980
Anal. Chem. 1980, 52, 980-982
of the range of enzyme electrodes to substrate concentrations in excess of K M is possible. Table I indicates that to achieve deviation from linearity of less than 0.05 loglo units a t bulk substrate concentrations equal to 100 times the Michaelis constant, over 1000 enzyme units/cm3 are required for Khl < 1 mM. Such a n electrode should be linear to nearly 100 mM. Enzyme electrodes with activities of 50-100 units/cm3 are more realistic. We expect that linearity to 10 mM for KM equal to 1 m M can be achieved. One should note that the required amount of enzyme decreases as the diffusivity of the substrate decreases. This implies t h a t use of a highly cross-linked gel to support the enzyme might be desirable. But, as we have shown previously (7), electrode response time will increase in proportion to the decrease in diffusivity. Thus there is a trade-off between linearity and response time. The same is true of the thickness of the enzyme layer, i.e., a thick layer promotes linearity and increases the response time. In summary, i t has been shown that the linear dynamic range of potentiometric enzyme electrodes can be extended beyond KM when the enzyme immobilized in the membrane surrounding the sensor is of sufficiently high activity. In a practical sense, this illustrates the point that only enzymes capable of being immobilized while retaining a high specific activity hold promise for use in routine applications. Furthermore, since stabilization of the immobilized enzyme is still a problem, significant deterioration of the linear dynamic range of the sensor with time is implied. This suggests the need for frequent recalibration and assessment of the time dependent of the sensor's dynamic range.
LITERATURE CITED Rechnitz, G. A.; Kobos, R. K.:Riechei, S . J.; Gebauer. C. R. Anal. Chim. Acta 1977, 9 4 , 357. D'Orazio, P.; Meyerhoff, M. E.; Rechnitz, 6. A. Anal. Chem. 1978, 50, 1531. Guilbautt, G. G. In "Comprehensive Analytical Chemistry", Vol. 8, Svehla, G., Ed.; Elsevier: Amsterdam, 1977. Blaedel, W. J.: Kissei, T. R.; Boguslaski, R. C. Anal. Chem. 1972, 4 4 , 2030. Mell, L. D.: Maloy, J. T. Anal. Chem. 1975, 47, 299. Tran-Minh, C.: Brown, G. Anal. Chem. 1975, 4 7 , 1359 Carr, P. W. Anal. Chem. 1977, 49, 799. Villadsen, J. V.; Stewart, W. E. Chem. Eng. Sci. 1967, 22, 1483. Karanth, N. G.; Hughes, R. Chem. Eng. Sci. 1974, 2 9 , 197. Ferouson. N. B.: Finlavson. B. A. Chem. €no. J . 1970. 1 . 327. Serih, R.'W. Int. J . Num. Math. Eng. 1975: 9 , 691. Whiting, L. F.; Carr, P. W. Anal. Chem. 1978. 50, 1997. Whiting, L. F.; Carr. P. W. J . €lectroana/. Chem. 1977, 8 1 , 1. Johnson, L. W.; Ries, R . D. "Numerical Analysis"; Addison-Wesley: Reading, Mass., 1977. Dennis, J. E. P Mor& J. E. SIAM Rev. 1977, 19, 46. Brent, R. P. SIAM J . Num. Anal, 1973, 10, 327. Bowers, L. D.; Carr, P. W. Anal. Chem. 1976, 48, 544A. Guilbault. G. G. "Handbook of Enzymatic Methods of Analysis"; Marcel Dekker: New York, 1977.
James E. Brady P e t e r W. Cam*
Department of Chemistry University of Minnesota Minneapolis, Minnesota 55455
RECEIVED for review September 17,1979. Accepted February 4,1980. This project was partially supported by the University of Minnesota Computer Center and a grant from the National Science Foundation (CHE 78-17321).
