Correspondence Anal. Chem. 1996, 68, 1062-1066
Analysis of a Reactive Dimethylenedihydrothiophene in Methylene Chloride by Low-Temperature Atmospheric Pressure Ionization Mass Spectrometry Jentaie Shiea,* Wen-Shyang Wang, Chin-Hsiung Wang, Ping-Shu Chen, and Chin-Hsing Chou
Department of Chemistry, National Sun Yat-sen University, Kaohsiung, Taiwan 804 The use of atmospheric pressure ionization at -78 °C for the analysis of a thermally unstable compound, 2,5dimethylene-2,5-dihydrothiophene, dissolved in methylene chloride is demonstrated with a home-built lowtemperature electrospray source connected to a quadruple mass analyzer. To prevent the analyte molecule from polymerizing at elevated temperatures, the sample solution was maintained at -78 °C in the dry-ice-cooled sample injection and sample loop units. The solution was delivered to the spray needle by compressing the nitrogen gas in a syringe which was connected to the sample loop by a Teflon tube. At such low temperature, a strong ion signal was obtained from the analyte molecule as a result of corona discharge ionization. Electrospray ionization (ESI) mass spectrometry has been proven to be very useful for analyzing not only large biomolecules1,2 but also small organic and inorganic compounds.3-7 Since most organic and inorganic compounds do not dissolve well in water, organic solvents such as n-hexane, benzene, chloroform, methylene chloride, acetonitrile, and methanol are used alone or mixed together with water for the analysis of such compounds by ESI.8-15 Most organic solvents freeze at temperatures far below 0 °C. For example, the melting point of methylene chloride (CH2(1) Smith, R. D.; Loo, J. A.; Edmonds, C. G.; Barinaga, C. J.; Udesth, H. R. Anal. Chem. 1990, 62, 882. (2) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.: Whitehouse, C. M. Science 1989, 246, 64. (3) Colton, R.; Traeger, J. C. Inorg. Chim. Acta 1992, 201, 153. (4) Colton, R.; Traeger, J. C.; Harvey, J. Org. Mass Spectrom. 1992, 27, 1030. (5) Straub, R. F.; Voyknser, R. D. J. Am. Soc. Mass Spectrom. 1993, 4, 578. (6) Poon, G. K.; Bisset, G. M. F.; Mistry, P. J. Am. Soc. Mass Spectrom. 1993, 4, 588. (7) Colton, R.; Tedesco, V.; Traeger, J. C. Inorg. Chem. 1992, 31, 3865. (8) Cole, R. B.; Harrata, A. K. Rapid Commun. Mass Spectrom. 1992, 6, 536. (9) Aubagnac, J.-L.; Munster, H.; Elguero, J.; Meutermans, W. Rapid Commun. Mass Spectrom. 1992, 6, 540. (10) Wilson, S. R.; Wu, Y. J. Chem. Soc., Chem. Commun. 1993, 784. (11) Jaquinod, M.; Leize, E.; Potier, N.; Albrecht, A.-M.; Shanzer, A.; Van Dorsselaer, A. Tetrahedron Lett. 1993, 34, 2771. (12) Van Berkel, G. J.; McLuckey, S. A.; Glish, G. L. Anal. Chem. 1991, 63, 1098. (13) Duffin, K. L.; Henion, J. D.; Shieh, S. S. Anal. Chem. 1991, 63, 1781. (14) Hiraoka, K.; Kudaka, I. Rapid Commun. Mass Spectrom. 1992, 6, 265.
