Design and Optimization of a Corona Discharge Ion Source for

line tip and discharge needle relative positioning, curtain gas and makeup gas flow interactions, ion chamber temperature, and elution pressure of ana...
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Anal. Chem. 1996, 68, 1924-1932

Design and Optimization of a Corona Discharge Ion Source for Supercritical Fluid Chromatography Time-of-Flight Mass Spectrometry Iulia M. Lazar and Milton L. Lee*

Department of Chemistry, Brigham Young University, Provo, Utah 84602-4672 Edgar D. Lee

Sensar Corporation, 1652 West 820 North, Provo, Utah 84601

The interfacing of capillary column supercritical fluid chromatography (SFC) to time-of-flight mass spectrometry (TOFMS) through atmospheric pressure chemical ionization (APCI) was investigated. An ion source chamber and a new, flexible, and efficient transfer line from the SFC to the TOFMS system were designed to accommodate the requirements of this study. Ionization of analytes was performed using a corona discharge needle. The interface was equipped with two multiple-axis translation stages for positioning of the transfer line tip and the discharge needle inside the ion chamber. The investigations were oriented toward the optimization of parameters which have a strong effect on the intensity and stability of the analyte signal, including background stability, corona discharge needle positioning in the ion source, transfer line tip and discharge needle relative positioning, curtain gas and makeup gas flow interactions, ion chamber temperature, and elution pressure of analytes from the SFC system. Supercritical fluid chromatography (SFC) is a well-suited technique for the separation of high molecular weight and thermally labile compounds. The continuous growth in need for detailed and accurate information about the nature and structure of separated compounds from chromatographic methods requires the development of sophisticated analytical detection tools. The mass spectrometer, with its high sensitivity and selectivity, has become the preferred chromatographic detector. On-line coupling of SFC to a variety of mass spectrometers has been described in numerous papers. Several types of interfaces have been reported in the literature, some being modified LC-MS interfaces and some being specially developed for SFC-MS. SFC-MS interfacing started with the molecular beam interface,1 as described by Randall and Wahrhaftig. Further developments included direct fluid introduction,2,3 moving belt,4 thermospray,5 and particle beam6 interfaces. Electron impact and chemical ionization, including charge exchange, spectra were obtained. As an alterna(1) Randall, L. G.; Wahrhaftig, A. L. Anal. Chem. 1978, 50, 1703. (2) Smith, R. D.; Felix, W. D.; Fjeldsted, J. C.; Lee, M. L. Anal. Chem. 1982, 54, 1883 (3) Smith, R. D.; Udseth, H. R. Anal. Chem. 1987, 59, 13. (4) Berry, A. J.; Games, D. E.; Perkins, J. R. J. Chromatogr. 1986, 363, 147. (5) Berry, A. J.; Games, D. E. ; Mylchreest, I. C.; Perkins, J. R.; Pleasance, S. Biomed. Environ. Mass Spectrom. 1988, 15, 105. (6) Edlund, P. O.; Henion, J. D. J. Chromatogr. Sci. 1989, 27, 274-282.

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tive ionization method, SFC-MS with fast atom bombardment (FAB) has also been reported.7 Along with further improvements in the above-mentioned techniques, recent papers have described SFC-MS interfacing with atmospheric pressure chemical ionization.8-15 Ionization of the analytes was achieved using either a corona discharge needle8-12 or electrospray.13,15 APCI has three important advantages in SFC-MS interfacing over traditional, lowpressure, high-vacuum ionization systems. First, the mass spectrometer vacuum system does not have to handle the continuously increasing mobile-phase flow rates delivered during an SFC pressure or density program. Second, heat transfer to the SFC capillary restrictor can be better achieved at higher ion source pressures. Third, the SFC-MS experimental setup is much simplified, allowing relatively independent operation of the SFC and MS systems, including easy column and restrictor changing without affecting the MS operation. In this paper, we report the development of an on-line SFC-TOFMS interface and discuss some preliminary results obtained during optimization studies. Since the time-of-flight mass spectrometer used in this study is a commercial device and was designed specifically for permanent gas analysis, the present research was conducted using rather low molecular weight compounds instead of compounds typically analyzed by SFC. As a consequence, we focused the research undertaken here on phenomena specific to the ion source which are not greatly affected by the molecular weight of the analytes. EXPERIMENTAL SECTION Reagents. Standard solutions were prepared in HPLC grade methylene chloride or chloroform. Pyridine was purchased from J. T. Baker (Phillipsburg, NJ); trans-1,2-cyclohexanediol, 2-ethylaniline, quinoline, methyl benzoate, 1-naphthol, 1,4-naphtho(7) Matsuura, K.; Takeuchi, M.; Nojima, K.; Kobayashi, T.; Saito, T. Rapid Commun. Mass Spectrom. 1990, 4, 381-383. (8) Huang, E.; Henion, J. D.; Covey, T. R. J. Chromatogr. 1990, 511, 257-270. (9) Anacleto, J. F.; Ramaley, L.; Boyd, R. K.; Pleasance, S.; Quilliam, M. A.; Sim, P. G.; Benoit, F. M. Rapid Commun. Mass Spectrom. 1991, 5, 149155. (10) Matsumoto, K.; Nagata, S.; Hattori, H.; Tsuge, S. J. Chromatogr. 1992, 605, 87-94. (11) Tyrefors, L. N.; Moulder, R. X.; Markides, K. E. Anal. Chem. 1993, 65, 2835-2840. (12) Thomas, D.; Sim, P. G.; Benoit, F. M. Rapid Commun. Mass Spectrom. 1994, 8, 105-110. (13) Sadoun, F.; Virelizier, H.; Arpino, P. J. J. Chromatogr. 1993, 647, 351-359. (14) Pinkston, J. D.; Chester, T. L. Anal. Chem. 1995, 67, 650A-656A. (15) Pinkston, J. D.; Baker, T. R. Rapid Commun. Mass Spectrom. 1995, 9, 10871094. S0003-2700(95)00936-X CCC: $12.00