Test for Dehydrogenation in Gas Chromatography-Mass Spectrometry Systems Sir: During a recent synthesis of 2-hexyl-5-pentylpyrrolidine (a), the pheromone of the fire ants Solenopsis molesta and S. texanas ( I ) , we had occasion to compare the gas chromatograph/mass spectrum of our product (Figure 1) with that of the natural product run earlier (Figure 2). Gross differences are apparent that were traced to the presence in t h e former of overlapping spectra of the related pyrrolines b and c. Homologues of b and c had been encountered earlier
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as natural products in S. punctaticeps ( 2 ) ;their spectra are characterized by an abundant m / z 82 ion (iii) arising from &-cleavageof the rearrangement ion (ii). However, there were no such impurities in the synthetic sample of a. Under the 0003-2700/80/0352-0980$01.00/0
gas chromatographic conditions employed here [2-m, 3% SP-1000 (stabilized Carbowax) packed column, programmed 10"/min, see Refs. 1 and 2 for details], the pyrrolines separate easily from the pyrrolidines and even trace quantities would be easily detected. Clearly, dehydrogenation of a was occurring either in the ion chamber, jet separator, or the intervening valve of our system (LKB-9000). As expected, the spectra were not highly reproducible, more serious degradation being encountered a t low levels of samples. A sample admitted by direct insertion probe produced an excellent spectrum (Figure 3), absolving the ion chamber as the source of trouble. On the other hand, the jet separator and following valve had been replaced recently; earlier spectra were run on a system that had been in continuous operation for many years. Indeed, the spectrum of a improved considerably after a few weeks of further use of the mass spectrometer (Figure 4 ) ; several months later i t was indistinguishable from that obtained earlier with the natural product (Figure 2). We have observed that once this stage is reached, the extent of dehydrogenation no longer increases as the sample size is diminished to the nanogram level and the spectra become entirely reproducible. Sherman has observed a related loss of sample at low levels after installing a valve that prevents column bleed from entering the jet separator when the system is not in use (3). He suggests, and we agree, that a continual flow of column bleed (usually siloxane polymer) appears to be important in mainC 1980 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, MAY 1980 82
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taining passivation of the metal surfaces. As mentioned above, in our spectrometer a gold-plated bellows-type valve is located between the jet separator and ion source. An identical valve in the same housing separates a gold-plated heated inlet system from the source. To determine whether the valve rather than the jet separator might be the source of dehydrogenation, samples of a were admitted via the heated inlet system a t a time when the jet separator had become completely "passivated". With the inlet system and associated lines at 75 "C and the jet separator and valves a t their normal temperature of 285 O C , the spectrum was the same as shown in Figure 4, i.e., partial sample dehydrogenation was occurring. When the temperature of the valve was reduced to 100 "C, dehydrogenation was almost entirely eliminated. For various reasons, it is impractical to allow the valve to remain a t 100 "C for routine use, but this experiment tends to implicate the identical valve following the jet separator, rather than the separator itself, as the source of trouble in our system. In any case, it is clear that the pyrrolidine a is useful in disclosing such problems in mass spectrometer systems. I t appears to owe its unique sensitivity to dehydrogenation to the 2,5-dialkyl substituents on the pyrrolidine ring. T h e natural product is largely the trans isomer ( I ) ; however, synthetically prepared cis and trans isomers exhibited the same tendency to undergo dehydrogenation in our system. Adkins has discussed the exceptionally easy catalytic hydrogenation of 2,6-dialkyl substituted pyridines to the cispiperidines and ascribed the effect to inhibition of catalytic poisoning by the alkyl groups flanking the nitrogen ( 4 ) . Thenot et al. ( 5 ) have noticed a marked improvement in sensitivity for tris-(2,3-dibromopropyl)phosphateand aldosteronedi-TMS after coating the direct insertion probe glass sample vials with silicone polymer. They also note, without experimental details, conversion of nicotine to nicotyrine on the metal surface of a new transfer line. In view of our results, it may be that the improvement they observe is associated with distillation of the silicone coating onto critical metallic elements in the source region as well as passivation of active sites on the glass surface of the probe tip. We have had occasion t o test several commercial instruments with pyrrolidine a with results varying between the extremes represented by Figures 1 and 2. While the compound appears to constitute a delicate test for dehydrogenation in mass spectrometers, our experience indicates that
Anal. Chem. 1980, 52,982-983
982
the results obtained with a given system will vary depending upon the recent history of the apparatus as well as on its design characteristics. Samples of the pyrrolidine a are available from Henry M. Fales.
(5) Thenot, J. P.; Nowlin, J.; Carroll, D. I.; Montgomery, F. E.; Homing, E. C. Anal. Chem. 1979, 5 1 . 1101.
Henry M.Fales* William Comstock National Heart, Lung, and Blood Institute Building 10, 7N322 Bethesda, Maryland 20205 Tappey H. Jones Department of Entomology University of Georgia Athens, Georgia 30602
LITERATURE CITED (1) Jones, T. H.; Bium, M. S.; Faies, H. M. Tetrahedron Lett. 1979, (12), 1031. (2) Pedder, D. J.; Faies, H. M.; Jaouni, T; Blum, M. S.; McConneil, J.; Crewe, R. M. Tetrahedron, 1976, 32, 2275. (3) Sherman, W., Washington University School of Medicine, St. Louis, Mo. 631 10, personal communication, 1979. (4) Adkins, H. "Reactions of Hydrogen with Organic Compounds over Copper Chromium Oxide and Nickel Catalysts"; University of Wisconsin Press: Madison, Wis., 1937; pp 57, 66.
RECEIVED for review November 5, 1979. Accepted February 11, 1980.
