Corresfiondence Anal. Chem. 1995, 67, 1474- 1476
Atmospheric Pressure Chemical Ionization Interface for Capillary Electrophoresis/Mass Spectrometry Yasuaki Taka&,* Minoru Sakairl, and Hideaki Koizumi Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo 185, Japan
Capillary elecbrophoresis/atmospheric pressure chemical ionization mass spectrometry (CE/APCI-MS) is described. The interface between capillary electrophoresisand mass spectrometryconsists of an electrospray-typenebulizer, a vaporizer, and an atmospheric pressure chemical ionization source using a needle electrode. In this system, a solution is nebulized by electrospray, and vaporized samples are ionized by corona discharge followed by ion/ molecule reactions. Protonated caffeine molecules are obtained using CE/APCI-MS with sodium phosphate buffers. CE/APCI-MS is a very promising technique because the APCI process is not heavily affected by salts in CE buffers, allowing the use of a variety of CE buffers in CE/MS analysis. Recently, capillary electrophoresis (CE) has been recognized as a powerful tool for analysis of mixtures.' On-line combination of high separation efficiency in CE and identification potential in mass spectrometry @IS) is very promising. But, a direct connection between capillary electrophoresis and mass spectrometry is dficult because of incompatibility between the liquid phase separation technique and ion trajectory analysis in a vacuum. Therefore, one of the most important goals in capillary electrophoresis/mass spectrometry (CE/MS) is the development of interfaces between capillary electrophoresis and mass spectrometry.2.3 Two types of interfaces are currently in use for CE/MS: electrospray (or ion spray) and fast atom b~mbardment.~-~ In particular, high sensitivity detection of peptides and proteins has been achieved by electro~pray.~ However, several problems still exist in CE/MS. First, no present ionization technique can ionize all of the samples separated by CE. Second, the buffer composi~~~~
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(1) Monnig, C. A; Kennedy, R T. Anal. Chem. 1994,66,280R (2) Niessen, W. M. A; Tjaden, U. R; van der Greef, J.1. Chromatogr. 1993, 636, 3. (3) Smith, R D.;Wahl, J. H.; Goodlett, D. R; Hofstadler, S. A Anal. Chem. 1993,65, 574A. (4) Olivares, J. A; Nguyen, N. T.; Yonker, C. R; Smith, R D. Anal. Chem. 1987,59, 1230. (5) Lee, E. D.; Muck, W.; Henion, J. D.; Covey, T. R J. Chromatogr. 1988, 458, 313. (6) Suter, M. J. F.; Caprioli, R M. 1.Am. SOC.Muss Spectrom. 1992,3, 198. (7) Wahl, J. H.; Goodlett, D. R; Udseth, H. R; Smith, R D. Electrophoresis 1993,14,448.
1474 Analytical Chemistry, Vol. 67, No. 8, April 15, 1995
Differential pumping region
Electrospray probe
I MS 2nd aperture
Figure I. Schematic diagram of the CWAPCI-MS interface.
tions that are available for electrospray are so limited that the extraordinary potential of CE, which includes miceller electrokinetic chromatography (MEKC)! has not been suffciently utilized for CE/MS analysis. Therefore, we expect various types of CE/ MS interfaces, based on different ionization methods, to become available for a wide range of applications. In the case of liquid chromatography/mass spectrometry (LC/ MS) using multiatmospheric pressure ionization (MI),structures of several interfaces that have different ionization modes are very similar and the multi-API method can analyze a wide variety of samples by simply exchanging interfaces? Therefore, we have examined an atmospheric pressure chemical ionization (APCI) mode for the CE/MS interface. In this paper, we describe basic characteristics of capillary electrophoresis/atmosphericpressure chemical ionization mass spectrometry (CE/APCI-MS). EXPERIMENTAL SECTION Figure 1 shows a schematic diagram of the CE/APCI-MS experimental system. A Hitachi (Tokyo, Japan) M-1000 type quadrupole mass spectrometer equipped with the CE/MS interface was used. The CE instrument and the CE/MS interface used in this work were constructed in our laboratory. The interface consisted of an electrospray-type nebulizer, a vaporizer, and an APCI ion source. In the commercially available APCI interface (8) Terabe, S.; Otsuka, IC; Ichikawa, IC; Tsuchiya, A; Ando, T. Anal. Chem. 1984,56, 111. (9) S W , M.; Yergey, A L. Rapid Commun. Mass Spectrom. 1991,5, 354.
