Asymmetrical Emitter Geometries for Increased Range of Stable

All experiments were performed using a P/ACE MDQ capillary electrophoresis system with a modified capillary cartridge (Beckman Coulter Inc., Brea, CA)...
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Anal. Chem. 2010, 82, 8377–8381

Letters to Analytical Chemistry Asymmetrical Emitter Geometries for Increased Range of Stable Electrospray Flow Rates E. Jane Maxwell, Xuefei Zhong, and David D. Y. Chen* Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1 Because electric field distribution is determined by emitter size and shape, sprayer tip geometry determines the optimum liquid flow rate that can be processed by the electrospray ionization interface. Electric field is the highest at the sharpest edge of an electrode; therefore, for a beveled tip, the field is highest at the very tip, and for a conventional symmetrically tapered tip, the field is the highest around the rim of the electrode. Electrospray performance as a function of flow rate was investigated using both continuous infusion and peak-based analysis. The sharpest symmetrical emitter gave the most stable electrospray ionization (ESI) at flow rates e0.10 µL/min, while beveled emitters provided significantly better performance at expanded flow rates up to 1 µL/min. The use of beveled emitters offers the potential for increased versatility in electrospray ionization interfaces. One of the challenges in designing effective interfaces for coupling liquid chromatography (LC) or capillary electrophoresis (CE) with mass spectrometry (MS) is that, in order to process effluent from a variety of separations, the interface must work over a wide range of flow rates. Particularly for capillary electrophoresis, these flow rates range from near 0 nL/min, when there is no electroosmotic flow (EOF) present, to a few hundred nanoliters per minute when there is a strong EOF. However, most electrospray emitters are designed to give optimal performance over a relatively narrow range of flow rates, determined primarily by the inner and outer diameters of the sprayer tip.1 When coupling electrospray ionization (ESI)-MS online to LC or CE, the flow rate of electrospray is forced to match that of the separation. As such, it is important to match the optimal flow rate and electrospray potential for a particular ESI emitter with the flow rate of the separation technique to maintain acceptable signal stability and sensitivity. This type of investigation is often carried out using a fixed value for the electrospray potential,2 which may not completely characterize the performance of the emitter. Another common practice is to adjust the spray potential at each flow rate in order to maintain a stable cone-jet electrospray, which * To whom correspondence should be addressed. Tel: 604 822 0878. Fax: 604 822 2847. E-mail: [email protected]. (1) Maxwell, E. J.; Chen, D. D. Y. Anal. Chim. Acta 2008, 627, 25–33. (2) Ishihama, Y.; Katayama, H.; Asakawa, N.; Oda, Y. Rapid Commun. Mass Spectrom. 2002, 16, 913–918. 10.1021/ac1017953  2010 American Chemical Society Published on Web 09/27/2010