© 1996 American Chemical Society

Figure 1. Schematic of the SFC-TOFMS system: (1) SFC system, (2) transfer line, (3) heated ion source, (4) corona discharge needle, (5) interface plate, (6) nozzle, (7) lens system, (8) cryogenic pump, (9) ion pulse plate, (10) grounded focus grid, (11) acceleration grid, (12) flight tube, (13) final grid, and (14) electron multiplier.

quinone, and phenanthrene were obtained from Aldrich (Milwaukee, WI); 2-ethylhexanoic acid was obtained from Chem Service (West Chester, PA); caffeine was obtained from Mann Research Laboratories (New York, NY); methylene chloride was purchased from Fisher Scientific (Pittsburgh, PA); and chloroform was obtained from Mallinckrodt (Chesterfield, MO). Instrumentation. A schematic diagram of the instrumental setup is shown in Figure 1. Analytes separated in the SFC system were transported through a transfer line to the API source, where ionization occurred. The ions were adiabatically expanded into the mass spectrometer vacuum chamber through a very small orifice, forming a supersonic beam. Ions were subsequently pulsed from the supersonic beam into a perpendicular field-free drift tube and detected using an electron multiplier. A detailed description of a similar TOFMS is given in a previous paper.16 Chromatography. Supercritical fluid chromatography was carried out using a Lee Scientific Model 501 SFC system (Dionex, Sunnyvale, CA). Short length (3-5 m × 50 µm i.d.) SE-54-coated (0.2 µm film thickness) fused silica capillaries with on-column integral restrictors, and SFC grade carbon dioxide (Mountain Air Gas, Salt Lake City, UT) were used for the SFC experiments. Chromatography was performed at 120 °C oven temperature using either 150 atm constant pressure or pressure programming from 150 to 220 atm at 10 atm min-1. Conditions were chosen to ensure fast analyte elution from the separation column. Sample injection was achieved using a helium-actuated DC14W internal sample injector (Valco, Houston, TX) fitted with a 0.06 µL rotor, with dynamic split set at 1:10 split ratio. Pressure restriction for maintaining supercritical conditions along the SFC column was accomplished by preparing on-column integral restrictors with a Model M100 manipulator (Polymicro Technologies, Phoenix, AZ). The restrictors allowed for a 0.7-1 mL min-1 mobile phase flow rate. The carbon dioxide flow through the column and the split was measured using uncalibrated flowmeters (Gilmont Instruments, Barrington, IL). As a safety precaution, one should make certain that all connections to high-pressure gas cylinders and equipment are properly made and that the equipment is able to withstand supercritical carbon dioxide pressures. (16) Sin, C. H.; Lee, E. D.; Lee, M. L. Anal. Chem. 1991, 63, 2897-2900.

