Anal. Chem. 1991, 63,2995-2999
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Measurement of Gas-Phase Ultraviolet-Visible Absorption Spectra of Thermally Labile Molecules with a Pulsed Rapid Heating Technique for Sample Vaporization Jian-Yun Zhang, Davinder S. Nagra, and Liang Li*
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2 INTRODUCTION Gas-phase UV-visible spectra have proven useful in many areas such as electronic transition studies of molecules with relatively higher resolution compared with that in a solution or solid state (1).The absorption spectrum can also provide valuable information for the selection of optimal wavelengths for laser-based detection methods such as multiphoton ionization mass spectrometry (2) and laser-induced fluorescence gas-phase spectroscopy (3-9). Among the laser-based detection methods, supersonic jet spectroscopy (SJS) (10, 11) is shown to be a powerful technique for chemical analysis such as isomer identification and molecular structural analysis. Recently, laser-induced fluorescence and multiphoton ionization jet spectroscopy have been extended to the studies of biological molecules (2, 12-14). However, in SJS,in order to obtain a jet-cooled wavelength spectrum, some prior knowledge of the general absorption contours of the sample molecule in the UV-visible region in the gas phase is generally required to locate the original transitions. For volatile and thermally stable molecules, gas-phase spectra can be easily obtained by heating the sample in a gas cell in a spectrophotometer. However, for thermally labile biochemicals, conventional heating cannot be applied, since decomposition will occur upon heating before a spectrum can be obtained. Although solution-phase spectra for these molecules can be obtained, the spectra are usually different from the gas-phase spectra. This is particularly true for biological molecules which often consist of functional groups such as OH and COOH. The solvent and pH shifts for these molecules can be quite large. In some cases, over 10-nm shifting is observed (15). This shift clearly causes some problems for locating the original transitions in laser-based high-resolution spectroscopy in light of the fact that the laser beam is monochromatic and wavelength resolution is very high (e.g., X0.02 nm for SJS). Thus, methods for obtaining UV spectra in the gas phase are needed. Recently, Li and Lubman have developed a pulsed laser desorption method for sample volatilization in a diode array spectrophotometer for the measurement of gas-phase UV spectra of biological molecules (15). In this technique, a pulsed COz laser is used for vaporizing biochemicals at atmospheric pressure. Gas-phase W spectra for a variety of biochemicals were obtained and studied. These spectra were proven to be extremely valuable for locating the original transitions or 0-0 transitions in SJS for tyrosine-related molecules, indoles, and catecholamines (13, 14). In this paper, we describe a simple and inexpensive alternative method for obtaining gas-phase UV-visible spectra of biochemicals. This technique involves the use of a pulsed rapid heating (PRH) method for sample volatilization. The PRH method has been recently developed for the sample vaporization in the vacuum in a multiphoton ionization mass spectrometer (16).In this study, we wish to demonstrate that this PRH technique, producing a heating rate of -lo6 K/s, can be used to vaporize intact biochemicals at atmospheric pressure without any significant thermal decomposition. I t is shown that UV spectra of biochemicals such as tyrosine, dopa, norepinephrine, and tryptophan can be obtained with this experimental setup. It is our hope that this simple method of obtaining gas-phase UV-vis spectra of thermally labile molecules can be readily adopted by investigators interested 0003-2700/91/0363-2995$02.50/0
in the studies of these molecules by high-resolution laser spectroscopy.
