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Anal. Chem. 1989, 61, 2516-2523
Laser Mass Spectrometric Analysis of Compounds Separated by Thin-Layer Chromatography Alan J. Kubis, Kasi V. Somayajula, Andrew G.Sharkey, a n d David M.Hercules*
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
The combhation of thin-layer chromatography (TLC) and laser mass spectrometry (LMS) Is establlshed as a potentlally powerful analytical t a l q u e . LMS Is used to detect the separated compomb directly from the polyamkle TLC plate. One can analyze dkectly from polyamide because it does not alter compound Identiffcatlon, and polyamtde does not interfere with the mass spectrum owing to its low mass fragment ions (loo.
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F&we4. Negative ion spectra from histidhe: (a)from zinc, &focused (laser focal plane 50 pm behind sample), laser energy 2.8 llJ; @) from TLC plate, focused at sample, laser energy 12.8 pJ.
Typically the intensity of the molecular ion species from TLC plates is the same or leas than that for spectra obtained from a zinc surface. Exceptions to this seem to be purine derivatives; several gave more intense molecular ion species from TLC plates than from zinc. In general, matrix effects caused large changes in both relative and absolute intensities of ions formed in LMS (26). The absolute intensities of the analyte peaks varied less when obtained from a TLC plate than from a zinc substrate. This may be due to the fact that the analyte is more homogeneously dispersed in the TLC matrix than on zinc, giving more reproducible conditions from shot to shot. Combined TLC-LMS Experiments. Polynuclear Aromatic Hydrocarbons. Several PAHs were separated on polyamide TLC plates to demonstrate interfacing of TLC with LMS. In all cases the molecular ion was the most intense peak and could be used to determine the positions of compounds on the TLC plate. Figure 5 shows a plot of the absolute intensity of the molecular ions versus distance along the path of development for three PAHs: coronene, 2-methylanthracene, and rubrene. Although there is a certain amount of scatter in the molecular ion intensities, the peaks of all three components could be clearly seen. Using a step size of 250 km gives enough data points that Rf values of 0.27,0.52, and 0.79 can easily be calculated for coronene, 2-methylanthracene, and rubrene, respectively. The values are the same as those obtained by using W fluorescence, except for coronene, which gave an R, value of 0.23 with fluorescence. Coronene shows tailing in this separation, which made defining ita shape and calculating an R value difficult when UV fluorescence was used. In Figure 5 the plot of intensity of the molecular ion versus position for coronene clearly shows the tailing, and an accurate R, value can be calculated, even though visually no
ANALYTICAL CHEMISTRY, VOL. 61, NO. 22, NOVEMBER 15, 1989
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clear peak is defined by UV fluorescence. LMS has the advantage of being able to distinguish between tailing of a single compound and overlapping of two or more compounds to produce a broad TLC peak. Hence, mutually interfering compounds can be distinguished from poor transfer of a single compound along the plate, minimizing confusion in identifying different components of a mixture. In the case of coronene, no peaks other than the M+ a t mlz 300 could be observed between 4.0 and 10.5 mm along the TLC plate, clearly indicating that the broad peak was due to tailing. Purine Derivatives. Figure 6 shows a plot of peak intensity versus distance along the separation path of four purine derivatives: guanine, adenine, purine, and Smethyladenine. Rf values of 0.00, 0.11, 0.29, and 0.90 can be calculated for the
