Anal. Chem. 1987, 59, 537-539
PEIQ-NH2 layer
Flgure 1. Cross-sectional diagram of the artificial enzyme electrode showing dialysis membranes (a), gas-permeable hydrophobic membrane (b), internal sensing element (c), Rlllng solution (d), and reference element (e). Portions of b, c, d, and e constltute the Orion Model 95-02 pC0, electrode. lOOr
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8), e.g., Mg2+and Mn2+,which must be added to the sample or buffer for proper operation; the artificial enzyme has no such requirements. Second, the artificial enzyme has a broad pH profile with optimum (6) at pH 4.5, which corresponds to the ideal operating range of the pCOz electrode. The natural enzyme has its pH optimum between pH 7 and 8, where the pCOz electrode is of marginal utility. The artificial enzyme sensor is quite selective. Since the catalytic action of the synzyme is thought (6) to involve Schiff base formation between the amino function of the PEIQ-NHz polymer and the keto group of the substrate, a response to other keto acids or other diacids might be expected. In our study of 13 likely interferents, we found a mild response to 8-ketoglutaric acid and an even lesser response to the diethyl ester of P-ketoadipic acid, but no measurable response to the a or y keto acids or the other six diacids. These findings suggest that synthetic or artificial enzymes could be advantageously employed for the construction of catalytic biosensors where long lifetimes and operating simplicity are required. It appears possible that artificial enzymes could be synthesized to meet specific biosensor needs for application in biotechnology and biomedicine.
ACKNOWLEDGMENT We thank I. M. Klotz for his generous gift of artificial enzyme materials. Registry No. PEIQ-NH2,68053-21-4;oxalacetic acid, 32842-7.
LITERATURE CITED
01
I
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I
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4
3
2
J
-Log [oxalacetate] , M
Figure 2. Potentiometricresponse of the artificial enzyme electrode to its primary substrate in pH 4.5, 0.05 M cltrate buffer at 25 O C : freshly prepared electrode (O),after 2 months (O),after 3 months (O), after 4 months (O),aher 5 months (A),and after 6 months (H).
enzyme electrodes is shown in Table I. The oxalacetate biosensor using the artificial enzyme showed two other advantages over its natural enzyme counterpart. First, the natural enzyme requires cofactors (7,
(1) Bowers, L. D. Anal. Chem. 1988, 58, 513A-530A. (2) Rechnltz. 0.A.; Rlechel, T. L.; Kobos, R. K.; Meyerhoff, M. E. Science 1978, 199, 440-441. (3) Rechnk, G. A.; Arnold, M. A.; Meyerhoff, M. E. Nature (London) 1979, 278, 466-467. (4) Caras, S. D.; Petelenz, D.; Janata, J. Anal. Chem. 1985, 5 7 , 1920- 1923. ( 5 ) Klotz, I. M. Ann. N . Y . Acad. Sci. 1984, 434, 302-320. (6) Spetnagel, W. J.; Klotz, I . M. J . Am. Chem. SOC. 1978, 9 8 , 8199-8204. (7) Horton, A. A.; Kornberg, H. L. Blochlm. Blophys. Acta 1964, 89, 381-383. (8) Barman, 1.E. Enzyme Handbook; Springer-Verlag: Berlin, Heidelberg, New York, 1969, Vol. 11, p 703.
RECEIVED for review May 19,1986. Accepted September 23, 1986. The financial support of the National Institutes of Health Grant GM-25308 is gratefully appreciated.
High-Temperature Sample Holder for Fast-Atom Bombardment Mass Spectrometry of Molten Materials Robert J. Doyle, Jr. Chemistry Division, Naval Research Laboratory, Washington, D.C. 20375-5000 Fast-atom bombardment (FAB) mass spectrometry has shown wide application in the analysis of intractable and low-vapor-pressure compounds (1). The particle bombardment technique has been successfully applied to solid samples, samples dissolved in a liquid matrix, and samples deposited on a solid substrate. Ions are sputtered or desorbed from the sample surface by a beam of high-energy atoms and are subsequently identified by mass spectrometry.
