Anal. Chem. 1999, 71, 4413-4417
Application of Direct Electrospray Probe To Analyze Biological Compounds and To Couple to Solid-Phase Microextraction To Detect Trace Surfactants in Aqueous Solution Chih-Pin Kuo and Jentaie Shiea*
Department of Chemistry, National Sun Yat-Sen University, Kaohsiung 804, Taiwan
This work presents two novel direct electrospray probes (DEP) to generate an electrospray without using a capillary and/or syringe pump. One of the DEPs is simply a copper coil connecting to a high-voltage power supply. The sample solution is deposited on the coil by a micropipet and the electrospray is subsequently generated at the tip of the copper coil after high voltage is applied to it. Another DEP is constructed by inserting two parallel optical fibers through the copper coil. The two fibers extend one end of the copper coil by 1 cm. Electrospray is generated at the tip of the fibers through the solution predeposited on the copper coil as the high voltage is applied on the copper coil. The ES mass spectra of myoglobin in liquid or solid phases can be obtained using this DEP-MS. Coupling the DEP to a solid-phase microextraction fiber is extremely easy, and a trace amount (in ppb range) of surfactants (Triton X-100) in the aqueous solution are selectively concentrated and detected. Fenn et al. developed electrospray mass spectrometry (ESMS) in 1984, providing an effective means of analyzing not only large biomolecules but small organic and inorganic compounds as well.1-4 A conventional ES device uses a stainless steel tubing or a coaxial arrangement of fused-silica and stainless steel capillaries. In practice, a syringe pump delivering a low flow through a fused-silica capillary column feeds the sample solution to a syringe needle, which is maintained at a high voltage. In many cases, ES-MS is combined with separation techniques such as high-performance liquid chromatography (HPLC), capillary electrophoresis, (CE) and even gas chromatography (GC).5-9 How(1) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4451. (2) Aleksandrov, M. L.; Gall, L. N.; Krasnov, V. N.; Nikolaev, V. I.; Pavlenko, V. A.; Shkurov, V. A. Dokl. Akad. Nauk SSSR 1984, 277, 379. (3) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whithouse, C. M. Science 1989, 246, 64. (4) Jayaweera, P.; Blades, A. T.; Ikonomou, M. G.; Kebarle, P. J. Am. Chem. Soc. 1990, 112, 2452. (5) Bruins, A. P.; Covey, T. R.; Henion, J. D. Anal. Chem. 1987, 59, 2642. (6) Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B. Anal. Chem. 1985, 57, 675. (7) Smith, R. D.; Olivares, J. A.; Nguyen, N. T.; Udseth, H. R. Anal. Chem. 1988, 60, 436. (8) Olivares, J. A.; Nguyen, N. T.; Yonker, C. R.; Smith, R. D. Anal. Chem. 1987, 59, 1230. (9) Lee, C. Y.; Shiea, J. Anal. Chem. 1998, 70, 2757. 10.1021/ac990049r CCC: $18.00 Published on Web 09/03/1999
© 1999 American Chemical Society
ever, ES-MS is also conventionally used to analyze large numbers of pure samples. An example is the samples derived from combinatorial chemistry.10-12 The synthetic products are normally quite pure, thereby eliminating the need for further chromatographic separation. In this case, an ES device capable of rapidly switching the sample solution is required. Moreover, use of a small spraying capillary (e.g., nanospray) risks a plugging problem when a contaminated sample is analyzed (e.g., the solution containing catalyst in fine particles).13,14 Our recent work demonstrates that electrospray can be generated simply by applying a high voltage to a small copper ring with ∼1 µL of the sample solution predeposited on it.15 There is no plugging problem and the sample switching is nearly immediately because the capillary is not used. The analyte dissolved in 100% water (containing 1% acetic acid) can also be electrosprayed. Although the syringe pump and the capillary are not used, the ES mass spectra of the proteins obtained by this direct electrospray probe (DEP) are exactly the same as those by conventional ES generated from a capillary.15 This technique can be applied to rapidly characterize components in purified samples. However, properly applying an aqueous solution on the small copper ring sometimes is difficult, particularly when the surface of the copper ring is contaminated with grease. This difficulty is because the strong surface tension of the droplet prevents it from evenly spreading on the ring. In light of this problem, this work presents two novel electrospray probes capable of easily accommodating the sample solution. About 2-5 µL of the sample solution can be deposited on the probes causing the analyte ion signals to last approximately 2-3 times longer than that of a single copper ring. Solid-phase microextraction (SPME) has been widely applied in analytical and environmental chemistry since Pawliszyn developed it in 1989.16-21 Interfacing work on SPME has been thoroughly investigated by coupling it with gas chromatography/ (10) Kyranos, J. N.; Hogan, J. C. Anal. Chem. 1998, 389, 9A. (11) Dunayevskiy, Y.; Vouros, P.; Carell, T.; Wintner, E. A.; Rebeck, J. Anal. Chem. 1995, 67, 2906. (12) Hegy, G.; Gorlach, E.; Richmond, R.; Bitsch, F. Rapid Comm. Mass Spectrom. 1996, 10, 1894. (13) Emmett, M. R.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1994, 5, 605. (14) Wilm, M. S.; Mann, M. Int. J. Mass Spectrom. Ion Processes 1994, 136, 167. (15) Hong, C. M.; Lee, C. T.; Lee, Y. M.; Kuo, C. P.; Yuan, C. H.; Shiea, J. Rapid Commun. Mass Spectrom. 1999, 13, 21.