four compounds, respectively. A step size of 500 pm was needed to fully define the peaks. To trace the separation shown in Figure 6, the TLC plate was offset 2 cm during analysis because the sample stage used has a range of only 3.1 cm, and the separation took 3.5 cm to complete. The Rf values for purine and 3-methyladenine are the same as those measured when iodine vapor is used to visualize the spots, within experimental error. No Rf values could be obtained by iodine for adenine and guanine due to overlap. Adenine and guanine can be resolved by using their (M + H)+ions, and their Rf values can be calculated. Figure 7 shows eight spectra taken from 0 to 3.5 mm at 0 . 5 " intervals along the TLC plate. The protonated molecular ion for guanine at mlz 151 decreases in intensity as the protonated molecular
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ion for adenine at mlz 135 increases. When one is analyzing an unknown, this information would be valuable for finding a section of the TLC plate having only one component. This region could then be further analyzed to induce fragmentation in the mass spectra and gain structural information to aid in compound identification. The intensities of fragment ions and molecular ion species for a single compound w ill increase and decrease together. For example, in Figure 7 the peak seen at mlz 119 is a fragment ion of adenine because the relative intensity change follows that of the protonated molecular ion. Therefore, one can clearly establish that the elongated spot on the TLC plate arises from two components, and their spectra can be obtained independently of each other. Also, it is clear that the peak at mlz 119 is a fragment of the ion at m/z 136. It has been demonstrated that interfacing laser mass spectrometry with thin-layer chromatography is a useful technique for obtaining both molecular weight and structural information about compounds on a TLC plate. R, values
measured by TLC-LMS are the same as those measured by other means. No special sample preparation is needed for TLC-LMS, avoiding the loss of material and introduction of contaminants that are possible with other TLC-MS combinations. The limit of detection of TLC-LMS is a t the picogram level or below. Because TLC-LMS is a microprobe technique, it has the ability to perform mapping of the TLC plate to obtain spatial information about compound distribution. This is especially important when separation is not complete, as demonstrated for adenine and guanine. The microprobe capability of LMS can be used to determine if tailing is real or due to a mixed spot. The examples shown demonstrate that TLC-LMS has a wide variety of potential applications. A large number of organic molecules can be desorbed from a TLC plate by LMS, and by use of appropriate separation conditions, difficult mixtures can be analyzed. This information can augment retention information from the separation itself, helping to identify unknown compounds. These advantages make
Anal. Chem. 1989, 61, 2523-2528
TLC-LMS a potentially very useful analytical technique.
LITERATURE CITED McFaddsn' w' TenhnlquesOf Combhsd chrome@FWhy'Mess Specirescopy: AppVcedkns In olgank Ana&ls; Wlley: New York, . . . e
iaia.
Modem RaCilces ofchrrwnatosplsphy; Kkland, J. J., Ed.; Wlley: New York, 1971. Arplno, P. J.; Gukchon, 0.Anal. Chem. 1981, 57(7), 882A-70lA. Covey, T. R.; Lee, E. D.; Brulns, A. P.; Henlon, J. D. Anal. Chem. 1988, 58, 1451-148lA. -1 of chrometCgrephy-WnC H@ Pwfomance Thin L a p chrometoprephy; Ziatkle, A., Kaiser, R. E., Eds.; Elsevfer Sclentlfic: Amsterdam, 1977; Vol. 9. Kalser, R. Chem. Brly. 1989, 5 , 54-81. Jacob. J. J . chrometog. Scl. 1975, 13, 415-422. Dwden, D. A.; Jwrlo, A. V.; Davls, B. A. Anal. Chem. 1980, 52, 1815-1820. ScheHers, S. M.; Verma, S.; Cooks, R. G. Anal. Chem. 1983, 55, 2280-2288. Warner. M. Anal. Chem. 1967, 59, 47-48A. Unger, S. E.; Vlncze, A.; Cooks, R. 0.Anal. Chem. 1981, 53, 978-981. DiDonato, G. C.; Busch, K. L. Anal. Chem. 1988. 58, 3231-3232. Chang, T. T.; Ley, J. O., Jr.; Francel, R. J. Anal. Chem. 1984, 58, 109-1 1 1. Stanley, M. S.; Busch, K. L. Anal. Chlm. Acta 1987. 194, 199-209.