Introduction of a sample into a FAB ion source is achieved with a FAB probe with a sample holder affixed to the probe tip. Conventional FAB probes are provided with electrical contacts that permit resistive heating by passing a current through the sample holder. The requirement of a large surface area on a FAB sample holder results in a low-resistance conductor that can provide only limited heating with a standard probe heater or an ion source heater power supply.
Thls article not subject to U S . Copyright. Published 1987 by the American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987
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For example, the FAB sample holder on the ZAB-2F mass spectrometer (VG Analytical, Ltd.) can be heated to approximately 45 "C with the 2.5 A maximum current available from the ion source heater power supply. Reported here is a new high-temperature sample holder, of simple design, that enables the mass-spectrometric analysis of adhesive samples above their melting points without modification of the ion source heater power supply, the FAB probe, or the FAB ion source. Figure 1 shows a sample holder designed for high-temperature applications. The sample holder is designed specifically for a ZAB-2F FAB probe but can easily be modified to fit any FAB probe that is provided with electrical contacts. The holder is supported by two 1.4-mm-diameter stainless steel posts 10 mm in length. The posts are press fitted into the existing sample-holder sockets in the ceramic tip of the FAB probe. One post is fitted with a 2.8-mm-0.d. ceramic insulator 5 mm in length. The sample holder is formed from an 8.7 mm X 5.4 mm rectangle of 0.01-in. stainless-steel shim stock. The rectangle is bent 90" to form a 3.2 mm X 5.4 mm sample surface and spot welded to the noninsulated post as shown in Figure 1. The filament consists of an appropriate length of 0.127-mm-diameter tungsten wire coiled to a 0.53 mm i.d. and spot welded to each post. Power is supplied to the filament by the standard ZAB-2F source-heater power supply capable of floating at 10 kV. The ZAB-2F FAB ion source provides current to the FAB sample holder via two spring-loaded contacts that connect with two screws on the ceramic probe tip when the probe is inserted into the source. Source-heater power is diverted to the FAB probe tip by changing the supply connections located externally on the source-housing flange. The maximum sampleholder temperature was 610 O C , as measured by a thermocouple. A t this temperature the current and voltage were measured at 2.5 A and 9.1 V, respectively. The performance of the high-temperature sample holder was evaluated by using B203(boron trioxide). B203is an example of a hygroscopic glass that reacts readily with ambient water vapor. The rapid formation of B203-H20 reaction products, the most abundant of which is B(OH)3(boric acid), makes this material extremely difficult to work with (2, 3). Unless rigorous preparation and handling procedures are implemented, the B(OH)3contaminant will be present both on the surface of and within the glass sample. However, even if the glass sample is introduced into the ion source region of a spectrometer without water contamination, the water vapor present in the ion source at typical FAB operating pressures (10-6-10" torr) is sufficient to provide a continuously renewable source of B(OH), contaminant to the surface of the glass sample. In addition, the presence of water at the glass surface results in the formation of protonated boron oxides which complicate the mass spectrum and prevent accurate isotope ratio measurements. A sample of vitreous B203was prepared by heating B203 crystals at 650 "C in air (mp of B,03, 450 "C). The sample holder was dipped into the melt to coat the surface, cooled to room temperature, and inserted into the mass-spectrometer