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Cl2) is -95 °C. If such solvents are used in ESI, then, it is possible to obtain the mass spectra at very low temperatures. The success of such a technique would be particularly important for chemists working on thermally unstable compounds. Since many thermally unstable compounds decompose or polymerize at room temperature, they are usually stored in dry ice or in a liquid nitrogen bath.16,17 Presently, the structural characterization of such compounds can only be done by low-temperature NMR.16,17 Good mass spectra of such compounds have never been reported. The reason is that none of the ionization techniques used in mass spectrometry can efficiently generate ions at such low temperatures. In this paper, we report the mass spectra of a volatile and reactive compound, 2,5-dimethylene-2,5-dihydrothiophene18 (DMDHT), from dry-ice-cooled CH2Cl2 solution. Owing to its high reactivity at elevated temperatures, DMDHT oxidized or polymerized rapidly in the electron impact ionization and chemical ionization sources. It was also found that DMDHT is too volatile to be analyzed by FAB. The mass spectra were obtained on a home-built low-temperature electrospray source, where the temperature of the sample solution was maintained at -78 °C during the experiment. The possible ionization mode responsible for the formation of the ion in the source will also be discussed. EXPERIMENTAL SECTION 2,5-Dimethylene-2,5-dihydrothiophene (DMDHT) was prepared by flash vacuum pyrolysis (FVP) of 5-methylthenyl benzoate at 600 °C and 0.01 Torr according to the procedure previously described.17,18 Once generated, the compound was cold-trapped in a collecting tube by liquid nitrogen. The compound was subsequently dissolved and stored in a CH2Cl2/dry ice bath. The structure of the compound was confirmed by low-temperature NMR.18 The yield of the FVP reaction was estimated to be 90%. In this study, the reported concentration of DMDHT in CH2Cl2 was based on this estimate. (15) Huang, S.-W.; Shiea, J. Proceeding of the 42nd ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May 29-June 3, 1994; ASMS: East Lansing, MI, 1994; p 994. (16) Colton, R.; Traeger, J. C.; Tedesco, V. Inorg. Chim. Acta 1993, 210, 193. (17) Chou, C.-H.; Trahanovsky, W. S. J. Am. Chem. Soc. 1986, 108, 4138. (18) Huang, C.-S.; Peng, C.-C.; Chou, C.-H. Tetrahedron Lett. 1994, 35, 4175. 0003-2700/96/0368-1062$12.00/0
© 1996 American Chemical Society
Figure 1. Schematic diagram of low temperature electrospray ion source. I, solution delivering unit; II, sample injection and storage unit; III, spray unit; A, B, and C, finger-tight fittings; D, syringe pump; E, 50 mL gas-tight syringe; F, Teflon tube (2 mm i.d., 8 cm long); G1 and G2, three-way tees; H, stainless steel tube (500 µm i.d., 5 cm long); J, fused silica capillary column (100 µm i.d., 375 µm o.d.); K, Teflon tubes (1 mm i.d., 10 cm long); L, acrylic box (8 cm × 8 cm × 5 cm) filled with fine dry ice powder; M, high-voltage power supply).
A low-temperature ion source was built to obtain mass spectra from the methylene chloride sample solution at -78 °C (Figure 1). The source is comprised of three partssthe solution delivering unit (I), the sample injection and storage unit (II), and the spray unit (III). The configuration of the spray unit is exactly the same as that in an electrospray source. Actually, if the cooling system is not used, good electrospray mass spectra of myoglobin (in aqueous solution) can be obtained with this setup. To prevent oxidation and polymerization of DMDHT during analysis, the temperature of the source was maintained at -78 °C by enclosing it in an acrylic box (L; 8 cm × 8 cm × 5 cm) which was filled with fine dry ice powder. The temperature inside box L was measured by a temperature probe (Hanna, HI 93530; effective temperature range from -200 °C to 1370 °C). We first tried to use a syringe drive design to transfer the sample solution from the reservoir to the ion source. However, the syringe plunger shrank and leaked at -78 °C. To solve this problem, a sample injection and storage unit was built (II). This unit is comprised of two three-way tees (G1 and G2), Teflon tubes (F and K), and two finger-tight fittings (B and C). Two three-way tees were glued together face-to-face using epoxy cement. About 0.5 mL of the cold sample solution was transferred from the sample reservoir into a sample loop (Teflon tube F; 2 mm i.d.; 8 cm long), which connects G1 and G2. This was simply done by withdrawing the sample solution through C by a 5 mL gas-tight syringe connected at B. During this operation, fitting A was kept tightened to prevent the sample solution from flowing into syringe E. After the Teflon tube F was filled with the sample solution, fittings B and C were plugged, and the gas-tight syringe was removed. The next step was to deliver the sample solution to the spraying needle. First, fitting A was opened. This connected the threeway tee G1 to a 50 mL gas-tight syringe E by a Teflon tube (K; 1