EXPERIMENTAL SECTION Figure 1shows the experimental setup for the measurement of gas-phase UV-vis absorption spectra with a pulsed rapid heating method for sample vaporization. A commercial UV-vis photodiode array spectrophotometer (Hewlett-Packard 8450A) is employed to detect the gas-phase species. The PRH probe has been described elsewhere (16). Briefly, it consists of a solenoid, a heating probe, and a water cooling jacket. A solenoid is used to drive a long stainless-steel plunger. A steel base is connected to one end of the plunger so that an electromagnetic force generated inside the solenoid is able to drive the plunger. ' h o springs are used to hold the plunger so that it can move freely for about 1 cm in either direction. In this study, a 1.5-mm-diameter thermocoax heating coil is bent into a U-shape and silver-soldered to the other end of the plunger. The heating coil is directly used as the tip of the heating probe. Because the solenoid cannot be operated at high temperatures, a water-cooling jacket constructed of copper is screwed into the solenoid to isolate the hot probe from the solenoid. The electrical circuit of the power supply used for driving this solenoid is the same as the one used for the solenoid nozzle (17). This power supply is triggered by a pulse from a pulse generator (HP 8005A) and is capable of generating a 400 V pulse with a duration of -35 M. In this experiment, an -170-V pulse is used to drive the solenoid. The vaporization process is performed under normal atmospheric conditions. The PRH probe vaporizes the sample coated on a 10-mm-diameterceramic probe, generating a plume of vapor. The light beam from the spectrophotometer is positioned about 1 mm above the PRH probe so as to intersect the maximum amount of vapor without blocking the light beam from the probe. The entire absorption spectrum in the W-vis region is obtained within 18. The adual sample cell used to concentrate the sample plume is constructed of glass with four circular holes, and the sample holder and the PRH probe are placed -8 mm apart (see Figure 1). The contact times between the sample and the probe is -210 jts, producing a heating rate of -lo6 K/s, as reported previously (16). About 1mg of sample is generally used and placed onto the ceramic probe by either wetting or dissolving the compound in methanol and coating on the surface with the use of a spatula. A fresh sample is always availabe for vaporization by simply rotating the sample probe. The PRH probe is operated at 1Hz to allow the spectrophotometer to record and store the entire spectrum in 1s. Up to 25 spectra can be stored in the memory of the spectrophotometer in each run. The temperature of the heating probe is optimized for each sample. Depending upon the melting point of the sample, the temperature is generally in the range 150-300 "C. Since no significant absorptions are present in the visible region for the compounds studied herein, the data are acquired in the UV region only. In the studies of the heating process, laser desorption/supersonic jet multiphoton ionization (LD/SJMPI) mass spectrometry is used for the analysis of the sample vapors generated at atmospheric pressure by the PRH technique and by the conventional heating method. To collect the sample vapor, a liquid nitrogen cooled stainless steel sample probe is placed near the vaporization region. The gas-phase sample molecules are condensed onto the probe, which is then inserted into the vacuum chamber of the LD/SJMPI mass spectrometer for COz laser desorption with 266-nmlaser ionization. The mass spectrometer has been described in detail elsewhere (18). The slow heating method employed in this work is performed by wrapping a quartz cell with several turns of a coaxial heating wire. The sample is put inside the quartz cell, which is then placed 0 1991 Amerlcan Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 24, DECEMBER 15, 1991
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Pulsed Rapid Heating Probe
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Figure 1. Schematic diagram of the experimental setup.
in the light path of the spectrophotometer, and the quartz cell is slowly heated. The heating rate is estimated to be -2 "C/s. Again, for the analysis of the sample vapor, the vapor is collected at the top of the quartz cell using the procedure outlined above and analyzed by LD/SJMPI. All chemicals were purchased from Sigma Chemical Co., St. Louis, MO, and used without further purification.
RESULTS AND DISCUSSION As shown in earlier studies (16),our PRH method is capable of generating intact molecules without significant thermal
decomposition in the vacuum for MPI mass spectrometry. The PRH method described herein generates gas-phase molecules at atmospheric pressure, similar to laser desorption with a spectrophotometer for obtaining gas-phase UV spectra (15). Figure 2 shows the gas-phase UV spectra of tyrosine, dopa, norepinephrine, and tryptophan obtained by the pulsed rapid heating technique. These spectra are virtually identical to laser desorption results obtained previously (15),as was the case for other compounds studied. These results suggest that the PRH technique is an attractive, inexpensive method over laser desorption for obtaining gas-phase spectra for thermally labile or high melting point compounds. However, the similarity between the UV spectra obtained by LD and PRH does not rule out the possibility that thermally decomposed products may be generated by both techniques. Moreover, we found that the UV spectrum of tryptophan obtained by the PRH method is similar to that obtained by the slow heating method, yet it is well-known that tryptophan is thermally labile (12,15). Thus the next question we have to address is whether we are generating gas-phase parent neutral species, thermally decomposed products, or both by the PRH method. Here, a laser desorption/supersonic jet multiphoton ionization mass spectrometer is used to address this question. Parts A and B of Figure 3 show the LD/SJMPI mass spectra of pure tryptophan and the sample collected from the vapor generated by the PRH method. As parts A and B of Figure 3 indicate, the two mass spectra are essentially identical, with a strong molecular ion peak of tryptophan and a major fragment ion at m/z 130. Note that the relative peak intensity between m/z 130 and 204 is strongly
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Figure 2. Gas-phase UV spectra of (A) tyrosine, (B) dopa, (C) norepinephrine,(D) tryptophan obtained by using the pulsed rapid heating method for sample volatilization in a diode array spectrophotometer.
