Anal. Chem. 2007, 79, 1564-1568
Microemulsion Electrokinetic Chromatography with On-Line Atmospheric Pressure Photoionization Mass Spectrometric Detection Markus Himmelsbach, Manuela Haunschmidt, Wolfgang Buchberger, and Christian W. Klampfl*
Institute of Analytical Chemistry, Johannes Kepler University, Altenbergerstrasse 69, A-4040 Linz, Austria
In the present paper we report, for the first time, the successful on-line coupling of microemulsion electrokinetic chromatography (MEEKC) with mass spectrometric (MS) detection using an atmospheric pressure photoionization interface. Microemulsions (MEs) including mostly volatile ingredients and classical MEs based on nonvolatile buffer components and sodium dodecyl sulfate (SDS) as surfactant were compared with respect to their compatibility with MS detection. The investigations performed revealed that MEs with up to 3% SDS and buffers containing sodium borate can be employed without significant suppression of the MS signals. A test mixture of nine substances could be separated by MEEKC using a ME consisting of 0.8% octane, 2% SDS, 6.6% butanol, and 90.6% of 20-mmol ammonium hydrogencarbonate buffer (pH 9.5). Operating the MS instrument in the MS2 mode provided improved signal/noise ratios for analytes leading to characteristic MS-MS transitions. Thereby, limits of detection ranging between 0.5 (carbamazepine) and 5 µg mL-1 (phenacetin) could be obtained. Micellar electrokinetic chromatography (MEKC)1,2 and microemulsion electrokinetic chromatography (MEEKC)3,4 are powerful electroseparation methods that combine electrophoretic separation principles with chromatographic ones, in particular the distribution of the analytes between the electrolyte and micelles (in the case of MEKC) or microemulsion (ME) droplets (in the case of MEEKC). By this, they allow extension of the field of application of electroseparation methods toward the analysis of uncharged analytes. In addition, the separation of substances showing identical ionic radius/charge ratios, which cannot be resolved using simple capillary electrophoretic techniques, can be achieved employing these two techniques. MEEKC can be seen as a further development of MEKC with the micelles being replaced by ME droplets. This leads to an increase in the size of the separation window as well as the ability to separate even highly hydrophobic * To whom correspondence should be addressed. E-mail: christian.klampfl@ jku.at. Fax: +43/732/2468/8679. (1) Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111-113. (2) Pappas, T. J.; Gayton-Ely, M.; Holland, L. A. Electrophoresis 2005, 26, 719734. (3) Watarai, H. Chem. Lett. 1991, 231, 391-394. (4) Altria, K. D.; Mahuzier, P. E.; Clark, B. J. Electrophoresis 2003, 24, 315324.
1564 Analytical Chemistry, Vol. 79, No. 4, February 15, 2007
compounds that would permanently remain in the micellar phase in MEKC.5 Therefore, the major field of application of MEEKC can be found in the fields of the analysis of nonpolar substances,6-8 but reports describing MEEKC separations of polar analytes can also be found in the literature.9 For a long time, a principal disadvantage of these two methods was their obvious incompatibility with mass spectrometric (MS) detection. Whereas the coupling of capillary electrophoresis with MS detection has faced increasing attention over the last 15 years, this trend is much less expressed in the case of the combination of electroseparation methods including pseudostationary phases like MEKC or MEEKC with MS. Focusing on MEEKC in particular, no report describing the combination of this electroseparation method with MS detection can be found in the literature up to now.10-12 Major reasons for that are the fact that these techniques are mostly used to separate analytes that are nonpolar and thereby not easily ionized employing the electrospray ionization (ESI) source, the frequent use of nonvolatile borate and phosphate buffers in MEKC and MEEKC, and even more problematic the need of charged or uncharged surfactants to form the micelles. The most widely employed surfactant in the case of negatively charged micelles or ME droplets is sodium dodecyl sulfate (SDS), a substance that is well-known for its strong suppression effect in ESI-MS.13 Whereas several approaches, including the use of volatile surfactants such as perfluorinated carboxylic acids,14 the partial filling method,15 and the use of high molecular mass surfactants,16,17 have been tested toward the coupling of MEKC with ESI-MS detection, no reports describing the hyphenation of