Anal. Chem. 2004, 76, 6456-6460
Porous Polymer Monolith Assisted Electrospray Terry Koerner, Kiera Turck, Laurie Brown, and Richard D. Oleschuk*
Department of Chemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6
Coupling low-flow analytical separation instrumentation such as capillary electrophoresis, capillary electrochromatography, nano-HPLC, and microfluidic-based devices with electrospray ionization mass spectrometry has yielded powerful analytical tools. However, conventional coupling methodologies such as nanospray suffer from limitations including poor conductive coating robustness, constant clogging, complicated fabrication processes, and incompatibility with large flow rate regimes. This study demonstrates that robust nanospray emitters can be fabricated through the formation and utilization of a porous polymer monolith (PPM) at the end of a fused-silica capillary. Stable electrosprays can be produced from capillaries (75-100-µm i.d.) at a variety of flow rates (50-1000 nL/ min) without the need to taper the capillaries by etching or pulling. The PPM is photopatterned to be present only near the capillary exit aperture using conditions that generate pore sizes similar to those seen with nanospray tips. The porous nature of the PPM aids in developing a stable electrospray generating a single clearly visible Taylor cone at relatively high flow rates while at low flow rates (18 MΩ Milli-Q water (Millipore, Bedford, MA). Butyl acrylate, ethylene glycol dimethacrylate, 1-propanol (HPLC grade), 1,4-butanediol, 3-(trimethoxysilyl)propyl methacrylate, 2-acrylamido-2-methyl-1-propanesulfonic acid, benzoin methyl ether, O-(2-aminopropyl)-O′-(2-methoxyethyl)polypropylene glycol 500 (PPG), and cytochrome c were all purchased from Sigma-Aldrich (Oakville, ON, Canada) and used without purification. Glacial acetic acid and methanol (HPLC grade) were purchased from Fisher Scientific (Ottawa, ON, Canada) and were also used without purification. Preparation of Polymer Monolith. Fused-silica capillaries were obtained from Polymicro Technologies (Phoenix, AZ) and were supplied with either a UV-transparent coating (360-µm o.d., (11) Figeys, D.; Ning, Y.; Aebersold, R. Anal. Chem. 1997, 69, 3153-3160. (12) Bings, N. H.; Wang, C.; Skinner, C. D.; Colyer, C. L.; Thibault, P.; Harrison, J. D. Anal. Chem. 1999, 71, 3292-3296. (13) Meng, Z.; Qi, S.; Soper, S. A.; Limbach, P. A. Anal. Chem. 2001, 73, 12861291. (14) Xue, Q.; Foret, F.; Dunayevskiy, Y. M.; Zavracky, P. M.; McGruer, N. E.; Karger, B. L. Anal. Chem. 1997, 69, 426-430. (15) Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 1997, 69, 1174-1178. (16) Licklider, L.; Wang, X.; Desai, A.; Tai, Y.; Lee, T. D. Anal. Chem. 2000, 72, 367-375.
75-µm i.d.) or supplied with a polyimide coating (360-µm o.d., 100µm i.d.). In the case of the polyimide-coated capillary, a small portion of the coating needed to be removed with a flame to create a UV-transparent window to enable the activation of the radical initiator used in the polymerization reaction. For PPM fabrication, the inside of the capillaries was first derivatized with 3-(trimethoxysilyl)propyl methacrylate to provide sites needed to anchor the polymer monolith to the capillary wall.17 Briefly, the capillary was first washed with 1 M NaOH for 30 min at a flow rate of 1.0 µL/ min; if not stated, all flow rates for the preparation of the capillaries were set to 1.0 µL/min, using a model 11 55-1199 syringe pump (Harvard Apparatus, Saint-Laurent, PQ, Canada). The capillary was then rinsed with water for 30 min and afterward with a silane solution, consisting of 1 mL of 3-(trimethoxysilyl)propyl methacrylate in 20 mL of a 5 mM acetic acid solution, for 60 min. The capillary was then rinsed for 30 min with the ternary porogenic solvent mixture that was to be used for the preparation of the polymer monolith, which consisted of 10 wt % water and 90 wt % of a mixture of 1-propanol (60 wt %) and 1,4-butanediol (40 wt %). At this point, the capillaries could be stored in the porogenic solvent until they were needed. A monomer mixture was prepared in a manner similar to Peters et al.18 by combining butyl acrylate (59.4 wt %), ethylene glycol dimethacrylate (40.0 wt %), 2-acrylamido-2-methyl-1-propanesulfonic 0.6 wt %), and benzoin methyl ether (0.5% with respect to the monomer wt). A prepolymerization mixture, consisting of porogenic solvent (60 wt %) and the monomer mixture (40 wt %), was flushed through a pretreated capillary for 5 min and then the capillary was placed under the UV light to initiate the