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mass spectrometry (GC/MS).16-19 Recently, Pawliszyn et al. successfully coupled SPME to high-performance liquid chromatography- electrospray ionization mass spectrometry (HPLC/ESMS).22,23 The interface (or desorption chamber) for SPME and HPLC uses a six-port injection valve, in which the loop is replaced by a three-way tee. Although a large volume (70 µL) of the desorption chamber may incur a great dilution problem, it has been demonstrated that the SPME-LC/ES-MS is useful in determining water-soluble, semi- and nonvolatile organic compounds that are difficult to be detected by GC/MS.22,23 In this work, the direct electrospray probe is coupled with SPME to selectively concentrate and rapidly detect trace amounts of surfactants in water without chromatographic preseparation. Only 2 µL of the desorbed solvent is needed. The results demonstrate that SPME-DEP-MS is a relatively simple means of determining trace amount of nonvolatile surfactants from the aqueous solution. EXPERIMENTAL SECTION All chemicals used in this work were purchased from Sigma or Aldrich and used without further purification. Figure 1 schematically depicts the DEP and SPME-DEP. The DEP presented in Figure 1a is simply a copper coil mounted on an acrylic plate. The plate was held on a XYZ-translation stage, and the tip of the copper coil was located approximately 1-2 cm in front of the ion sampling orifice of an atmospheric pressure ionization mass spectrometer. The internal diameter of the coil is ∼375 µm, and the diameter of the copper wire is 125 µm (Figure 1b). About 2-5 µL of the sample solution was transferred onto the copper coil by a micropipet to generate the electrospray from the probe. The power supply (Glassman, EH10R10) was subsequently switched on to supply 4500 V to the copper coil for the aqueous solution. Figure 1c illustrates the second type of DEP, which consists of two parallel optical fibers (100 µm in diameter and 5 cm long) inserted into a copper coil (1.5 cm long). The two optical fibers were bound together by inserting them into a small Teflon tube located at one end of the copper coil. When a liquid sample was analyzed, the sample solution was deposited on the copper coil. The solution moved along the channels between both sides of the two optical fibers and an electrospray was generated at the tip of the fibers as the high voltage was applied onto the copper coil. One of the SPME-DEP assemblies was constructed by circling the copper wire on the SPME graphite fiber (0.3 mm in diameter and 2 cm long) for 12 turns (Figure 1d). The SPME-DEP assembly was then exposed to the aqueous phase (containing 10-9 M Triton X-100) and stirred for 60 min. The SPME-DEP was then mounted on an acrylic plate held on a XYZ-translation stage. Approximately 2 µL of the desorption solution (50% H2O/MeOH with 1% of acetic (16) Pawliszyn, J. Solid-Phase Microextraction: Theory and Practice; WileyVCH: New York, 1997. (17) Arthur, C. L.; Pawliszyn, J. Anal. Chem. 1990, 62, 2145. (18) Eisert, R.; Pawliszyn, J. Crit. Rev. Anal. Chem. 1997, 27, 103. (19) Arthur, C. L.; Killam, L. M.; Buchholz, K. D.; Pawliszyn, J.; Berg, J. R. Anal. Chem. 1992, 64, 1960. (20) Li, S.; Weber, G. Anal. Chem. 1997, 69, 1217. (21) Berg, J. R. Am. Lab. 1993, 25, 18. (22) Moeder, M.; Popp, P.; Pawliszyn, J. J. Microcolumn Sep. 1998, 10, 225. (23) Moder, M.; Loster, H.; Herzschuh, R.; Popp, P. J. Mass Spectrom. 1997, 32, 1195.