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Flgure 2. Portion of the FAB mass spectrum of B,O,. (A) sample temperature was 30 O C . (B) sample temperature was 550 "C. All spectra were obtained at 8 kV accelerating voltage and are the summation of 100 scans. source housing via the FAB probe. Figure 2A shows a portion of the FAB maw spectrum of vitreous Bz03obtained with an unheated sample holder (30 "C)and an 8 kV primary beam of Xe atoms. The calculated abundance ratios of the isotopic ions of [B507]+,obtained by using a value of 20% loB vs. 80% llB, (m/z are 81:100:5012 for [l1B5O7]+( m / z 167), [11B410B07]+ 166), [11B310B207]+ ( m / z 165), and [11Bz10B307]+ ( m / z 164), respectively. The measured abundance ratios from Figure 2A are 88:100:51:14. A minor contribution to the apparent abundance of [11B507]+is due to the isotopic ions of [HB5O7]'+ ( m / z 168) and [H2B507]+( m / z 169). The abundance ratios of the isotopic ions of [B&]'+ ( m / z 138,139,and 140) are masked by the isotopic ions of [HB40s]+. The calculated abundance ratios of the isotopic ions of [HB406]+ are 100:99:37:6 for [Hl1B4O6]+ ( m / z 141), [H11B310BOs]+( m / z 140), [H11Bz10B06]+( m / z 139), and [H11B1030e]+ ( m / z 138), respectively. The measured abundance ratios are 80100.5017, which indicate significant contributions by the isotopic ions of [B406]*+. Figure 2B shows the corresponding mass spectrum of liquid B2O3 at a melt temperature of 550 "C.The measured abundance ratios of the isotopic ions of [B507]+ are now 84:100:51:13, which is in better agreement with the calculated values. [HB507]*+( m / z 168) and [H2B507]+( m / z 169) are no longer detected in signifcant abundance. The most striking feature of Figure 2B is the absence of [HB4OS]+and its isotopic ions. The abundance ratios of the isotopic ions of [B4O6]'+ can now be measured. They are 100:97:38:7 for [11B406]*+ ( m / z 1401, [*1B310B06]'+ ( m / z 1391, ["Bz'oB206]'+ (m/z 1381, and [11B10B306]"( m / z 138), respectively-in good agreement
Anal. Chem. 1987, 59, 539-540
with the calculated ratios of 100:9937:6. It should be noted that subsequent cooling of the Bz03 sample to ambient temperature is accompanied by the reformation of protonated boron oxides. This process is observed while the sample surface is continuously bombarded by the primary Xe beam during cooling. If, after the sample is cooled, the primary beam is turned off for a few minutes and then turned on, a sharp increase (about 200%) in the relative abundances of protonated boron oxides is observed. The enhanced abundances will decrease within a few seconds until equilibrium abundance ratios (Figure 2A) are reestablished. The experiment demonstrates that adsorbed HzO and B,03-Hz0 reaction products are continuously and rapidly renewed on the surface of the sample. The high-temperature FAB sample holder is useful in identifying ions of hygroscopic samples by their isotopic abundance ratios by eliminating interfering protonated or hydrated species that would be detected at the same nominal mass-to-charge ratios. It is of particular utility in identifying high-mass, low-abundance ions that cannot be separated from
539
interfering species by high-resolution techniques. Equally important, the high-temperature capability enables the use of tandem mass spectrometry (MS/MS) techniques such as collision-induced dissociation to obtain structural information about ions, such as [B&]'+ ( m / z 140), which cannot be separated from interfering ions ( [H11B310B06]+,m / z 140) by the low-resolution, parent-ion selection inherent in MS/MS. While the example presented here involved the use of a vitreous material, the technique should be useful with any sample that does not undergo thermal decomposition and maintains its adhesive properties above its melting point.
LITERATURE CITED (1) Barber, M.; Bordoli, R. S.; Elliot. G. J.; Sedgewlck, R. D.; Tyler, A. N. Anal. Chem. 1982, 5 4 , 645A-657A. (2) Golubkov, V. V.; Tltol, A. P.; Vasilevskaya, T. N.; Poral-Koshlts, E. A. Fiz. Khlm. Stekla 1977, 3 , 312-315. (3) Mackenzie, J. D. J. Phys. Chem. 1959, 6 3 , 1875-1070.
RECEIVED for review August 20, 1986. Accepted October 10, 1986.