polymerization reaction. To control the region of polymerization, all UV-transparent areas of the capillary were masked where the PPM was not desired. The polymerization process was monitored by observation under a microscope. After the polymerization was complete (10-30 min), the capillary was flushed with porogenic solvent for 30 min. Capillaries were stored in the porogenic solvent and then simply rinsed with a buffer solution before use. Instrumentation. All experiments were performed on an API 3000 triple quadrupole mass spectrometer (MDS-Sciex, Concord, Canada) fitted with a nanoelectrospray source (Proxeon, Odense, Denmark) consisting of a x-y-z stage and two CCD camera kits to aid in the positioning of the capillary. A micro-Tee union (Scientific Products, Toronto, ON, Canada) was used to couple the solution transfer line, the electrospray capillary, and the electrode necessary to supply the electrospray voltage. A syringe was filled with the solution to be analyzed and fitted to the transfer line of the micro-Tee union. The entire assembly was fixed to the x-y-z stage, and the capillary was directed to the entrance of the mass spectrometer with the aid of CCD cameras. In most experiments, the capillary was maintained ∼5 mm from the orifice of the MS. The ES voltage was supplied through a liquid junction by connecting the MS power supply to a platinum electrode inserted within the micro-Tee, Figure 1. All spectra were acquired in positive ion mode with a scan range of 300-1200 m/z at 1 Hz with a flow of room-temperature nitrogen curtain gas set at 1 L/min. Samples of a PPG standard (0.13-1.3 µM) and cytochrome (17) (a) Hjerte`n, S. J. Chromatogr. 1985, 347, 191-195. (b) Schweitz, L.; Andersson, I.; Nilsson, S. Anal. Chem. 1997, 69, 1179. (18) Peters, E. C.; Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1998, 70, 2288-2295.
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Figure 1. Schematic diagram representing our instrumental nanoelectrospray configuration. Expanded regions represent the liquid junction, the ESI open tube, and PPM-enhanced capillaries. SEM inset picture shows the PPM at the end of the capillary and the small pores to act as nanospray emitters.
c (1.0 µM) were prepared in a solution consisting of 50% aqueous methanol and 1% acetic acid with respect to the total volume and were diluted to desired concentration before analysis. RESULTS AND DISCUSSION Given the analytical potential of a robust interface between the mass spectrometer and low-flow separation techniques such as nanoLC, CE, and microfluidic devices, many academic and industrial research groups have been actively pursuing such couplings. For nanoLC and CE applications, the coupling has focused on the development of an interface that possesses a stable conductive coating or liquid junction to apply the electrospray voltage to a nanospray capillary.19 We have developed a promising method to facilitate the coupling between microfluidic, nanoLC, and CE applications to the mass spectrometer that relies on the simple formation of a PPM at the end of a capillary or microfluidic device. PPMs have been used extensively in chromatographic applications because of the ease with which they can be prepared and the facile way in which their physical properties, such as pore size and hydrophobicity, can be modified.18 The small pores in the PPM (inset, Figure 1) can divide the flow in the capillary into smaller streams creating the potential of multiple electrospray emitters. Others have shown that multiple electrospray ionization (ESI) orifices are beneficial on a larger scale and for larger flow rates,20 with an array of polymeric orifices that create smaller Taylor cones. The reduced size of the Taylor cones will produce smaller droplets that have a larger surface area-to-volume ratio, increasing the analyte concentration at the droplet surface, and ultimately requiring less desolvation. A stable electrospray involves balancing both the surface tension of the solution and the Coulombic forces of the applied electric field. To reduce the hydrophilic surface area of fused silica, others have used pulled capillaries or a hydrophobic surface treatment to reduce the wetting characteristics of fused silica.14,20 but the introduction of a large hydrophobic surface, created by the PPM, may produce a similar effect and improvement in the electrospray process. The validity of PPMs (19) Gelpi, E. J. Mass Spectrom. 2002, 37, 241-253. (20) Tang, K.; Lin, Y.; Matson, D.; Kim, T.; Smith, R. Anal. Chem. 2001, 73, 1658-1663.
