Anal. Chem. 2006, 78, 8162-8164
Atmospheric Pressure Photoionization for Ionization of Both Polar and Nonpolar Compounds in Reversed-Phase LC/MS Damon B. Robb and Michael W. Blades*
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia, Canada, V6T 1Z1
Atmospheric pressure photoionization can provide high ionization efficiency simultaneously to both polar and nonpolar compounds delivered in reversed-phase solvent. The method to achieve this utilizes toluene as a dopant and simply requires that the solvent flow be limited so that reactions between toluene photoions and the organic component of the solvent are not driven to completion. Under these conditions, toluene photoions remain in the source for ionizing nonpolar compounds via charge exchange (electron transfer), while protonated solvent ions are available for proton-transfer reactions with polar molecules. The reagent ion mixture is then suitable for ionizing a wide range of both polar and nonpolar compounds. The critical effect of solvent flow rate is demonstrated here with results for a test analyte, 9-methylanthracene, which may be ionized by either charge exchange or proton transfer. For a solvent of 50:50 methanol/water (v/v), lowering the flow from 200 to 50 µL min-1 results in a 10× increase in charge exchange ionization efficiencys further flow reductions provide even greater enhancements. This method is compatible with sample delivery by direct infusion and micro- and narrow-bore LC, as well as conventional LC using a flow splitter. The two most commonly used ionization methods for LC/MS, electrospray ionization and atmospheric pressure chemical ionization, both work best for compounds containing a polar functional group, which can accept or donate a proton. Neither of these methods is generally effective for ionizing nonpolar compounds. Atmospheric pressure photoionization (APPI) with a dopant, on the other hand, is capable of ionizing both polar and nonpolar compounds through proton transfer and charge exchange reactions, respectively.1 Reagent ions for proton transfer are conveniently produced via photoionization of toluene in the presence of a reversed-phase solvent such as methanol or acetonitrile, since toluene ions react with these solvents through a clustering process that ultimately produces protonated solvent reagent ions.2-5 The reagent ions for charge exchange ionization, however, are the * Corresponding author. E-mail:
[email protected]. (1) Robb, D. B.; Covey, T. R.; Bruins, A. P. Anal. Chem. 2000, 72, 36533659. (2) Koster, G.; Bruins, A. P. Mechanisms for ion formation in LC/MS by Atmospheric Pressure Photo-Ionization (APPI). Proceedings of the 49th ASMS Conference on Mass Spectrometry and Allied Topics; Chicago, IL, May 2001.
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dopant photoions themselves. Accordingly, for the charge exchange ionization pathway to be most effective, the dopant photoions must not be lost through reactions with the solvent. Because of the reactivity of toluene ions with methanol and acetonitrile, the use of toluene as a dopant to ionize nonpolar compounds delivered in these solvents is inefficient at conventional LC flow rates (g200 µL min-1), and alternate methods have been pursued. One alternative is to use normal-phase solvents such as hexane and chloroform, which generally do not eliminate the toluene photoions.2,3,6 This approach is not ideal, though, because it is desirable to retain the use of reversed-phase LC methods. Another alternative is to use anisole as the dopant, since anisole photoions are stable in the presence of reversed-phase solvents and will then be available to serve as reagents for charge exchange ionization.7 However, for charge exchange ionization of the analyte to be possible, the dopant must have an ionization energy (IE) above that of the analyte, and anisole has a relatively low IE of 8.20 eV, limiting its usefulness. The purpose of this technical note is to describe how toluene (IE ) 8.83 eV) can in fact be used as a dopant with reversedphase solvents to promote efficient charge exchange ionization. The method exploits the fact that equilibria are involved in the overall reaction between toluene photoions and solvent neutrals,5 and simply requires that the solvent flow be limited so that the reaction is not driven toward completion. For example, at methanol flow rates below ∼25 µL min-1, toluene photoions are not entirely consumed and charge exchange ionization of nonpolar compounds can take place with high efficiency. Moreover, protonated methanol ions are also available for proton-transfer ionization of polar compounds. Hence, by taking the simple step of limiting the flow rate of the sample stream into the source, the APPI method becomes conducive to the ionization of both polar and nonpolar compounds and the option to use reversed-phase LC methods is retained. Note that this (near) universal ionization capability has recently been reported elsewhere, as part of the description of a (3) Kauppila, T. J.; Kuuranne, T.; Meurer, E. C.; Eberlin, M. N.; Kotiaho, T.; Kostiainen, R. Anal. Chem. 2002, 74, 5470-5479. (4) Kauppila, T. J.; Bruins, A. P.; Kostiainen, R. J. Am. Soc. Mass Spectrom. 2005, 16, 1399-1407. (5) Robb, D.; Blades, M. J. Am. Soc. Mass Spectrom. 2005, 16, 1275-1290. (6) Impey, G.; Kieser, B.; Alary, J.-F. The Analysis of Polycyclic Aromatic Hydrocarbons (PAHs) by LC/MS/MS using a New Atmospheric Pressure Photoionization Source. Proceedings of the 49th ASMS Conference on Mass Spectrometry and Allied Topics; Chicago, IL, May 2001. (7) Kauppila, T. J.; Kostiainen, R.; Bruins, A. P. Rapid Commun. Mass Spectrom. 2004, 18, 808-815. 10.1021/ac061276d CCC: $33.50
© 2006 American Chemical Society Published on Web 10/27/2006
Figure 1. Full scan mass spectrum of 9-methylanthracene (9-MA). The solvent was 50:50 methanol/water and the dopant was toluene. Both the solvent and dopant flow rates were 20 µL min-1. The labeled peaks correspond to the protonated methanol solvent dimer (S2H+), the toluene dopant radical ion (D+‚), the radical molecular ion (M+‚) of 9-MA, and the protonated molecular ion (MH+) of 9-MA.