MEEKC with MS can be found in the literature. (5) Klampfl, C. W. Electrophoresis 2003, 24, 1537-1543. (6) Hansen, S. H. Electrophoresis 2003, 24, 3900-3907. (7) Marsh, A.; Clark, B.; Broderick, M.; Power, J.; Donegan, S.; Altria, K. Electrophoresis 2004, 25, 3970-3980. (8) Huie, C. W. Electrophoresis 2006, 27, 60-75. (9) Siren H.; Karttunen A. J. Chromatogr., B 2003, 783, 113-124. (10) Schmitt-Kopplin, P.; Frommberger, M. Electrophoresis 2003, 24, 38373867. (11) Schmitt-Kopplin, P.; Englmann, M. Electrophoresis 2005, 26, 1209-1220. (12) Klampfl, C. W. Electrophoresis 2006, 27, 3-34. (13) Yang, L.; Lee, C. S. J. Chromatogr., A 1997, 780, 207-218. (14) Petersson, P.; Jo¨rnten-Karlsson, M.; Stalebro M. Electrophoresis 2003, 24, 999-1007. (15) Nelson, W. M.; Tang, Q.; Harrata, A. K.; Lee, C. S. J. Chromatogr., A 1996, 749, 219-226. (16) Ozaki, H.; Terabe, S. J. Chromatogr., A 1998, 794, 317-325. (17) Shamsi, S. A. Anal. Chem. 2001, 73, 5103-5108. 10.1021/ac061584b CCC: $37.00
© 2007 American Chemical Society Published on Web 01/20/2007
A major step forward was achieved with the introduction of the atmospheric pressure photoionization (APPI) source18,19 and its successful hyphenation with capillary electrophoresis,20,21 capillary electrochromatography,22 and finally also with MEKC.23,24 The fact that this ion source is relatively unsusceptible to suppression effects caused by either nonvolatile buffer ingredients such as borate or phosphate or surfactants such as SDS makes it the optimum choice for coupling MEKC with MS detection. This has been shown in some recent publications.23,24 Focusing on MEEKC, the starting point of its hyphenation with MS detection is even less favorable than in MEKC. First, the analytes separated with this technique are often highly nonpolar, leading to an insufficient ion yield in ESI-MS; second, the amount of SDS or other nonvolatile surfactants needed to form a ME is by far higher than the concentration usually employed in MEKC; finally, most MEs employed so far include borate or phosphate buffers. These might be some of the reasons why up to now no work reporting the successful combination of MEEKC with MS exists. In the present paper we describe, for the first time, the successful coupling of MEEKC with APPI-MS detection on the example of nine test substances,. Within this work, the effect of different types of ME, varying in composition with respect to surfactant and aqueous buffer used, on the signal intensities obtained for the selected analytes was investigated. EXPERIMENTAL SECTION Instrumentation. An Agilent 3D CE system (Agilent, Waldbronn, Germany), with a diode array detector was used for all experiments. MS detection was performed on an Agilent MSD SL ion trap mass spectrometer equipped with an APPI source. An Agilent G1607A coaxial sprayer was mounted onto the APPI source using a 36-mm APCI/APPI adapter (Agilent part G160768707). It should be noted that in this specific type of interface high voltages are applied to the MS orifice whereas the sprayer needle is grounded, a setup that greatly facilitates the coupling of electroseparation methods with MS. A syringe pump (model 22; Harvard Apparatus, South Natick, MA) was used to deliver the sheath flow and to perform infusion experiments. Materials and Reagents. Fused-silica capillaries (50 µm i.d. × 375 µm o.d.) obtained from Polymicro Technologies Inc. (Phoenix, AZ) were used throughout this work. New capillaries were cut to a total length of 90 cm and conditioned by flushing with 0.5 M NaOH for 10 min, followed by water, and then ME, each for 10 min. The following chemicals were employed in this study: ammonium hydrogencarbonate, sodium dodecyl sulfate, phenacetin (all Fluka, Buchs, Switzerland), ammonia solution, sodium hydroxide, boric acid, 1-butanol, 4-dimethylaminobenzaldehyde (DMAB) (all Merck, Darmstadt, Germany), pentadecafluorooctanoic acid, carbamazepine, quinoline, isoquinoline, (18) Robb, D. B.; Covey, T. R.; Bruins, A. P. Anal. Chem. 2000, 72, 3653-3659. (19) Syage, J. A.; Evans, M. D.; Hanold, K. A. Am. Lab. 2000, 32, 24-29. (20) Nilsson, S. L.; Andersson, C.; Sjoberg, P. J. R.; Bylund, D.; Petersson, P.; Jo ¨rnten-Karlsson, M.; Markides, E. Rapid Comunn. Mass Spectrom. 2003, 17, 2267-2272. (21) Mol, R.; de Jong, G. J.; Somsen, G. W. Electrophoresis 2005, 26, 146-154. (22) Zheng J.; Shamsi S. A. Anal. Chem. 2006, 78, 6921-6927. (23) Mol, R.; de Jong, G. J.; Somsen, G. W. Anal. Chem. 2005, 77, 5277-5282. (24) Somsen, G. W.; Mol, R.; de Jong, G. J. Anal. Bioanal. Chem. 2006, 384, 31-33.
