Osaka City Med. J . 1982, 2 8 , 145-148. 111) . . Matthes, S. A.; Farrell, R. F.; Mackle, A. J. Tech. f r o g . Rep-US, Bur. Mines 1983, No. 720. (12) Fernando, L. A.; Heavner, W. D.; Gabrlelll, C. C. Anal. Chem. 1986,
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(13) Fischer, Lynn, B. Anal. Chem. 1986, 58, 261-263. (14) Jassle, L. B.; Kingston, H. M. 1985 Pittsburgh Conference Abstracts, Paper 108A. (15) Jackwerth, E.; Gomlscek, S. Pure Appl. Chem. 1984. 56(4), 480-489. (16) Bock, R. A Handbook of Decomposition Methods in Ana/ytical Chemlstry; translated and revised by Marr, I. L.; Wlley: New York, 1979. (17) Moody, J. R.; Beary E. S. Talanta 1982, 2 9 , 1003-1010. (18) Reeder, D. J.; Sniegoskl, L. T.; Schaffer, R. Anal. Blochem. 1978, 86, 490-497. (19) Cheung, W. Stephen Microwaves Made Simple: Principles and AppllCatlOnS; Cheung, W. Stephen, Levien, Fredrlc H., Eds.; Artech House: Dedham, MA, 1985; Chapter 6. (20) Colllns, Michael J.; Cruse, Bernard W.; Goetchlus, Ronald, J. US. Patent 4 457 632, 1984. (21) “Teflon PFA Fluorocarbon Resins’’ Du font Tech. Bull. E-33272-3. (22) Internatbnal Critical Tables; Washburn, E. W., Ed.; McGraw-Hill: New York, 1928; Vol. 3, pp 304-305. (23) Stoeppler, M. K.; Muller, P.; Backhaus, F. 2.Anal. Chem. 1979, 297, 107-112.
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(24) Fassett, J. D.; Kingston, H. M. Anal. Chem. 1985, 57, 2474-2478. (25) Copson, D. A. Microwave Heatlng; The Avi Publishing Co., Inc.: Westport, CT, 1975. (26) Gerllng, E. E. Mlcrowave fnefgy Appl. Newsl. 1978, 7 1 , 20-27. (27) Gerllng, J. E. Gerllng Laboratories Report, 1980, No. 80-013. (28) Guy, A. W.; Lehmann, J. F.; Stonebridge, J. 6. Proc. I€€€ 1974, 62(1), 55-75. (29) Lehmann, J. F.; Guy, A. W.; Stonebrldge, J. B.; DeLateur, 6. J. I€€€ Trans. Microwave Theory Tech. 1978. MTT-26(8). 556-563. (30) Johnson, C. C.; Guy, A. W. f r o c . I€€€ 1972, 60(6), 692-718. (31) Mlnard, D. fhyslological and Behavioral Temperature Regulation ; Hardy, J. D.; Gagge, A. p.. Stolwljk, J. A. J., Eds.; Charles C. Thomas: Springfield, IL, 1970 Chapter 25. (32) Parker, V. B. Natl. Stand. Ref. Data Ser. ( U S .Natl. Bur. Stand.) 1985, NSRDS-NBS 2. (33) Parker, V. B. CRC Handbook of Chemistry and fhysics; 66th ed.; Weast, Robert C., Ed.; CRC Press: Cleveland, OH, 1985; p D-122. (34) Rubln, T. R.; Glauque W. F. J . fhys. Chem. 1952. 74, 800-804. (35) Kunzler, J. E.; Giauque, W. F. J . fhys. Chem. 1952, 74, 3472-3476.
RECEIVED for review May 12,1986. Accepted June 13, 1986. Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental procedure. 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 for the purpose.
Inductively Coupled Plasma Mass Spectrometric Detection for Multielement Flow Injection Analysis and Elemental Speciation by Reversed-Phase Liquid Chromatography Joseph J. Thompson’ and R. S. Houk*
Ames Laboratory-U.S. Department of Energy and Department of Chemistry, Iowa State University, Ames, Iowa 50011
The feasibility of using an inductively coupled plasma mass spectrometer as a muitieiement detector for flow injection analysis (FIA) and ion-pair reversed-phase liquid chromatography was investigated. Sample introduction was by uitrasonk nebulization with aerosol desolvation. Absolute detecton limits for FIA ranged from 0.01 to 0.1 ng for most elements using 10-pL injections. Over 30 elements were surveyed for their response to both anionic and cationic ion pairing reagents. The separation and selective detection of various As and Se species were demonstrated, yielding detection limits near 0.1 ng (as element) for ail six species present. Determination of 15 elements in a single injection with multiple ion monitoring produced shniiar detection limits. Isotope ratios were measured with sufficient precision (better than 2 % ) and accuracy (about 1% ) on eluting peaks of Cd and Pb to demonstrate that liquid chromatographyhductively coupled plasma mass spectrometry should make speciation studies with stable tracer isotopes feasible.
The toxological and biological importance of many metals and metalloids depends greatly upon their chemical form (1, 2). However, most measurement techniques for trace elemental analysis are suitable for determining only the total amount of individual elements and do not provide information ‘Present address: Department of Chemistry, sity, Muncie, IN 47306.
Ball State Univer-
0003-2700/86/0358-2541$01.50/0
about the chemical states present. Initial investigation of techniques for “elemental Speciation”began in the early 1970s ( 3 , 4 ) ,and research activity in this area has escalated steadily since then (5-18). Liquid chromatography (LC) is a particularly attractive aproach to speciation because it can be used to separate nonvolatile and thermally labile compounds. In particular, reversed-phase liquid chromatography (RPLC) has been employed to an increasing extent. It can be used to separate both ionic and nonionic components and shows greater resolution capabilities than ion exchange (19),meaning it is applicable to a broader range of species. Also, the R P packings are considerably cheaper than those used for ion exchange (20). Conventional detectors for LC, such as refractive index, ultraviolet, or conductance detectors, provide response that is more or less universal for various metal species. Thus element-specific or multielement detectors are also needed. Of the element-specific detectors, colorimetry (21,22),flame atomic absorption spectrophotometry (FAA) (5-9, and graphite furnace atomic absorption spectrophotometry (GFAA) (23) have found the most use. FAA has not exhibited sufficiently low detection limits as a chrpmatographic detector, except where preconcentration is possible (5, 7). GFAA exhibits useful detection limits but suffers from an inability to monitor the LC effluent in a continuous fashion (fractions must be taken, which is cumbersome even if the process is done automatically). Multielement detection of LC effluents offers additional advantages over other techniques. Sample introduction 0 1986 American Chemical Society