mm i.d., 10 cm long). The syringe was filled with about 30 mL of nitrogen gas. The syringe drive D slowly compressed the nitrogen gas in the syringe. The sample solution in sample loop F was then pushed forward to the spraying needle. The needle assembly consisted of a stainless steel tube (H; 500 µm i.d.) with a fused silica capillary column (J; 100 µm i.d.; 375 µm o.d.) inserted inside. At the needle tip, the end of the fused silica capillary column was about 2 mm recessed from the end of the stainless steel tube. No nebulizing gas is used. The flow rate of the sample solution was about 2 µL/min. The high voltage (5.3 kV) required for ionization was supplied through the stainless steel tube H by a high-voltage power supply (Glassman, EH10R10). The voltage used here was higher than that commonly used in ESI. Under this condition, the ionization mode in the source is somewhat different from ESI and will be discussed later. The ions generated by this low-temperature ion source were detected by a PE Sciex API 1 mass spectrometer. The operating parameters of the mass spectrometer for the low-temperature solution were similar to those used at room temperature. The temperature of the interface chamber in the mass spectrometer was held at 55 ( 1 °C. The mass scan rate was ∼2 s/scan. At least 10 consecutive scans were averaged to give the presented mass spectra. RESULTS AND DISCUSSION 2,5-Dimethylene-2,5-dihydrothiophene (DMDHT; C6H6S, 110 g/mol) is very volatile and also very reactive. The boiling point of DMDHT has never been reported, because at room temperature or at higher concentrations, DMDHT rapidly cyclizes to form polymers. The DMDHT also reacts easily with oxygen, water, acetonitrile, acetone, or methanol; therefore, once generated, the compound must be stored in nonpolar solvents such as CH2Cl2. Analytical Chemistry, Vol. 68, No. 6, March 15, 1996
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Figure 2. Mass spectrum of 2,5-dimethylene-2,5-dihydrothiophene dissolved in CH2Cl2 (0.01 M). During the sample analysis, the solution was maintained (A) at room temperature and (B) at -78 °C.
To prevent the occurrence of polymerization during storage, the concentration of DMDHT in CH2Cl2 as well as the temperature of the solution must always be kept low. In ESI, ions are formed at 1 atm without heating. Therefore, ESI mass spectrometry seems to be a very suitable technique for obtaining ion signals from thermally unstable compounds such as DMDHT. Figure 2A shows the mass spectrum of DMDHT dissolved in CH2Cl2 obtained at room temperature by our homebuilt ion source (i.e., without any cooling of the source). In this case, the source is not cooled by the dry ice, and CH2Cl2 instead of nitrogen gas was used to deliver the sample solution to the spraying needle. Only weak signal from protonated DMDHT ion at m/z 111 was observed. However, the intensities of the background peaks in the spectrum were high. By comparing the 1064 Analytical Chemistry, Vol. 68, No. 6, March 15, 1996
mass spectrum with that of pure CH2Cl2 (data not shown), it was found that the background peaks were all of CH2Cl2 origin. The onset electrospray voltage required to obtain an ion signal from the sample solution was about 5300 V. This value is ∼2000 V higher than that commonly used for electrospray ionization of aqueous solutions. Obviously, with such a high voltage, it is likely that ions will be formed mainly by a corona discharge instead of electrospray ionization.14,19,20 We have found that, with such high electric fields, the hydronium ion (H3O+) plus a series of water cluster ions [(H2O)nH+, n ) 2-40] are detected on the mass spectra, even though no solution flows through the electrospray capillary column. The hydronium ions and water cluster ions must (19) Hiraoka, K.; Kudaka, I. Anal. Chem. 1992, 64, 75. (20) Ikonomou, M. G.; Blades, A. T.; Kebarle, P. Anal. Chem. 1991, 63, 1989.
Figure 3. Mass spectrum of 2,5-dimethylene-2,5-dihydrothiophene dissolved in CH2Cl2 (0.25M). (A) The solution was kept at -78 °C during the sample analysis. (B) The solution was dried at room temperature and then redissolved in CH2Cl2.
originate from the moisture in the air (∼90% humidity in the laboratory).20,21 Protonated DMDHT ions can then be generated by a series of ion-molecule reactions between the hydronium or water cluster ions and DMDHT molecules (the same as that in atmospheric pressure chemical ionization). The chemical properties of CH2Cl2, such as surface tension and viscosity, are different at -78 °C and at room temperature. Therefore, we should expect to see a difference in the onset electrospray voltage for the ion signal between the two temperatures if the ions were generated by the electrospray ionization.22,23 (21) Good, A.; Durden, D. A.; Kebarle, P. J. Chem. Phys. 1970, 52, 222. (22) Kebarle, P.; Tang, L. Anal. Chem. 1993, 65, 972A.