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introduced into the capillary as ESI emitters was determined using fused-silica capillaries because a variety of inner diameters and PPM formulations could be prepared and tested inexpensively. Also capillaries were chosen with a sufficiently large outer diameter to mimic the large hydrophilic surface area of a typical glass microfluidic device (inset, Figure 1). The capillaries were masked off except for a small portion (1-2 mm) at the end to limit monolith formation to that region. The monomer solution was introduced into the capillaries via syringe, and then the capillary was placed under UV light to activate the polymerization. Polymerization conditions were chosen from the work of Peters et al.18 to form pores approximately 500 nm-1.0 µm in size. After the polymerization was complete, the capillaries were rinsed, using a syringe pump, to remove any unreacted material and then fitted into the liquid junction of the electrospray source, Figure 1. Given in Figure 2 are the electrospray results obtained from a constant infusion of a PPG solution (125 nM) at a variety of flow rates using a 75-µm-i.d. capillary with a PPM formed at the end. It can be seen from these results that an efficient electrospray can be obtained at a variety of flow rates with a PPM present at the end of the capillary. The stability of the electrospray is quite good even at flow rates lower than conventional electrospray processes (RSD ) 3%, 800 nL/min) and is only slightly less stable at flow rates nearing those obtained for conventional nanospray conditions (RSD ) 8%, 50 nL/min). This stability has enabled the acquisition of mass spectra with excellent signal-to-noise values with only a few femtomoles of material consumed, Figure 2. In addition, the intensity of the total ion current (TIC) remains relatively constant over the various flow rates studied, which is consistent with electrospray theory.3 Furthermore, flow rates of ∼50 nL/min are similar to those developed electroosmotically in microfluidic devices11 and show the potential of this method in microfluidic applications. These encouraging results were not exclusive to PPG standard samples but were also obtained for protein sample solutions at low concentration. Given in Figure 3 are the results obtained from a continuous infusion of a solution of cytochrome c (1.0 µM, 50% water/50% methanol with 1% total acetic acid). The results are consistent with those obtained from the PPG samples and show that a stable electrospray can be produced even at relatively low flow rates yielding good spectra (S/N ) 33) with very little sample consumed (∼30 fmol). The PPM-assisted electrospray is quite robust, allowing for little loss in performance even when the voltage is removed and then reapplied, Figure 4A. Following removal of the ESI voltage and its subsequent reapplication, the ion current was reestablished with minimal recovery time. Surface wetting is a significant problem when spraying directly from a microchip or open tube capillary. Large droplet formation at the end of capillary or microfluidic device has been shown to severely degrade separation performance resulting from large Taylor cone volumes. However, the presence of the hydrophobic PPM at the end of the capillary appears to limit surface wetting, causing the electrospray to emanate from the bore of the capillary only (Figure 4B) and not from the entire surface of the capillary end. This phenomenon resulted only at flow rates 200 nL/min and below. Above 200 nL/min, a Taylor cone that wetted the entire surface of the capillary end resulted.
Figure 2. TIC at a variety of flow rates obtained with a 75-µm-i.d. capillary for the constant infusion of a PPG solution (125 nM) with an applied voltage of +3.0 kV (A-D). The representative mass spectrum (E) was generated from averaging 6 s of the TIC (D) and represents ∼3 fmol of material. The expanded region around the peak at m/z 599.0 (F) shows excellent signal-to-noise ratio for this small amount of material.
Figure 3. TIC and representative mass spectrum of a PPM-assisted electrospray process of a constant infusion of cytochrome c (1.0 µM, 50% aqueous methanol with 1% acetic acid). These results were obtained with a 100-µm-i.d. capillary at a flow rate of 200 nL/min and an applied voltage of +3.0 kV. The mass spectrum shows good signal-to-noise ratio (S/N ) 33 for the peak at 883.9) with only 30 fmol of material consumed.
Figure 4. PPM-assisted electrospray TIC trace for constant infusion of PPG at 100 nL/min and applied voltage of 3.0 kV using a 100-µm-i.d. capillary. (A) shows the stability of the spray by turning the voltage off and back on to regenerate the electrospray process. (B) Photomicrograph showing the electrospray generated with a 100 nL/min flow rate, which produced a mist emanating from the capillary core rather than the usual Taylor cone.
The reproducibility of the fabrication method for the PPMpatterned capillaries was determined by comparing the electrospray performance for a series of three capillaries manufactured with the same monomer solution and polymerization conditions. Performance was assessed by directly comparing the TIC for a
1.25 µM PPG solution sprayed with each of the capillaries. Reproducibility in fabrication was quite good where capillaries made from the same monomer solution had little deviation (e8%) in both the TIC performance and signal-to-noise ratio. One of the difficulties in using a conventional nanospray capillary is the Analytical Chemistry, Vol. 76, No. 21, November 1, 2004
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Figure 5. Comparison of TIC obtained from an open tube (B, D) and PPM-assisted (A, C) electrospray at two capillary internal diameters (100- and 75-µm i.d.). The results were obtained with a constant infusion of PPG, 1250 nM in (A, B) and 125 µM in (C, D), at 200 nL/min and an applied voltage of +3.0 kV.
tendency for the small inner diameter (