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Figure 1. (a) Schematic diagram of direct electrospray probe-mass spectrometry (A, electrical cable; B, C, and D, acrylic boxes; E, alligator clip, pressing D will open the clip; F, copper coil; G, XYZtranslation plate). The three direct electrospray probes used in this work are (b) a copper coil, (c) two optical fibers inserted through a copper coil, and (d) a SPME graphite fiber inserted into a copper coil. The sample solution (∼2 µL) is deposited on the copper coil by a micropipet. The dots on the probe represent the sample solution. The Taylor cone is also portrayed on the tip of each probe.
acid) was deposited onto the copper coil in the SPME-DEP by a micropipet. The high-voltage power supply connected on the probe was then switched on to produce an electrospray on the tip of the coil. The ES on the copper coil was roughly coaxial with the entrance of the ion sampling orifice of a quadrupole mass analyzer. Another type of SPME-DEP was constructed simply by replacing the optical fibers with SPME graphite fibers from the DEP presented in Figure 1c. The ions generated by the DEP were detected by a PE Sciex API 1 mass spectrometer. The temperature of the interface chamber in the mass spectrometer was maintained at 55 ( 1 °C. The mass scan rate was ∼2 s/scan, and 20 consecutive scans were averaged to produce the presented mass spectra. RESULTS AND DISCUSSION Our previous study indicated that the electrospray can be generated on a single copper ring predeposited with 1 µL of the sample solution.15 However, the ion signal only lasts for 45 s. The copper coil illustrated in Figure 1 has a higher surface area than a single copper ring, thereby making it possible to hold more sample solution on the probe. Actually, 5 µL of the sample solution can be held on the coil and the analyte’s signal lasts ∼3 times longer than that of a single copper ring. According to our observation through a microscope, the solution originally held on the copper coil tends to move away from the coil as the high voltage (positive) is applied. This is because the high voltage charges the surface of the solution and creates a repelling force between the solution and the electrode (i.e., copper coil). Increasing the voltage applied to the copper
coil makes the repelling force stronger; concurrently, the solution tends to be dragged back to the coil due to the strong surface tension of the solution. At a low voltage, the dragging force is stronger than the repelling (or leaving) force so that the solution remains on the probe. Increasing the voltage strengthens the leaving force applied onto the solution and deforms the surface of the solution (the droplet tends to elongate toward the anode). The surface of the solution breaks when the voltage reaches about 3200 and 4500 V for pure methanol and aqueous solutions, respectively. Stereomicroscopy results demonstrate that the residue solution on the copper coil contains many sharp ends pointing at the anode at the moment of surface breakage. The sharp ends immediately converge to exhibit a small Taylor cone and the electrospray is initiated from the center of the Taylor cone. Related investigations have confirm that deformation of the surface of a solution destablizes the droplets and eventually leads to uneven fission (i.e., emission of a fine droplet spray from the sample solution) at the droplet charge that is lower than the charge at the Rayleigh limit.24-27 This phenomenon can also account for why ES can be directly generated from the solution predeposited on the DEP. The exact flow rate of the solution during electrospray is difficult to measure because the copper coil is exposed to air. Therefore, part of the sample solution is lost by rapid evaporation. Since the electrospray lasts for 45 s for 1 µL of the sample solution predeposited on a copper ring, it can be estimated that the electrospraying rate on the DEP is ∼0.8 µL/min, assuming 40% of the sample solution is lost by evaporation. The mass spectra of the protein solutions analyzed by DEPMS are exactly the same as those by a conventional ES using a syringe pump and a capillary. Figure 2 displays the ES mass spectra of myoglobin (Figure 2a) and cytochrome c (Figure 2b) obtained by using the copper coil as the electrospray probe. The protein (10-6 M) is dissolved in pure water containing 1% acetic acid. The mass spectra reveal all of the ions with multiple charges up to +25 and +20 for myoglobin and cytochrome c, respectively. The adduct ions displayed on the mass spectra originate from [protein + acetic acid] adduct ions. No copper adduct ions are detected on the mass spectra. In addition, the probe is not washed between each sample application and no contaminating signals from previous sample are found. In this case, the sample switching is immediately. This is attributed to the fact that the analyte solution is difficult to retain on the copper coil with a rather small diameter. However, if necessary, the probe can be easily washed by either dipping the copper coil into a cleaning reagent or simply by flushing it with an acidic solution. The detection limit (∼10-7 M) of the DEP-MS closely resembles that of conventional ion spray using a capillary. This was estimated by using myoglobin as the analyte. Figure 1c depicts the second type of DEP developed in this work. Again, the sample solution (2 µL) is applied onto the copper coil by a micropipet. However, an electrospray from an extremely small Taylor cone at the tip of the two optical fibers is observed (24) Hager, D. B.; Dovichi, N. J.; Klassen, J.; Kebarle, P. Anal. Chem. 1994, 66, 3944. (25) Hager, D. B.; Dovichi, N. J. Anal. Chem. 1994, 66, 1593. (26) Kebarle, P.; Ho, Y. In Electrospray Ionization Mass Spectrometry; Cole, R., Ed.; John Wiley & Sons: New York, 1997; Chapter 1, p 3. (27) Gomez, A.; Tang, K. Phys. Fluids 1994, 6, 404.