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(m/z 117) if aslow heating rate is used (-2 "C/s). It should be noted that the gas-phase spectra for the indole-related molecules are very similar. Thus, in this case, it is difficult to observe in the UV spectrum if any thermal decomposition is taking place during the vaporization process. However, the mass spectrometric study suggests that we are indeed generating neutral gas-phase tryptophan species in the pulsed rapid heating process even under atmospheric pressure, since vaporization is clearly favored over decomposition at the faster heating rate (2G22). Another class of compounds we have studied are aromatic acids such as amino- and/or hydroxy-substituted benzoic acids. Many of these molecules are biologically significant. For example, a number of metabolites of catecholamines are aromatic acids (23). These molecules are generally believed to be thermally very labile (23). Indeed, we found that the UV spectra of the aromatic acids obtained by the PRH method are completely different from those obtained by the slow heating method. Figure 4 shows the UV spectra of 3amino-4-hydroxybenzoic acid in the gas phase with the use of the PRH method for sample vaporization and in methanol solvent. The absorption contours for these two spectra are essentially identical, although, as expected, the spectrum in methanol is shifted to longer wavelengths. Figure 5a shows the U V spectrum of the same compound obtained by the slow heating method. Note that this spectrum is different from that shown in Figure 4a, which indicates that structurally
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 24, DECEMBER 15, 1991
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different molecules are generated during these two heating processes. To analyze the molecules, the UV spectra of 2aminophenol in the gas phase and in methanol solvent are obtained and shown in Figure 5b,c. The spectrum of 2aminophenol (Figure 5b) is almost the same as that shown in Figure 5a. This result indicates that thermal decomposition is taking place during the slow heating of 3-amino-4hydroxybenzoic acid, and one of the major decomposed products might be 2-aminophenol. To further confirm that slow thermal heating of $amino4-hydroxybenzoic acid produces significant amounts of 2aminophenol, LD/SJMPI mass spectrometry is again employed to determine the species in the vapors generated by the slow heating and the PRH method. The mass spectra are shown in Figures 6 and 7. Figure 6 shows that the pulsed rapid heating technique produces only 3-amino-4-hydroxybenzoic acid in the gas phase with no thermal decomposition products. Figure 7 shows that with slow heating only the decomposed product, 2-aminophenol (m/z = 109))i s observed in the gas phase. This study indicates that although a mass spectrometer with the capability of detecting thermally labile molecules is useful for the determination of thermally decomposed products, a simple UV-vis spectrophotometer can still provide some information on whether there is any thermal decomposition during the heating process if the spectrum for the decomposed product is different from that of the intact molecule. More importantly, this study suggests that the PRH method is capable of generating gas-phase molecules under atmospheric pressure conditions for the measurement of UV-vis spectra of these very labile compounds.
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Figure 7. MPI mass spectra of (A) the pure solid sample of 2aminophenol (MW = 109) and (6) the sample collected from the condensed vapor after 3-amino-4-hydroxybenzolc acid is volatillzed in the atmosphere by using tJw slow heating method. The experlmentai parameters are the same as shown in Figure 3.