has been generally regarded as the optimal electrospray regime for MS detection.3 However, more recent studies have brought this assumption into doubt, suggesting that pulsating modes of electrospray may provide better sensitivity than the cone-jet mode.4 When the flow rate is determined by the electrospray process, as in some nanoESI applications, the applied potential and the inner and outer diameters of the emitter appear to be the most significant determining factors.5,6 For fused-silica emitters, the outer diameter of the tip acts as the base for the Taylor cone, such that the wall thickness can significantly affect the optimal flow rate range.7 For emitters constructed from stainless steel, or other materials that have a lower wettability than silica, the base of the Taylor cone may be restricted to the inner diameter of the emitter orifice under low flow rate conditions,8,9 allowing them to operate at lower flow rates than fused silica emitters of similar dimensions. For both stainless steel and fused silica emitters, it has been demonstrated that reducing the wall thickness leads to better stability at lower flow rates.2,10 Sprayers with a beveled tip may provide an alternative to small diameter symmetrical emitters because of the sharp tip and the gradual increase in cross-section area. Her and coworkers demonstrated that beveled glass emitters provided improved spray stability at low flow rates.11-14 Although beveling does not affect the inner diameter of the emitter, it generates an electric field distribution that causes the Taylor cone to form at the sharpest point of the beveled surface. We (3) Page, J. S.; Kelly, R. T.; Tang, K.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2007, 18, 1582–1590. (4) Marginean, I.; Kelly, R. T.; Prior, D. C.; LaMarche, B. L.; Tang, K.; Smith, R. D. Anal.Chem. 2008, 80, 6573–6579. (5) Smith, K. L.; Alexander, M. S.; Stark, J. P. W. Phys. Fluids 2006, 18, 092104-092101-092104-092107. (6) Ryan, C. N.; Smith, K. L.; Alexander, M. S.; Stark, J. P. W. J. Phys. D: Appl. Phys. 2009, 42, 155504–155511. (7) Cech, N. B.; Enke, C. G. Mass Spectrom. Rev. 2001, 20, 362–387. (8) Shui, W.; Yu, Y.; Xu, X.; Huang, Z.; Xu, G.; Yang, P. Rapid Commun. Mass Spectrom. 2003, 17, 1541–1547. (9) Gibson, G. T. T.; Mugo, S. M.; Oleschuk, R. D. Mass Spectrom. Rev. 2009, 28, 918–936. (10) Schmidt, A.; Karas, M.; Du ¨ lcks, T. J. Am. Soc. Mass Spectrom. 2003, 14, 492–500. (11) Chen, Y. R.; Tseng, M. C.; Her, G. R. Electrophoresis 2005, 26, 1376–1382. (12) Tseng, W. C.; Chen, Y. R.; Her, G. R. Anal. Chem. 2004, 76, 6306–6312. (13) Tseng, M. C.; Chen, Y. R.; Her, G. R. Electrophoresis 2004, 25, 2084–2089. (14) Chang, Y. Z.; Chen, Y. R.; Her, G. R. Anal. Chem. 2001, 73, 5083–5087.

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Figure 1. Different shapes of stainless steel electrospray emitters investigated: symmetrically tapered electrospray needle (1), blunt tapered tip (2), sharp tapered needle (3), 30° bevel tip made from 2 (4), 45° bevel tip made from 2 (5), 35° bevel tip with a smaller surface area (6).

recently presented a CE-MS interface using a stainless steel hollow needle with a beveled sprayer tip for low flow rate CE operations.15 Observations of the Taylor cone varying in size in response to changes in flow rate have lead us to hypothesize that the beveled geometry may have the potential to provide stable ESI over a wider range of flow rates than symmetrical emitters. In the interest of quantifying the effect of emitter geometry, this work presents a comparison of electrospray performance as a function of flow rate for six different conductive emitters, including sharp, blunt, and beveled tips. EXPERIMENTAL SECTION Chemicals and Materials. Individual amino acid standards were purchased from Sigma-Aldrich (St. Louis, MO). Formic acid and methanol were purchased from Fisher Scientific (Nepean, ON, Canada). All chemicals were of analytical grade or better and were used without further purification. The background electrolyte of 0.2% (v/v) formic acid and 50% methanol in water was filtered through a 0.45 µm pore size membrane prior to use. A sample solution of 20 µM each of proline, threonine, isoleucine, and arginine was prepared by diluting the amino acid standards in the background electrolyte. This sample solution was used in all experiments. Fused silica capillary (75 and 50 µm ID, 365 µm OD) was purchased from Polymicro Technologies (Phoenix, AZ). The six custom microfabricated stainless steel electrospray needles studied are shown in Figure 1. Emitters 4 and 5 were modified in-house from #2 tips by grinding at an angle to achieve the desired bevels (30° and 45°, respectively). Emitter 6 was beveled during the microfabrication process in order to achieve a 35° angle with a significantly smaller tip surface area than