Two-Laser Induced Selective Infrared-Visible Fluorescence for Thin Film Analysis Sir: We wish to report a novel laser technique which has applicability to thin film analysis. The concept of this technique is to use a CW visible or ultraviolet laser to stimulate visible fluorescence from a thin fluorophor film,and a chopped low power infrared laser to selectively excite characteristic infrared absorption bands in the sample. The optical detection system monitors the resulting modulated visible fluorescence signal. We attribute this signal to a thermally perturbed fluorescence yield, not to a true double resonance effect. Infrared double resonance techniques are common in the gas phase ( I ) , and visible-ultraviolet pulse-probe techniques yield important kinetic parameters in condensed phases (2). However, visible-infrared double resonance techniques in condensed phases are restricted due to the fast vibrational relaxation times involved ( 3 ) . Nonselective thermal effects have been initiated by previous workers using high-power lasers, followed by selective mass spectrometric ( 4 ) or optical (5) analysis. Selective multistep excitation is used in the laser-ion mass spectrometer, which uses a high-power laser and a mass spectrometer to obtain both wavelength and m / e information (6). In contrast, this work reports the use of a low-power laser to initiate a selective thermal effect, followed by sensitive fluorescence analysis. Currently the limit of detection is on the order of 1-5 pg (2-10 nmol), which begins to compare with FTIR techniques (7,8) but does not yet approach laser optoacoustic techniques (9). The advantage of this two-laser technique over conventional fluorescence is the enhancement in selectivity obtained to the extent that the infrared absorption spectrum characterizes the sample. Ultimately i t is expected that optical detection with fluorescence techniques will improve the sensitivity beyond that obtainable by FTIR techniques. An advantage of this two-laser technique over a single laser technique such as optoacoustic spectroscopy is the potential to use additional selectivity parameters for mixture analysis (e.g., fluorescence as well as infrared characteristics for each component). A Lexel Model 96 argon ion laser was the fluorescence excitation source. Typically the wavelength was 488.0 nm, with 1 W m W unfocused power incident on the cell. A grating tuned, 2-m CW COPlaser was the selective infrared excitation source. Numerous lines of the 9.4 p (00°1-0200) and 10.4 p (00°1-10°O) bands were selected, with 7 W as a typical output power. The COz laser was not focused; average power actually incident on the cell was approximately 2.5 W, distributed over approximately 0.5 cm2. 0003-2700/80/0352-0982$01 .OO/O
An Ithaco 383 variable speed chopper was used to modulate the COPlaser beam. Detection was with an Amperex XP2020 photomultiplier tube and a PAR HR-8 lock-in amplifier; Schott long pass filters were used to block scattering from the argon ion laser beam. All chemicals were obtained from commercial sources and used without further purification. Initial experiments were performed in a light-tight sample chamber containing two NaCl windows mounted a t Brewster's angle. The two laser beams were brought in nearly colinear, overlapping throughout the length of the sample chamber. Fluorescence was monitored a t right angles to the two excitation beams. Various solutions were admitted into the sample chamber, which was then evacuated to dryness and a few millitorr and heated up to approximately 140 "C. Subsequent experiments demonstrated that neither heating nor reduced pressure had any appreciable effect on the signal. Consequently, only one NaCl window was used thereafter, open to the atmosphere and a t room temperature. For qualitative work a small quantity (51mg) of fluorophor was placed on the window and dissolved with a few drops of chloroform; after drying, the resulting film was irradiated. For semiquantitative work a solution of known concentration was carefully added dropwise to the window. The total amount of material thus deposited as a thin film was accurately known, but the film thickness was not very uniform. Figure 1 illustrates the selectivity achieved with this technique. A sample of rubrene was irradiated continuously a t 488 nm. When the COPlaser was tuned off an absorption band at 9.50 p (P(14)),no modulated signal was apparent. However, when tuned to coincide with an infrared absorption band a t 9.71 p (P(38)),a large modulated signal appears. The infrared absorption of C 0 2 laser irradiation by rubrene could be obtained in this fashion. Similar results were also obtained for coumarin 7 and rhodamine 6G perchlorate. A goal of this work was to enhance the selectivity of fluorescence techniques for mixture analysis. The infrared absorption spectrum of coumarin 7 and rubrene are sufficiently dissimilar such that selective excitation of a mixture of these two compounds was possible. For example, irradiation of the mixture at 9.21 (R(32))resulted in a signal attributable solely to coumarin 7; irradiation a t 9.71 (P(38))resulted in a signal attributable solely to rubrene. Irradiation a t 10.51 p (P(12)),where neither compound absorbs, resulted in no signal. Similar results were obtained with other COz lines. Thus, although the fluorescence characteristics of coumarin C
1980 American Chemical Society