0003-2700/95/0367-1474$9.00/0 0 1995 American Chemical Society
for LC/MS, effluents from Lc are nebulized by heating or by a pneumatic nebulizer.loJ1 However, we felt it was djf6cult to establish stable nebulization of the CE buffer solution using these techniques because of the low flow rate (0-0.1 pL/min) within the capillary. Therefore, we used a sheath-flow-assisted electrospray-type nebulizer to obtain fine droplets in the APCI mode. The nebulizer was very similar to that reported by Smith et a1.12 One end of the fused-silica capillary tube (50 pm i.d., 150pm o.d., and approximately 40 cm long) was inserted into the electrospray probe (a 0.25 mm i.d. and 0.40 mm 0.d. stainless steel capillary). The electrospray probe was maintained at 2.8 kV by a external power supply. The details of the electrospray-type nebulizer have been described el~ewhere.'~J~ A syringe pump (Model 44, Harvard Apparatus, Natick, MA) was used to deliver the sheath flow at a flow rate of 5 pWmin. The sheath fluid used here was pure methanol. The fine droplets produced by the electrospraytype nebulizer was introduced into the vaporizer. The vaporizer consisted of a stainless steel block, which had an opening (5 mm i.d. and 60 mm long), that could be uniformly heated to 300 "C by cartridge heaters. A needle electrode was located between the vaporizer and a sampling aperture, and high voltage of 3.0 kV was applied to the needle electrode to produce corona discharge. Vaporized samples and solvent molecules were ionized by the corona discharge resulting in ion/molecule reactions. The ions produced at atmospheric pressure were introduced into the mass spectrometer through a differential pumping region. The first and second apertures (0.25 mm i.d.) were heated to 120 "C with ceramic heaters. The center axis of the electrospray probe, the vaporizer, and the sampling apertures were aligned. The distance between the end of the electrospray probe and the first aperture was about 80 mm. A drift voltage of 30 V was applied between the first and second apertures to dissociate cluster ions by collisions.10 The mobile phases were pure water (PH 5.6) and sodium phosphate buffers (20 and 40 mM, pH 6.6). The sample was caffeine, which was purchased from Sigma Chemical Co. (St. Louis, MO) and used as received. The sample concentration used was M. The injections were performed by placing the anode end of the capillary in the sample solution and raising this solution 6 cm for 10 s. A net CE voltage of 10 kV was applied to the capillary for all experiments. RESULTS AND DISCUSSION
One of the most important goals in CE/MS is to be able to use buffers that include high concentrations of nonvolatile salts. We have investigated the background mass spectrum of sodium phosphate buffer solutions for CE to compare the characteristics of the APCI mode and the commonly used ESI mode.13J4 Figure 2 shows the background mass spectrum obtained with (a) the APCI mode and (b) the ESI mode when a 20 mM sodium phosphate buffer was used. A net CE voltage of 10 kV was applied to send the buffer solution to the interface by electroosmosis.In our preliminary experiments for the APCI mode, we have changed the flow rates of the sheath flow from 1 to 10 pL/min to obtain (10) Sakairi, M.;Kambara, H. Anal. Chem. 1988, 60, 774. (11) Thomson, B.A;Danylewych-May,L. Proceedings of the 31st Conference on Mass Spectromety and Allied Topics, Boston, 1983;p 852. (12) Smith, R D.;Barinaga, C. J.; Udseth, H. R Anal. Chem. 1988, 60,1948. (13) Takada, Y.; Nakayama, K.; Yoshida, M.; Sakairi, M. Rapid Commun. Mass Spectrom. 1994, 8, 695. (14) Takada, Y.; Nakayama, K; Yoshida, M.; Sakairi, M. Anal. Sci. 1994, 10, 713.