emitters 4 and 5. The dimensions of all needles are listed in Table 1. Instrumentation. All experiments were performed using a P/ACE MDQ capillary electrophoresis system with a modified capillary cartridge (Beckman Coulter Inc., Brea, CA). Time programs were created using Beckman Coulter’s 32 Karat software in order to carry out reproducible injections and infusions of the analyte solution. Detection was carried out using a Micromass Q-TOF-1 mass spectrometer (Waters, Milford MA) operating in TOF-MS mode. The potential and temperature of the MS inlet cone were set to 20 V and 100 °C, respectively. Data processing was performed using MassLynx 4.0 software. The standard Micromass ESI interface was removed and replaced by a CE-ESI-MS interface developed in our laboratory.15 For pressure infusion ESI-MS experiments, the orthogonal port of the tee union, normally used to deliver a modifier solution, was closed with a polyetheretherketone (PEEK) plug. The relationship of flow rate as a function of applied pressure was calibrated by measuring the time required for an analyte plug to reach the MS detector over a range of applied pressures. A linear best fit of the resulting plot was then used to determine the pressure required for a desired flow rate. Electric Field Calculation. Calculations of the electric field strength with various tip geometries and voltages were carried out using COMSOL Multiphysics 3.4 software (electromagneticselectrostatics module, COMSOL Inc., Los Angeles CA).16 Models were created using the measured dimensions of the emitter tips and of the mass spectrometer inlet. RESULTS AND DISCUSSION Continuous Infusion ESI-MS Evaluation Conditions. The sample solution containing four amino acids (20 µM each) was continuously infused while the flow rate was varied from 4 to 0.1 µL/min over 9 steps in 2 min intervals, while holding the electrospray potential constant. The potential at the capillary inlet was matched to the ESI potential in order to ensure that no electroosmotic flow was present during the experiments. The ion intensity during the second minute of each flow-rate step was used to calculate the mean and the standard deviation of the MS signal. This experiment was repeated at 0.2 kV intervals over the working range of electrospray potentials for each needle. The resulting contour plots of average signal and the signal-to-noise ratio (SNR) as a function of flow rate and electrospray potential are shown in Figure 2A,B, respectively. Because all of the tested amino acids, proline, threonine, isoleucine, and arginine showed similar trends; only the signals from arginine were plotted in Figure 2.

Table 1. Dimensions of the Stainless Steel Emitters Involved in the Investigation geometry 1 2 3 4 5 6

taper blunt taper sharp 30° bevel 45° bevel 35° bevel a

shaft diameter (mm)

orifice diameter (mm)

taper anglea (degrees)

bevel angleb(degrees)

face area (mm2)

0.64 0.72 0.72 0.72 0.72 0.72

0.08 0.10 0.05 0.10 0.10 0.08

16 14 14 14 14 16

0 0 0 30 45 35

0.01 0.05 0.007 0.04 0.07 0.01

With respect to needle cross-section. b With respect to needle axis.

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Figure 2. Average signal of arginine (A) and signal-to-noise ratio (B) as a function of flow rate and electrospray potential. Emitter geometry for each plot is indicated in the top right corner. Sample: 20 µM each of proline, threonine, isoleucine, and arginine in 0.2% formic acid and 50% methanol.

The limited working flow rate ranges of geometries 1 (tapered) and 2 (blunt taper) can be clearly seen, as neither provides a stable electrospray (SNR > 10) at flow rates greater

than 400 nL/min. The sharp tip (emitter 3) provided stable electrospray signals over a wider range of flow rates and was the only emitter for which SNR did not decrease at flow rates Analytical Chemistry, Vol. 82, No. 20, October 15, 2010

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Table 2. Relative Peak Height as a Function of Flow Rate for the Six Emitters Studied relative peak height (%)a geometry 1 2 3 4 5 6 a

taper blunt taper sharp 30° bevel 45° bevel 35° bevel

0.15 µL/min

0.40 µL/min

1.0 µL/min

ESI potential (kV)

maximum electric field (×107 V/m)

68 ± 6 46 ± 9 75 ± 15 98 ± 15 86 ± 6 100 ± 7

39 ± 9 39 ± 7 42 ± 7 72 ± 8 76 ± 5 69 ± 8

7±2 12 ± 4 13 ± 4 21 ± 5 36 ± 5 30 ± 8

3.2 3.8 3.0 3.6 3.4 3.2

1.5 1.4 1.9 1.7 2.0 1.8

± one standard deviation.