Electrothermal Atomic Absorptlon Spectrometry wlth Improved Tungsten Tube Atomizer Kiyohisa Ohta* Department of Chemistry, Mie University, Mie, Tsu, 514,Japan Syang Yang Su Department of Chemistry, Virginia Commonwealth University, 1001 West Main Street, Richmond, Virginia 23284 In recent years, a metal microtube atomizer has been demonstrated to be an excellent atomization device for atomic absorption spectrometry (1-5). As compared with flame or graphite tube atomization systems, the significant advantages for atomic absorption spectrometry with a metal tube atomizer are higher sensitivity, no carbide formation, lower background emission from atomizer surface, no absorption arising from carbon particles, long lifetime, better reproducibility, and lower costs for instrumentation and maintenance. Chakrabarti et al. (7,8)reported that peak absorbances and integrated absorbances of relatively involatile elements increased exponentially with increasing heating rates and that relatively volatile elements increased in the peak absorbances with heating rates. Therefore, to improve sensitivity and detection limits further, it is necessary to heat an atomizer to its maximum temperature a t an increased rate and/or develop a new adequate tube atomizer having a higher atomization efficiency and a longer residence time for neutral atoms. In this study, we report a novel metal tube atomizer made from small inner diameter (1.5 mm) tungsten tubing. The atomizer is evaluated for various elements.
EXPERIMENTAL SECTION Apparatus. The apparatus, including a microcomputer used
in the present work, has been described in a previous publication (2). The absorption signal from an amplifier and the output signal from a photodiode measuring atomizer temperature were monitored on a memoriscope (Iwatsu MS-5021) and were simultaneously fed into and processed by a microcomputer (Sord M223). The temperature of the atomizer was calibrated against the photodiode voltage by using an optical pyrometer (Chino Works). A tungsten tube atomizer (40 mm long, 1.5 mm i.d., and 0.05 mm wall) made from a high-purity tungsten foil (99.95% Goodfellow
Metals, Ltd.) is shown in Figure 1. Two legs made from molybdenum sheets (0.3 mm thickness) support the tube at both ends. A 0.3 mm diameter hole was drilled at the midpoint of the tube to enable a sample to be injected into the atomizer. When an absorption signal was measured, the hole was closed with a movable cover to confine the analyte vapor. There were two pinhole apertures in front of and behind the atomizer to provide a narrow beam of light about 1.0 mm in diameter and to remove background emission from the atomizer surface. Hollow-cathode lamps and electrodeless discharge lamps (Hamamatsu photonics KK.) were used as light sources. The atomic resonance lines for measuring absorption signal are listed in Table I. The atmosphere in the absorption chamber was purged by a mixture of argon and hydrogen gases, the ratio of the flow rates of which varied from one element to another as shown in Table I. The addition of hydrogen to the argon purge gas serves to protect the atomizer from oxidation by traces of oxygen in argon and also gives a favorable effect on the electrothermal atomization of some elements (9). The best flow ratio of purge gas for each element was chosen. Reagents. Stock solutions (1mg/mL) were prepared as nitrate salts, except for antimony (tartarate), arsenic (trioxide), and selenium (oxide), in 0.1-6 N acid. The solutions for measuring atomic absorption were diluted from the stock solutions with distilled-deionized water just before use. All chemicals used were of analytical grade purity. Procedure. A total of 50 NLof sample solution was pipetted, 10 p L at a time, into the tungsten tube atomizer. Each of the five 10-rL subportions was individually dried at 350 K for 10 s. After the last subportion was dried, the sample introduction port was covered, and then the temperature of the atomizer rapidly raised to 2570 K under the optimized conditions.
RESULTS AND DISCUSSION Compared to a molybdenum microtube atomizer previously described (4),the improved tungsten tube atomizer described here was assembled from a large piece of tubing (40 mm as
0003-2700/S7/0359-0539$01.50/00 1987 American Chemical Society