Figure 1. Chemical structures of the analytes.
3-methylquinoline, 6-methylquinoline, metoprolol, and pentoxifylline (all Sigma-Aldrich, St.Louis, MO). All chemicals had a purity of >98% and were used with 18-MΩ Milli-Q purified water (Millipore, Bedford, MA). Methanol, toluene, and acetone were purchased from J.T. Baker (Deventer, The Netherlands). The buffers for the ME were prepared by dissolving 20 mmol of ammonium hydrogencarbonate or 20 mmol of boric acid in 1 L of 18-MΩ water and adjusting the pH to 9.5 with ammonia solution or sodium hydroxide solution, respectively. MEs were prepared as follows: 2 or 3 g of surfactant (SDS or pentadecafluorooctanic acid) and 6.6 g of 1-butanol were mixed. Subsequently, 0.8 g of oil phase (n-octane) and 90.6 or 89.6 g of buffer were added, and the mixture was placed in an ultrasonic bath for 30 min to obtain a clear solution. The resulting ME was filtered through a 0.45-µm membrane filter before use. All analytes were dissolved in methanol giving 10 mg mL-1 stock solutions. Working standards were prepared daily prior to use in ME for MEEKC experiments and in sheath liquid with 1% ME for infusion experiments. Chemical structures of the substances included in the test mixture selected for this study are depicted in Figure 1. MEEKC-APPI-MS Conditions. The microemulsion systems with 2% SDS were used as background electrolytes for MEEKCAPPI-MS experiments. A separation voltage of +30 kV was used. Sample injection was performed by application of a sequence of 10 s 50 mbar of the sample followed by 10 s 50 mbar of the background electrolyte. The capillary temperature was thermostated to 25 °C. APPI-MS was carried out in the positive ion mode. The vaporizer temperature was set to 200 °C, the drying gas was operated at 180 °C and 2 L min-1, and the nebulizer pressure was set to 10 psi. The CE was coupled to the MS instrument (operated in the positive-ion modes throughout this work) via a coaxial sheath flow interface employing a sheath liquid consisting of methanol/water/acetone (75/25/5) at a flow rate of 15 µL min-1. All substances were detected as protonated species [M + H]+. Analytical Chemistry, Vol. 79, No. 4, February 15, 2007
1565
After each day of work, a white precipitate (mainly SDS) had to be removed from the MS spray shield. Apart from that, no further extra cleaning operations were necessary. RESULTS AND DISCUSSION ME and Sheath Liquid Composition. First, infusion experiments were performed to investigate the influence of different MEs as well as sheath liquid compositions on the MS signals obtained for the chosen solutes. For this reason, the analytes were dissolved in the ME and diluted 100-fold with sheath liquid, a factor that comes close to the situation encountered in MEEKC when a sheath liquid flow rate of 10-15 µL min-1 is employed. The final concentration was 2 µg mL-1 for each solute, and the infusion rate was set to 15 µL min-1. Starting point was a ME that was specially adapted toward maximum compatibility with MS detection, in particular by using a volatile buffer (ammonium carbonate) and perfluorinated octanoic acid as surfactant. Subsequently, these volatile ingredients were exchanged by the nonvolatile species employed in the commonly used ME compositions step by step. This led to MEs including 2 or 3% SDS and sodium borate or ammonium carbonate as buffer component. These investigations revealed that all tested ME compositions were compatible with APPI-MS and only minor differences were observed during the infusion experiments when the ME composition was changed. When APPI is used for ionization, signals can be increased significantly by adding so-called dopants.18 Acetone and toluene are most frequently used to enhance the ionization of analytes in APPI-MS, although reports describing the use of other substances for this purpose, such as anisole,25 can be found in the literature. The easiest way to introduce the dopant in the case of MEEKCAPPI-MS is to add it to the sheath liquid. Amounts of 5% acetone or toluene were added to the sheath liquid (methanol/water 75/ 25), and their effect on the signal intensities obtained was investigated. The following conclusions could be deducted from these experiments: first, without a dopant, the capillary voltage had to be optimized carefully to achieve maximum signal intensities, while in the case of a sheath liquid including either acetone or toluene, only minor differences in intensities over a wide range of MS capillary voltages were observed; second, for several analytes, signal intensities obtained with the sheath liquid including a dopant were 1.6 (DMAB) to 2.5 (carbamazepine, pentoxifylline, phenacetin) times higher than those without dopant. For the quinolines and metoprolol, identical maximum peak intensities were achieved with and without the dopant. Figure 2 shows a typical plot displaying the dependence of signal intensities on the MS capillary voltage applied with and without a dopant on the example of phenacetin. Similar observations have already been reported in several earlier papers.26-28 A possible explanation for the advantage of dopant-assisted APPI over dopant-free ionization can be that ionized species are produced over a larger volume if a dopant is present. As a consequence of this, protonated analyte (25) Kauppila, T. J.; Kostiainen, R.;. Bruins A. P. Rapid Comunn. Mass Spectrom. 2004, 17, 808-815. (26) Takino M.; Daishima S.; Nakahara T. J. Chromatogr., A 2003, 1011, 6775. (27) Takino M.; Daishima S.; Nakahara T. Rapid Comunn. Mass Spectrom. 2003, 18, 383-390. (28) Hanold, K.; Fischer, S. M.; Cormia, P. W. H.; Miller, C. E.; Syage, J. A. Anal. Chem. 2004, 76, 2842-2851.