Figure 2. Positive ES mass spectra of (a) myoglobin and (b) cytochrome c, obtained by the DEP illustrated in Figure 1b. The sample solution was prepared by dissolving the protein in the water containing 1% acetic acid (10-6 M).
when high voltage is applied to the sample solution through the copper coil. It appears that as the high voltage was applied onto the sample solution in the reservoir (i.e., the copper coil), the surface of the solution would be charged and a slight amount of the solution would be forced to leave the reservoir. The solution leaving the reservoir moves along the channels between both sides of the optical fibers by the capillary action to the tip of the fibers. Electrospray is initiated as the solution reached the tip of the optical fibers. Herein, the high voltage deemed necessary for ES is conducted through the solution. This phenomenon resembles that of nanospray, microchip ES, multiple-channel ES, and other designs described elsewhere.13-14,28-31 The length of the optical fibers used in this work is 5 cm. The ES mass spectra of the same samples obtained by using the DEP described in Figure 1c are exactly the same as those in Figure 2 (data not shown). The high voltage (28) Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 1997, 69, 1174. (29) Xue, Q.; Foret, F.; Dunayevskiy, Y. M.; Zavracky, P. M.; McGruer, N. E.; Karger, B. L. Anal. Chem. 1997, 69, 426. (30) Xue, Q.; Dunayevskiy, Y. M.; Foret, F.; Karger, B. L. Rapid Commun. Mass Spectrom. 1997, 11, 1253. (31) Shiea, J.; Wang, C. H. J. Mass Spectrom. 1997, 32, 247.
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Figure 3. (a) Total ion chromatogram of the acebutolol analyzed by the DEP illustrated in Figure 1c. The ES was generated from the acidic solution (0-1.4 min), and then, the high-voltage power supply was switched off and the analyte (in powder form) was placed between the channel between the two optical fibers (1.4-1.8 min). The high voltage was applied to the sample solution again (1.8-4 min). The ES mass spectrum recorded after 2.5 min is illustrated as the inset in (a). (b) Total ion chromatogram of myoglobin (in solid phase) analyzed by the DEP illustrated in Figure 1c. The experimental procedures resemble those described in (a). The ES mass spectrum of myoglobin is illustrated as the inset.
(3800 V) required for electrospray from the tip of the optical fibers is ∼700 V less than that from a copper coil. Moreover, the electrospray from the DEP in Figure 1c appears to be more stable than that in Figure 1b. Again, exposure of the sample solution to air makes it difficult to calculate the exact flow rate of the solution due to electrospray. However, the duration of the electrospray of a 2-µL solution is ∼2 min. The DEP presented in Figure 1c can also be used to analyze the solid sample. The sample powder (∼0.2 mg) is placed between the two optical fibers located ∼0.5 cm outside the copper coil. Five microliters of the methanol solution (50% H2O/MeOH with 1% acetic acid) is then deposited onto the copper coil by a micropipet. Applying high voltage to the copper coil forces the methanol solution to move along the channels between the two fibers while the electrospray is generated at the end of the fibers as well. As the methanol solution flows through the optical fibers, a portion of the analyte is dissolved and carried to the end of the fibers and the analyte signals are detected. Figure 3a summarizes the analysis of solid acebutolol by the DEP-MS. The mass analyzer is continuously scanned, and the total 4416 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999
Figure 4. Positive ES mass spectra of the solutions containing Triton X-100 with the concentrations of (a) 10-6 and (b) 10-9 M, as obtained by DEP-MS and (c) 10-9 M obtaining by SPME-DEP-MS.