However, the experimental results show that the required onset voltage was not affected by the temperature and remained about 5300 V. The current flow between the spray needle and the counter electrode was measured in both room- and low-temperature conditions. It was found that the current flows measured under both conditions were high and similar (∼1500-2000 nA). This finding further supports the idea that the ions generated at such high voltages originate from the corona discharge. It was also found that the ion signals obtained at low temperatures were much more stable and stronger than those obtained at room temperature. Figure 2B shows the mass spectrum of DMDHT in CH2Cl2 obtained at -78 °C. It can be seen that a very strong (23) Brunnix, E.; Rudstam, G. Nucl. Instrum. Methods 1961, 131, 131.
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protonated DMDHT ion (m/z 111) signal was observed in the mass spectrum. There are two major reasons why the signal of protonated DMDHT ion was enhanced at low temperature. First, at room temperature, DMDHT may undergo fast polymerization, and only a fraction of the DMDHT may remain as monomer in the solution. In contrast, the polymerization is very slow at very low temperatures and low concentrations. However, it was found when the concentration of DMDHT in the CH2Cl2 solution was increased by 25 times, polymerization of DMDHT occurred even at -78 °C. The mass spectrum was then dominated by the dimer and trimer ions (Figure 3A). The second explanation for the observation of low analyte signal intensity at room temperature may be the high volatility of the sample solution. It is very possible that, under such a low flow rate (2 µL/min), the volatile molecules will be evaporated at the spray needle tip. Under this condition, the gasous molecules will be easily diffused away from the ionization area, and weak ion signal will be obtained. However, at -78 °C, the volatility of the solution decreases dramatically; the effect of decreasing ion signal with rapid diffusion of sample molecules at the needle tip will then become less significant, and a stable and strong ion signal is obtained. Although the nebulizing gas was not used in this study, desolvation seemed not to be a problem since no analyte/solvent clusters were detected in the spectra. The main reason is the high volatility of CH2Cl2, which will facilitate rapid evaporation of methylene chloride from the droplet. The temperature in the interface chamber was kept at 55 °C; this might also help the desolvation. Although the reactivity of the DMDHT molecule is high at elevated temperatures, the molecules survived the passage through the interface chamber. This may be due to the very short residence time of the rapidly drifting analyte molecule in the heated interface chamber. The presence of a nitrogen curtain
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gas in the chamber may also help to prevent the DMDHT from reacting. During storage, DMDHT reacts with oxygen in the air to form oxides. Even when stored in a degassed CH2Cl2 solution, DMDHT reacts gradually with traces of oxygen originating from the surrounding air. The mass spectrum of DMDHT, which was first dried in air at room temperature and then redissolved in CH2Cl2, showed that the major ion was due to protonated oxidized compound (m/z 143) (Figure 3b). The signal from protonated DMDHT ion was very weak in this sample. In this paper, it is shown that strong and stable protonated ion signals can be obtained for very reactive compounds such as DMDHT at -78 °C. However, the ion source built for this study can only be operated at a fixed low temperature. A variabletemperature source would be more useful for studying structural changes of the thermally reactive compound at different temperatures. It is also known that the melting points of several mixed solvent systems are far below -78 °C; by dissolving the analyte in such solvents, an ion source with an even lower temperature can be built to study the reaction of thermally unstable compounds at such low temperatures. Liquid nitrogen gas instead of dry ice could be used in such a design to achieve the lower temperature condition. Both tasks are now under exploration in our laboratory. ACKNOWLEDGMENT We thank the National Science Council of the Republic of China for financial support (NSC 82-0208-M-110-078). Received for review December 19, 1995.X
October
16,
1995.
Accepted
AC951037I X
Abstract published in Advance ACS Abstracts, February 1, 1996.