1566 Analytical Chemistry, Vol. 79, No. 4, February 15, 2007
Figure 2. Effect of the MS capillary voltage on the signal intensity obtained for phenacetin using a sheath liquid with 5% acetone as dopant (2) and without dopant (9).
molecules may also be distributed over a larger volume, making their transfer into the MS less dependent from optimum MS capillary voltage parameters. Anyhow, more in-depth investigations (that would exceed the scope of this paper) on this issue would be necessary before a definite explanation for this phenomenon may be provided. Comparing the performance of the two dopants employed, acetone proved to be the better choice for all analytes except pentoxifylline with signal intensities that were on average 18% higher than those obtained with toluene. APPI Source Parameters. Proper vaporization and nebulization of the eluate is required before the sample molecules can be ionized by the high-energy photons from the krypton lamp. Other parameters affect the efficiency of their transport into the mass analyzer. For this reason, the optimum values for the following parameters were determined by infusion experiments using sheath liquid containing 1% ME and 2 µg mL-1 concentrations of each analyte: vaporizer temperature, drying gas flow, and drying gas temperature. First, the influence of vaporizer temperatures in the range of 160-310 °C on the signal intensities obtained for the nine test solutes was analyzed. Up to a temperature of 200 °C, a strong increase in signal intensities was observed for all solutes, followed by a slight decrease above a temperature of ∼240 °C. Optimum values were found to be 2 L min-1 and 180 °C. MEEKC-MS and MEEKC-MS/MS Experiments. In the next step, actual MEEKC-MS experiments were performed on the basis of the findings from the infusion experiments. Two additional aspects had to be taken into account. First, ME including a higher percentage of SDS led to high currents. Due to the current limitation given for the CE-APPI interface (maximum 50 µA), reduced separation voltages resulting in unfavorably long analysis times had to be employed. For this reason, only ME with 2% SDS was used for the MEEKC-APPI-MS experiments. Second, in MEEKC, organic modifiers often are added to manipulate the distribution behavior of the analytes between the ME droplets and the aqueous phase.5 During experiments employing MEs with different types of organic modifiers, frequent clogging of the separation capillary was observed. This may be explained by the increased volatility of the aqueous phase in the presence of organic solvents, leading to a precipitation of the surfactant and buffer ingredients due to the high temperatures present in the APPI vaporizer. Therefore, only MEs without organic modifier could
Figure 3. MEEKC-APPI-MS extracted ion electrochromatograms of a standard solution containing 100 µg mL-1 of each analyte. Experimental conditions: ME, 0.8% n-octane, 2% SDS, 6.6% butanol, 90.6% 20 mM ammonium hydrogencarbonate (pH 9.5); injection pressure, 50 mbar 10 s sample followed by 50 mbar 10 s ME; separation voltage, + 30 kV, detection, positive ion mode scan; sheath liquid flow rate, 15 µL min-1; sheath liquid, methanol/water/acetone (75/25/5). Peak assignments see Figure 1.