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(15) Fide, J. W.; DIDonato, G. C.; Busch, K. L. Rev. Scl. Instrum. 1986, 57(9), 2294-2302. (18) Novak, F. P.; W L , Z. A.; Hercules, D. M. J . Trace Mlcroprok, Tech. 1985, 3(3), 149-183. (17) Novak, F. P.; Hercules, D. M. Anal. Lett. 1965, 18(A4), 503-518. (16) . . Helnen. H. J.: Meler. S.: Voat. H.: Wechsuna. -. R. Int. J . Mess Smctrom. Ion phvs. 1S83,~47.-19-22. (19) Kraft, R.; Butmer, D.; Franke, P.; Etzold, 0. Bkmed. EnvWn. Mess specbwn.1987, 14, 5-7. (20) Kraft, R.; Otto, A.; Zopfl, H. J.; Etzold, G. B&med. EnvWn. Mess Spectrom. 1987, 14, 1-4. (21) Cheng. W. C.; Lee, M. L.; Chou, C. K.; Lee, S. C. Anal. Bkchem. 1983, 132, 342-344. (22) Jost, W.; Heuck, H. E. Anal. Blochem. 1983, 135, 120-127. (23) Armstrong, D. W.; McNeeiy, M. Anal. Lett. 1979, 12(A12), 1285-1291. (24) Hercules, D. M.; Novak, F. P.; Vlswanadham, S. K.; WIk, 2. A. Anal. Chim. ACte 1967, 195, 61-71. (25) Kubs, A. J. M.S. Thesis, University of Pbburgh, 1988. (28) Karas, M.; Bachman, D.; Bahr, U.; Hlllenkamp, F. Int. J . Mess Spectrm. IOn Processes 1987, 78, 53-88.
RECEIVED for review June 26,1989. Accepted July 14,1989. This work was supported, in part, by the National Science Foundation under Grant CHE-84-11835.
Elimination of Unexpected Ions in Electron Capture Mass Spectrometry Using Carbon Dioxide Buffer Gas L. J. Sears and E. P. Grimsrud*
Department of Chemistry, Montana State University, Bozeman, Montana 5971 7
The hlgh-preswre electron capture (HPEC) ma88 spectra of tetracyanoethyiene (TCNE), tetracyanoquinodimethane (TCNO), wrfluoro-p-benzoquinone (fluoranil), perchloro-pbenzoquinone (chloranll), and perchioro-5,l-bis( cyciopentadbne) (pentac) are drown to be greatly shrpll(kd when carbon dloxlde, rather than methane, is used as the buffer gas. Them compounds have previously been shown to be partkularly rutrc.ptibie to reaction with gabphase or surface-bound radkal specks, which are prevalent in an Ion source contalnlng methane. Through these secondary processes and subaeqwni electron capture (EC) reactions, urtexmed knr of m a w Intenstty are observed wWh the use of CH, buffer gas. Wkh COObuffer gas these unusual tons are eliminated, and only ions that can be expialned in terms of slmple resonance and dissoclatlve EC processes are observed. The hlgh k v d of sendtlvity normally expected of HPEC mrr# spectrometry k also maintained wtth CO, buffer gas. Other non-hydrocarbon buffer gases, including helium, argon, xenon, and nitrogen, are found to yield greatly diminished senrltlvlty relative to that observed with CH, and CO,.
INTRODUCTION The high-pressure electron capture (HPEC) ion source has demonstrated extraordinarily high chemical specificity and sensitivity in the mass spectrometric (MS) analysis of numerous molecules of environmental and biomedical importance (1-3). In HPECMS negative ions are generally formed in thermal electron capture (EC) reactions by either the dissociative or resonance EC mechanisms (4,5), shown in re0003-2700/89/0381-2523$01.50/0
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knowledge exists concerning these two EC mechanisms, and HPEC mass spectra can be readily interpreted or predicted for a given molecule when these reactions,alone, are operative. An inspection of the literature reveals, however, that HPEC spectra are often not explainable in terms of the simple dissociative and resonance EC reactions alone (6-11). In order to account for the HPECMS spectra reported in several studies, it has been necessary to suggest the occurrence of various secondary processes often involving the participation of gas-phase (6) or surface-bound (10) free radicals. These reactive species originate from the e-beam irradiation of the buffer gas and are thought to react with the analyte molecule prior to electron capture. The terminal negative ions thereby formed often include all or part of the reactive free radicals along with all or part of the anal@ molecule. Thw secondary reactions invariably complicate the spectra of molecules that undergo these reactions and can greatly confuse their identification by HPECMS. In this paper we report recent attempts in our laboratory to eliminate secondary reactions of free radicals in a highpressure ion source while maintaining the high level of sensitivity normally achieved by HPECMS. Since previous studies have indicated the participation of hydrocarbon-derived free radicals in secondary ion source processes (6-IO), we will focus here on the use of several non-hydrocarbonbuffer gases-helium, argon, xenon, nitrogen, and carbon dioxide; and we will compare the results obtained with those observed 0 I989 Amerlcan Chemical Society