20 for flow rates from 100 to 1500 nL/min. The trend for these needles is also more predicable, with wide regions where signal remains high regardless of changes in flow rate or ESI potential and fewer regions where the spray behavior changes abruptly. The results do somewhat justify the previously mentioned common practice of evaluating ESI performance using only a fixed electrospray potential, since for several of the investigated tip geometries the best SNR across the range of flow rates can be achieved within a narrow range of ESI potentials. However, there are cases where the optimal potentials for high and low flow rates differ significantly. Due to the number of combinations of voltages and flow rates tested, the analysis of each tip geometry required an entire day. Therefore, the different tips were evaluated on different days, making it difficult to correct for possible interday drift of the mass spectrometer detector sensitivity. This is the rationale for including signal-to-noise ratio as the basis of comparison in Figure 2, in addition to raw signal. The results appear to support the use of the beveled needles when analytes are continuously introduced to the ESI source. However, the use of continuous infusion of analyte makes it impossible to differentiate between analyte and background signal at a particular m/z. Peak-Based ESI-MS Evaluation. In order to verify if the results obtained by continuous infusion are also applicable for transient analyte plugs, a peak-based evaluation was also carried out. A time program was created using the 32 Karat software in order to automatically inject and elute sample plugs at three different flow rates in an alternating fashion while carrying out ESI-MS detection. Sample plugs (10 s injection at 1.0 psi) were pushed through the capillary and into the sprayer tip using hydrodynamically generated flow rates of 1.0, 0.40, and 0.15 µL/min (3.9, 1.5, and 0.6 psi, respectively, in a 75 µm ID, 85 cm long capillary), with the optimal spray potential at each flow rate determined in advance. This cycle of three injections followed by hydrodynamic infusion at decreasing flow rates was (15) Maxwell, E. J.; Zhong, X.; Zhang, H.; Zeijl, N. v.; Chen, D. D. Y. Electrophoresis 2010, 31, 1130–1137. (16) Zhong, X.; Yi, R.; Holliday, A. E.; Chen, D. D. Y. Rapid Commun. Mass Spectrom. 2009, 23, 689–697.

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repeated in triplicate, such that nine peaks were detected for each needle. The average and standard deviation of the peak heights and areas were calculated for each flow rate. Results obtained within a single day were normalized, in order to allow interday comparisons regardless of instrumental drift, and the experiment was repeated over six days. Table 2 shows a comparison of the normalized peak heights for each tip at 0.15, 0.40, and 1.0 µL/min. Consistent with the findings of the continuous infusion experiments, all six tips showed the greatest sensitivity at the lowest flow rate examined. The three beveled tips also showed the greatest sensitivity over the entire flow rate range. There does not appear to be a statistically significant difference between the beveled tips at any of the flow rates investigated by this method. Additionally, the optimal electrospray potentials for the peak-based evaluation were, in some cases, slightly lower than those observed during the continuous infusion analysis. Electric Field Simulation Results. In order to better understand the results of the investigations, the electric field around the various emitter tips were calculated using COMSOL Multiphysics. Table 2 shows the maximum electric field (Emax) at the emitter tip, using the optimal ESI potentials from the peak-based evaluation. Because the electric field depends on both the applied potential and the sharpness of the conductive surface, the blunt tip (emitter 2) had the lowest electric field (Emax ) 1.4 × 107 V/m), despite having the highest ESI potential (3.8 kV). This is ∼30% less than emitter 5 (45° bevel, 3.4 kV), which had the highest Emax ) 2.0 × 107 V/m. However, there is no correlation between the results in Table 2 and Emax, since the sharp tip (emitter 3), which has the second highest value of Emax, showed poor performance similar to the blunt tip. It should be noted that the physical model used in the simulations is based on the sprayer tip geometry and does not take into account the thickness of the fluid layer and electrostatic properties of the Taylor cone or droplets. CONCLUDING REMARKS The different sprayer tips used for this investigation demonstrate that optimum solution flow rates for ESI are related to both the size and specific geometry of the sprayer. While the size of a tapered sprayer tip can directly influence the optimum flow rate it can process, beveled sprayer tips can generally sustain stable ESI over a wider range of volumetric flow rates. The limited geometries available for this investigation prevent us from characterizing more precisely the dimensions that are most important in choosing a successful geometry

for electrospray. There are also variables that contribute to ESI performance that are not within the scope of this investigation, particularly the properties of the solution, including chemical composition, viscosity, volatility, and surface tension, and the geometry of the MS inlet. However, our observations support the hypothesis that beveled tips can effectively increase the working flow rate range of an electrospray emitter, while offering performance similar to or better than that of symmetrically tapered emitters even at low flow rates.

ACKNOWLEDGMENT This work was supported by grants from Beckman Coulter Inc. (Brea, CA, USA) and the Natural Sciences and Engineering Research Council of Canada.

Received for review July 6, 2010. Accepted September 10, 2010. AC1017953

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