be employed throughout this study. A principal consequence from this behavior is the fact that no highly nonpolar substances that would not partition between the ME droplets and the aqueous phase were included in the test mixture investigated in this work. The nebulizer pressure is an important parameter with respect to both ionization efficiency for the solutes and stability of the MEEKC system. For this reason, this parameter was optimized within the confines of 5 and 15 psi. If the nebulizer pressure is too low, the spray and the transport of the compounds toward the MS orifice may not be within the optimum range, resulting in less favorable signal/noise ratios. On the other hand, if the pressure is too high, the contact between the CE capillary and the sheath liquid can be disturbed, causing problems with the MEEKC current. The application of a nebulizer gas flow also results in a suction effect at the end of the separation capillary, inducing a hydrodynamic flow leading to peak broadening and reduced chromatographic resolution. Optimum signal intensities were observed with a nebulizer pressure set to 10-15 psi. Because at 15 psi peak broadening and a loss of resolution were observed, a nebulizer pressure of 10 psi was selected for the MEEKC-APPIMS experiments. Higher values such as 25 psi resulted in an unstable current, making proper MEEKC runs impossible. The sheath liquid flow rate was investigated in a range from 10 to 25
µL min-1. From 10 to 15 µL min-1, the signal intensity increased for all analytes, whereas they remained stable or decreased slightly from 15 to 25 µL min-1. Therefore, the sheath liquid flow rate was set to 15 µL min-1. MEs including 2% SDS and aqueous buffers based on sodium borate and ammonium carbonate were employed for the separation of the selected test mixture. No significant differences with respect to signal intensities were observed, but the use of ammonium carbonate resulted in improved peak shapes and a better resolution whereas the ME including the borate buffer provided a somewhat shorter analysis time. In Figure 3, an MEEKC-APPI-MS run using the carbonate-based ME is depicted. As can be seen from this figure, separation of all analytes was possible and sufficient signal/noise ratios with limits of detection (3× baseline noise) in the range of 7 (DMAB) and 50 µg mL-1 (carbamazepine) could be obtained for all compounds under investigation. Further improvement of the signal/noise ratio is possible when the instrument is operated in the MS2 mode. As no characteristic MS2 transitions could be observed for the quinolines, this class of compounds was excluded from the MS2 experiments. Figure 4 shows a MEEKC-APPI-MS2 electrochromatogram obtained for a set of five analytes. Using this approach, limits of detection between 0.5 (carbamazepine) and 5 µg mL-1 Analytical Chemistry, Vol. 79, No. 4, February 15, 2007
1567
Figure 4. MEEKC-APPI-MS2 extracted ion electrochromatograms of a standard mixture containing 10 µg mL-1 pentoxifylline, phenacetin, DMAB, metoprolol, and carbamazepine. For experimental conditions, see Figure 3. For peak assignments, see Figure 1.
(phenacetin) could be obtained. Variations in retention times and peak areas (n ) 4) were less than 5% for retention times and between 1.4 (pentoxifylline) and 19% (phenacetin) for peak areas. CONCLUSIONS The investigations described in this paper demonstrate that the on-line coupling of MEEKC and MS detection is possible. Comparison of MEs especially designed with respect to MS compatibility (all ingredients are volatile) and classical MEs based on nonvolatile buffers and up to 3% SDS revealed that even with the use of the latter type of MEs no negative effects such as ion suppression and source fouling are encountered when the APPI source was employed. Further investigations on the applicability of our approach for other types of MEs that are less commonly employed (different surfactants, oil-phase buffer systems) would also be of interest and may be the subject of future work. Some restrictions originate from the current limitation imposed by the CE-MS equipment employed and the need to use MEs without organic modifiers to avoid frequent capillary clogging. The first obstacle can be overcome either by the approach employed in this study, namely, the use of MEs with a maximum of 2% SDS,
1568 Analytical Chemistry, Vol. 79, No. 4, February 15, 2007
or by modification of the interface allowing a higher current limit. Whereas the current limitation of these interfaces is sufficient for most CZE-MS applications, demand for such modified interfaces may be stimulated by further work in the fields of MEKC-APPIMS or MEEKC-APPI-MS. Problems encountered with MEs including organic modifiers should be further investigated, as the need to use MEs with solely aqueous buffers significantly restricts the choice of analytes approachable by this new methodology. Signal/noise ratios obtained for the selected analytes were acceptable in the MS mode and could significantly be enhanced using the MS2 mode for analytes providing characteristic MSMS transitions. ACKNOWLEDGMENT The authors thank Dr. Gerard Rozing, Agilent Technologies (Waldbronn, Germany) for the loan of an APPI source.
Received for review August 24, 2006. Accepted December 19, 2006. AC061584B