Figure 2. Peak height as a function of solvent flow rate for (A) the dopant radical ion (m/z 92) and the protonated methanol dimer (m/z 65) and (B) the radical molecular ion (m/z 192) and the protonated molecular ion (m/z 193) of 9-MA. The solvent was 50:50 methanol/water, the dopant was toluene, and the dopant flow rate was fixed at 20 µL min-1.
capillary electrophoresis APPI-MS system utilizing a solvent makeup, but the importance of the solvent composition and flow rate was not mentioned.8,9 Here, the critical role of the solvent in determining the efficiency of the charge exchange ionization pathway is demonstrated for a model nonpolar analyte, 9-methylanthracene (9-MA), which may be ionized by either charge exchange or proton transfer. EXPERIMENTAL SECTION The test system used 50:50 methanol/water (v/v) as the solvent, toluene as the dopant, and 9-MA (IE ) 7.3 eV) as the analyte. A 15 mM stock solution of 9-MA was prepared in acetonitrile. The stock solution was diluted in the methanol/water test solvent to make a final 50 µM analyte solution. 9-MA was from Sigma-Aldrich (Oakville, ON, Canada). The organic solvents and the dopant were HPLC grade, from Fisher Scientific (Ottawa, ON, Canada). Deionized water was from an in-house generator. All chemicals were used as received. The analyte solution, solvent makeup, and dopant were delivered separately via syringe pumps from Harvard Apparatus (Holliston, MA). The analyte solution and solvent makeup were combined in a tee prior to delivery into the ion source. The analyte solution was introduced at a fixed rate, 4 µL min-1, so the mass flow rate of analyte was constant when the total solvent flow was varied. The dopant flow rate was fixed at 20 µL min-1. (8) Mol, R.; de Jong, G. J.; Somsen, G. W. Electrophoresis 2005, 26, 146-154. (9) Mol, R.; de Jong, G. J.; Somsen, G. W. Anal. Chem. 2005, 77, 5277-5282.
The APPI source was a first-generation PhotoSpray source from MDS Sciex (Concord, ON, Canada), designed for their API 100/300/3000 series of mass spectrometers.1 The ion source was powered by a custom HV supply (Electrical Services Shop, Chemistry Department, UBC), and the lamp current was set to 0.8 mA. The offset voltage applied to the source was 1.3 kV. For the results displayed here, the heated nebulizer temperature was 400 °C. The auxiliary and lamp gas flow rates were each 1.0 slpm, and the nebulizer gas flow rate was 1.9 slpm (70 psi). Liquid nitrogen boil-off was used for all the gases. The mass spectrometer was a prototype single-quadrupole instrument from MDS Sciex (ca. 1995), closely related to subsequent API 100 series instruments. The housing of the PhotoSpray source was compatible with the atmosphere-vacuum interface of the MS and was directly mounted without modification. The orifice plate and focusing ring voltages were set to 10 and 50 V, respectively, to minimize collision-induced dissociation. For the full scan spectrum (Figure 1), the scan range was 60-200 Da, the step size was 0.1 Da, the dwell time was 1 ms, and 10 scans were averaged. A multiple ion scan was used to obtain the charted data (Figure 2); for center m/z 65, 92, and 193 Da, the scan width was 5 Da, the step size was 0.1 Da, the dwell time was 10 ms, and 20 scans were averaged. RESULTS AND DISCUSSION Figure 1 is an APPI mass spectrum obtained using toluene as a dopant and 50:50 methanol/water as the solvent delivering the analyte, 9-MA. The total solvent flow rate was 20 µL min-1. The Analytical Chemistry, Vol. 78, No. 23, December 1, 2006
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spectrum features prominent signals at m/z 65 and 92 due to protonated methanol solvent dimers (S2H+) and toluene dopant radical cations (D+•), respectively. The high background signals at m/z 106-108 and 120 are attributed to impurities in the toluene. The spectrum also features two intense peaks for 9-MA: one at m/z 192 due to the radical molecular ion (M+‚) and another at m/z 193 due to the protonated molecular ion (MH+). No fragment ions from 9-MA are observed. The large signals at both m/z 65 and 92 show that reactions between the toluene ions and methanol do not proceed to completion under these low-flow conditions and that an abundance of reagents for both charge exchange and proton-transfer ionization can coexist in the source. As for the significance of the two 9-MA peaks, the intense M+‚ signal demonstrates the effectiveness of the charge exchange ionization pathway, while the strong MH+ peak indicates that the protontransfer ionization pathway is also effective. Thus, both the charge exchange and the proton-transfer ionization pathways are activated under these conditions, which means that this APPI method is capable of efficiently ionizing both nonpolar and polar compounds simultaneously. Figure 2A shows the effects of the solvent flow rate on the key reagent ions. At the lowest solvent flow tested, 10 µL min-1, the toluene ion signal (m/z 92) is very strong, about twice that of the protonated methanol dimer (m/z 65). The data confirm, however, that the toluene ions are rapidly consumed as the solvent flow is raisedsand the reaction equilibria are shifted5sand that the methanol-derived ions come to predominate. Clearly, the presence of methanol/water solvent is detrimental to the stability of the toluene charge exchange reagent ions, though the data indicate that some do survive at flow rates up to ∼50 µL min-1. Figure 2B shows the effects of the solvent flow rate on the ionization of 9-MA. These data indicate that the peak height of the M+• ion (m/z 192)sand thus the efficiency of the charge exchange ionization pathwaysdeclines steadily as the solvent flow is raised. Interestingly, the charge exchange ionization efficiency is not directly proportional to the quantity of charge exchange reagent ions exiting the ion source, since the signal for the toluene ions decays much more rapidly than the signal for the M+• ions. This means that under certain conditions it is possible for the charge exchange ionization pathway to remain fairly effective, even though there appear to be very few charge exchange reagent ions remaining in the source (e.g., see the data for 100 µL min-1 in Figure 2). We do not presently have an explanation for this nonintuitive finding. As for the MH+ ion (m/z 193), the data indicate that there is an optimum solvent flow rate for the ionization of 9-MA via proton transfer, in the range of 50-100 µL min-1. The decrease in proton-transfer ionization efficiency at lower flow rates is likely a result of the incomplete transformation of toluene ions to methanol-derived ions; however, even at the low flow of 10 µL min-1, the proton-transfer ionization efficiency
is only slightly less than at the optimum flow. At higher than optimum flows, on the other hand, the decrease in proton-transfer efficiency with increased solvent flow is believed to be due to effects stemming from the growth in size of the solvated reagent ion clusters.5 For some analytes, including 9-MA, the decline in proton-transfer efficiency at higher flows (>200 µL min-1) can be severe. To summarize, the data indicate that the efficiency of the charge exchange ionization pathway can be greatly enhanced by lowering the solvent flow rate so that the reaction between the charge exchange reagent ions and the solvent is not driven toward completion. The data also show that the efficiency of proton transfer is not substantially compromised by lowering the solvent flow rate. Thus, by tailoring the solvent flow to provide reagent ions for both the charge exchange and proton-transfer ionization pathways, APPI using toluene as a dopant can provide high sensitivity simultaneously to both nonpolar and polar compounds delivered in the reversed-phase solvent methanol. Addendum. Several additional parameters are noteworthy because of their effect on the method. Regarding the organic content of the solvent, it is primarily methanol which reacts with the toluene ions, not water.3 Accordingly, to maintain a given charge exchange ionization efficiency, the total solvent flow rate must be lowered as the organic content of the solvent is increased. Note also that acetonitrile may be substituted for methanol with little effect on the relationship between charge exchange ionization efficiency and solvent flow; in our experience, however, the protontransfer ionization efficiency is generally lower when acetonitrile replaces methanolsperhaps because of the higher gas-phase basicity of acetonitrile. As for the choice of dopant, benzene (IE ) 9.24 eV) may be used in place of toluene, to exploit its high IE and thereby extend the number of compounds amenable to charge exchange ionizationsthough workplace safety concerns must be addressed if benzene is to be used. Another important factor is the temperature of the source, since the reaction between dopant ions and solvent depends upon ion-solvent cluster formation.2,5 At higher source temperatures, the ion-solvent clusters are smaller for a given solvent flow rate and the loss of dopant photoions is impeded. Raising the source temperature is then another means of increasing the charge exchange ionization efficiency.10
(10) Cormia, P. H.; Fischer, S. M.; Miller, C. A. Analysis of Polyaromatic Hydrocarbons by Atmospheric Pressure Photoionization LC/MS. Proceedings of the 49th ASMS Conference on Mass Spectrometry and Allied Topics; Chicago, IL, 2001.
Received for review July 14, 2006. Accepted September 21, 2006.
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ACKNOWLEDGMENT The authors thank Peter Jacobs of NV Organon (Oss, The Netherlands) for the kind donation of the Photospray source, and David Chen and Don Douglas of the University of British Columbia (UBC) for providing access to the mass spectrometer. We also acknowledge funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) and UBC.
AC061276D