In conclusion, we have developed a pulsed rapid heating method as a means of vaporizing thermally labile biochemicals in a diode-array spectrophotometer for the measurement of the gas-phase UV-visible spectra. We have shown that this PRH technique with a heating of lo6 K/s can vaporize the sample molecules intact at atmospheric pressure without thermal decomposition. Multiphoton ionization mass spectrometry is employed for the identification of intact molecules and any thermally decomposed products that may be generated during the PRH process. Using 3-amino-4-hydroxybenzoic acid as a test compound, it is found that thermal decomposition is observed with a conventional heating method with a heating rate of about 2 'C/s, whereas the sample molecules are vaporized intact with the PRH technique. Registry No. Dopa, 59-92-7; Tyr, 60-18-4; Trp, 73-22-3; norepinephrine, 51-41-2; 3-amino-4-hydroxybenzoicacid, 1571-
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LITERATURE CITED (1) See for example: Innes, K. K.; Ross, 1. G.; Moomaw, W. R. J . hid. Speclrosc. lS88, 732, 492 and references cited thereln. (2) See for example: Lubman. D. M. Mass Spectrom. Rev. 1988, 7 , 535-554 and 559-592 and references cited thereln. (3) Zare, R. N.; Dadlglan, P. J. Science 1974, 785, 739. (4) Smalley, R. E.; Wharton. L.; Levy, D. H. Acc. Chem. Res. 1977, 70, 139. (5) Levy, D.H. Sclence 1981, 274, 263. (6) Warren, J. A.; Hayes, J. M.; Small, G. J. Anal. Chem. 1982, 5 4 , 138. (7) Imasaka, T.; Fuluoka, H.; Hayashl, T.; Ishibashl, N. Anal. Chim. Acte lS84, 756, 111. (6) Peplch, B. V.; Callls, J. B.; Burns, D. H.; Gouteman. M.;Kalman. D.A. A&. chem. 1986, 58. 2825. (9) Stiller, S. W.; Johnston, M. V. Anal. Chem. lS87, 59. 567. 110) . . Sin. C. H.: Tembreull.. R.:. Lubman. D. M. Anal. Chem. 1984, 56. 2776.
ANALYTICAL CHEMISTRY, VOL. 63,NO. 24, DECEMBER 15. 1991 (1 1) Hayes, J. M. chem. Rev. 1987, 87, 745 and references clted therein. (12) Riuo, T. R.; Park. Y. D.;Peteanu, L. A.; Levy, D. H. J. Chem. phvs. 1986, 84.2534. (13) Li, L.; Lubman, D. M. Rev. Sei. Instrum. 1988, 59, 557. (14) Li, L.; Lubmn. D. M. Anal. Chem. 1988, 60, 2591. (15) Li, L.; Lubman, D. M. Anal. Chem. 1987, 59, 2538. (16) Nagra, D. S.; Zhang. J. Y.; Li, L. Pulsed Rapid Heating Method for Volatilition of Biol~glcaiMolecules in Multiphoton Ionization Mass Spectrometry. Anal. chem. 1991, 63, 2188. (17) Sin, C. H.; Pang, H. M.; Lubman, D. M. Anal. Instrum. 1988, 17, 87. (18) Li, L.; Hogg, A. M.; Wang. A. P. L.; Zhang, J. Y.; Nagra. D. S. An.?/. chem. 1991, 63,974. (19) Grotemeyer, J.; Walter, K.; Boesi, U.; Schlag, E. W. Int. J . Mass
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Spectrom. Ion Processes 1987, 78, 69. (20) Beuhler, R. J.; Flanlgan, E.; Greene, L. J.; Friedman, L. J. Am. Chem. SOC. 1974, 96,3990. (21) Cotter, R. J. Anal. Chem. 1080, 52, 1589A. (22) Daves, G. D., Jr. Ace. Chem. Res. 1979, 12, 359. (23) Quentitethre Analjlsls of Catectwlamines and Related Compounds; Krstulovic, A. M., Ed.; John Wiley & Sons, Inc.: New York. 1986.
RECEIVED for review July 29,1991. Accepted September 30, 1991. This work was supported in part by the Natural Sciences and Engineering Research Council of Canada.