ion current (TIC) and the mass spectra are recorded by the mass spectrometer. The total ion chromatogram indicates that the TIC of the acidic solution is low but stable (0-1.4 min in Figure 3a) before the analyte powder is applied to the probe. The ion signals must originate from the acidic solution because the analyte is not added. The high-voltage power supply is then switched off and a slight amount of the solid analyte is placed between the two optical fibers. No ion current can be detected (1.4-1.8 min in Figure 3a). The high-voltage power supply is switched on again, and the TIC abruptly increases due to generation of a high concentration of the analyte and acetate ions by ES (1.8-4 min in Figure 3a). The inset in Figure 3a displays the ES mass spectrum of acebutolol obtained in this manner. The mass spectrum predominates with the protonated acebutolol ion (m/z 337). Figure 3b summarizes the analysis of myoglobin (in solid phase) by the DEP-MS. A slight amount of myoglobin powder is again placed between the two optical fibers, and the TIC increases abruptly after the high voltage is added to the copper coil. The ES mass spectrum clearly reveals multiply charged myoglobin ions (the inset in Figure 3b). The fact that most biological compounds dissolve well in the aqueous solution suggests that use of DEP to directly analyze
solid biological compounds is limited. However, as generally known, many chemical pollutants (e.g., surfactants and herbicide) are left in the aquatic environment in trace amounts. These chemicals must be concentrated on the surface of the solid adsorbers so that they can be detected by modern instruments. A DEP in a solid sample analysis can couple with SPME by inserting a SPME fiber into the space inside a copper coil (Figure 1d). The organic pollutants dissolved in the aqueous solution are then concentrated on the SPME fiber. The adsorbed organic pollutants can be easily desorbed by applying only a slight amount of the desorption solvent (2 µL) onto the copper coil. The desorption solution containing the organic pollutants is electrosprayed after the high voltage is applied to the copper coil. The SPME fiber inside the copper coil does not interfere with the electrospray. A trace amount of the surfactants in the aqueous solution was qualitatively determined by using SPME-DEP-MS. Figure 4 displays the ES mass spectra of Triton X-100 obtained by a DEPMS and a SPME-DEP-MS, respectively. Directly analyzing the sample solution (10-6 M) with a DEP-MS reveals the presence of ion signals from a series of ethoxamers (Figure 4a). As the concentration of Triton X-100 in the solution is decreased to 10-9 M, no analyte signal is detected by the DEP-MS (Figure 4b). This result is the same as previous investigation using conventional electrospray.22 However, use of a SPME-DEP-MS can obtain the ES mass spectrum of Triton X-100 (Figure 4c). Notably, only 2 µL of the desorption solution (H2O/MeOH (50%) with 1% acetic acid) is used to desorb the analytes from the SPME graphite fiber. The SPME-DEP can also be assembled simply by replacing the two optical fibers illustrated in Figure 1c with SPME graphite fibers. The analyte is adsorbed and concentrated on the graphite fibers as the SPME-DEP is immersed into the aqueous solution containing a trace amount of Triton X-100. The SPME-DEP
assembly is then removed from the solution and dried in the air. Two to three microliters of the desorption solution is added onto the copper coil. The adsorbed analytes are dissolved, and the ES mass spectra of the analyte can be obtained (data not shown) as the desorption solution moves along the channels between the two graphite fibers under a high electric field. Although not accomplished in this work, semiquantitative analyses for biological and environmental polymers with monodispersity can be achieved by adding internal standards into the desorption solution. This would be a worthwhile task for a future study. CONCLUSION This work demonstrates that an electrospray can be generated from the solution by two different types of the direct electrospray probes. The ES mass spectra of some biological compounds such as acebutolol and myoglobin in solid phase were obtained using the DEP-MS herein. Other analytical features of DEP are that the probes are free of maintenance and are also easy to clean and construct. In addition, trace amounts of nonvolatile and less polar surfactants in water can also be detected by coupling DEP with SPME. The construction of SPME-DEP is extremely easy, and no interface is required for the coupling while only 2-3 µL of the solvent is required to desorb the adsorbed molecules from the surface of the SPME graphite fiber. ACKNOWLEDGMENT The authors thank the National Science Council of Taiwan for financially supporting this research.
Received for review January 19, 1999. Accepted July 19, 1999. AC990049R
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