100
33 (CH30H) H+
0
(a)
1 10
50 mlz 23 Na+
(b)
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C
0 C
0 0
1 50
100
m/z Flguro 2. Background mass spectrum obtained with (a) the APCI mode and (b) the ESI mode. A 20 mM sodium phosphate buffer was used.
maximum ion intensity using caffeine as a model compound. Consequently, the flow rate of the sheath flow was set at 5 pW min. The background mass spectrum obtained with the APCI mode was very simple, and a protonated methanol peak (m/z = 33) was dominant. However, sodium ions (m/z = 23) were strongly indicated in the ESI mode. We must also point out that there were no differences in the background mass spectrum in the APCI mode with concentrations of up to 40 mM sodium phosphate in the CE buffers. We expect the APCI mode to be a powerful technique for analyzing samples that have strong proton af6nity in the gas phase such as amines, amino acids, antibiotics, and so on. In the APCI mode, sample molecules are ionized by corona discharge followed by ion/molecule reactions such as M SH+ MH+ S (M, sample molecule; S, solvent molecule). Since the solvent molecules in this work are methanol (see Figure 2), samples with stronger proton affinity than methanol will be easily ionized by the ion/molecule reactions. It is not clear why sodium ions cannot be detected with the APCI mode although we used the electrospray-type nebulizer. However, these salts in the buffer solution may be converted to gas phase ions by the electrospray-type nebulizer and neutralized at the stainless steel block vaporizer of the APCI interface. We investigated the innuence that salts in the CE buffers had on observed ion intensities using caffeine as a model compound. Figure 3 shows the relationship between relative intensities of the protonated caffeine molecules obtained with a selected ion monitoring mode and the concentration of sodium phosphate in the CE buffers. Analytical conditions and samples were the same for both the APCI mode and the ESI mode except for the sheath flow (the sheath liquid used for the ESI mode was composed of 1%formic acid in 50:50 water/methanol and delivered at a flow rate of 2 pL/min). The observed ion intensity without salts in the CE buffers was arbitrarily set at 100 for each mode. In the APCI mode, the ion intensities were not heavily affected by the
+
-
+
Analytical Chemistry, Vol. 67,No. 8, April 15, 1995
1475
(M+H)
p c1 Q)
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.-0 v)
E
-t
c)
\
1
i
ESI
0
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1
2 3 Time (min)
4
5
0
i
2
3 Time (min)
4
5
I
\
'
l
+
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Concentration (mM) Flgure 3. Relationshipbetween relative intensitiesof the protonated caffeine molecules and the concentration of sodium phosphate in the CE buffers obtained with the APCI mode and the ESI mode. The observed ion intensity without salts in the CE buffers was arbitrarily set at 100 for each mode.
sodium phosphate salts in the CE buffers. On the other hand, protonated caffeine molecules could not be observed when the 20 mM phosphate buffer, which is commonly used as a separation medium for CE, was used for the ESI mode. It is well-known that the ionization process of the electrospray is degraded by the presence of contaminant ions.I5 Therefore, buffer compositions available for CE/ESI-MS are limited to buffers with low concentrations of salts, and the high resolving power of CE has not been sufficiently utilized for CE/MS analysis. The results mentioned above show that the APCI mode will be more useful than the ESI mode when CE buffers include high concentrations of nonvolatile salts because the APCI process is not heavily affected by these salts, allowing the use of a variety of CE buffers in CE/MS analysis. As a result, we have succeeded in obtaining protonated caffeine molecules (m/z = 195) using CE/ MS with a phosphate buffer. Figure 4 shows the ion electropherogram of caffeine obtained with (a) the APCI mode and (b) the ESI mode; in both cases a 20 mM phosphate buffer was used. The amounts injected (2 pmol) generated a good signal/noise
Figure 4. Single ion electropherogram of caffeine obtained with (a) the APCI mode and (b) the ESI mode. A 20 mM sodium phosphate buffer was used, and the m/z was set at 195.
ratio with the APCI mode, although no ion peak was observed with the ESI mode. When the buffer contained no salts, signal intensity of the protonated caffeine molecules with the ESI mode was 3 times higher than that of the APCI mode. This is mainly because of large dead volumes in the APCI interface, which was primarily designed for LC/MS. The decrease of ion intensity caused by clogging of the apertures due to nonvolatile salts in CE buffers was not observed at for at least 1 day (operation time of 30 min x 10 runs). In conclusion, CE/APCI-MS is a very promising technique because the APCI process is not heavily affected by salts in CE buffers. This allows the use of a wider variety of CE buffers in CE/MS analysis. Further work will aim at obtaining higher sensitivity and coupling between MEKC and MS. Received for review December 5, 1994. February 9, 1995.@ AC9411740 @
1476 Analytical Chemistry, Vol. 67, No. 8, April 15, 1995
Accepted
Abstract published in Advance ACS Abstracts, March 1, 1995.