SFC-TOFMS Interface. Interfacing of the SFC to TOFMS was accomplished by designing and building a new transfer line and ion source chamber. Since it was difficult to build a device which could provide complete thermal insulation and avoid heat loss, the main idea in the transfer line design was to build it from materials with good thermal conductivity properties, which could minimize any local temperature nonuniformities. The transfer line (Figure 2) was built from 24 in. long, 0.04 in. i.d., 17 gauge stainless steel needle tubing (Hamilton, Reno, NV). While copper tubing has much better thermal conductivity and can be obtained with similar dimensions, the above-mentioned stainless steel was chosen for its chemical inertness and steel spring properties (ability to return to its initial shape after deformation). A copper braided sleeve was pulled over the stainless steel tubing and then was uniformly stretched out over the whole length of the tubing to give a tight fit on the surface. Resistive heating was supplied by wrapping a glass braided insulated thermocouple wire (type K, 30 gauge) over the copper braid. Since the themocouple wire actually provided a two-wire cable, the wires were welded together at one end, allowing the heating current to pass two times (back and forth) along the transfer line. The heater was tightly wound in a compressed coil so that the entire surface was continuously covered with the heating wire. The flat shape and thickness of the high-resistance thermocouple wire allowed for very uniform, equidistant wrapping around the stainless steel tubing. The heater was insulated from ambient temperature by several additional layers of a second copper braid, three Nextel 312 braided ceramic fiber sleeves (3/8 in. and 1/2 in. i.d.; Omega, Stamford, CT), a silicone heat insulating tubing (5/8 in. i.d. × 13/16 in. o.d.; Cole Parmer, Niles, IL), a third copper braid, and finally a 1.06 in. o.d. PTFE convoluted tube (Berghof/America, Concord, CA). The heating wire was placed between two copper braid layers, with the above-mentioned purpose of helping to spread local temperature gradients in order to achieve uniform heat distribution along the length of the transfer line. The third, outer layer of copper braid was sought to minimize local temperature variations induced by the ambient atmosphere. The ceramic fiber sleevings and the silicone tubing served the purpose of preventing heat loss, while the exterior convoluted PTFE tubing was used as a chemically inert, protective layer of the entire assembly. The temperature was monitored by three fine, Teflon insulated, J-type thermocouples (36 gauge; Omega, Stamford, CT) placed at the two ends and the middle of the transfer line, under the heating wire, on the first layer of copper braid. The temperature controller was an Omega CN 370 device. One thermocouple was used for transfer line temperature control, while the other two were just used for temperature monitoring. To minimize temperature gradients as much as possible, it was found useful to preheat the makeup gas in the oven and to supply it along the transfer line, inside the stainless steel tubing. A 7001000 mL min-1 makeup gas flow was found to be sufficient. The transfer line assembly was tightly secured in the SFC oven wall. The deleterious effects of carbon dioxide cooling during expansion in the restrictor region were avoided by supplementary heating of the transfer line tip. The tip was prepared from 0.030 in. i.d. × 1/16 in. o.d., 2 in. long stainless steel tubing, heated by a coil of Nichrome wire for about 80% of its length. Both heaters, on the transfer line and on the tip, were operated by home-built power supplies. An Omega CC high-temperature cement was deposited in a uniform, thin-layer coating on the stainless steel Analytical Chemistry, Vol. 68, No. 11, June 1, 1996

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Figure 2. Schematic of the SFC-TOFMS transfer line: (1) T-union, (2) stainless steel transfer line, (3) copper braid, (4) resistive heating, (5) ceramic braid, (6) silicone heat insulating tubing, (7) Teflon protective tubing, (8) J-type thermocouple, (9) heated tip, and (10) mini-T-union.

tip, and an uninsulated J-type thermocouple wire (36 gauge) was secured at the end of the tip with the same cement. Electrical insulation was provided by another layer of cement, coated over the thermocouple. Next, a Nichrome heating wire (32 gauge) was wrapped over the tip and protected by an additional cement coating. The tip temperature was controlled by a second Omega controller. The tip was connected to the transfer line by screwing both into a very small, home-built, stainless steel T-union. Introduction in the ion source of the entire makeup gas flow (necessary for optimal transfer line operation) was found to be detrimental to the analyte signal intensity, and the excess makeup gas was removed through the same mini-T-union and monitored with a flowmeter. The final setup resulted in a 1 in. thick, very flexible transfer line, which allowed easy positioning in the ion source. To perform the experiments, the SFC and TOFMS systems had to be positioned at 90° relative to each other. Finally, the transfer of the analytes to the ion source was simply accomplished by feeding the capillary SFC column from the oven, through the transfer line, until it reached the end of the transfer line tip. When the desired positioning of the capillary end was achieved, the capillary was secured with a nut and ferrule in the T-union in the oven. The ion source (Figure 3) was built from a hard, anodized aluminum block, 1.9 in. i.d. × 1.2 in. depth. Two oblong-shaped orifices, 5/8 in. × 3/4 in., were placed on the front cover of the source to allow variable positioning of the transfer line tip and the discharge needle inside the source. Precise positioning was achieved with two multiple-axis translation stages. The multiplestage micrometer used for needle positioning allowed for measurements only when the two axes, xn and yn, were arranged at an angle of about 60°, and not 90°. Nitrogen curtain gas was supplied between the nozzle and the interface plate to help decluster the analyte ions and to protect the nozzle from clogging. Curtain gas and makeup gas flows were controlled by needle valves and were monitored with flowmeters. The ion source was heated using a band heater (Heatcon, Bountiful, UT), tightly fit over the source wall. The ion source temperature was controlled using an RTD element and an Omega CN 3000 temperature controller. The RTD element was secured to the discharge needle holder so that its end was in close proximity to the region where expansion of the supercritical carbon dioxide occurred. The corona discharge needle was operated at 2.5 µA under constant current-controlled conditions. Mass Spectrometry. The mass spectrometer was a TOF 1000 (Sensar, Provo, UT) instrument. Ions were sampled through a 1926 Analytical Chemistry, Vol. 68, No. 11, June 1, 1996

Figure 3. Schematic of the corona discharge API source. (A) Top view and (B) front view. xc and yc ) axes for column positioning; xn and yn, axes for needle positioning. x defines the off-axis setting of the column tip/needle tip, y defines the distance between the nozzle and the column tip/needle tip.

30 µm nozzle and pulsed into the flight tube at 0.6 kHz repetition rate. Pulses of 300 V, 1.5 µs in width, were applied. RESULTS AND DISCUSSION SFC-TOFMS Interface Development. An ideal interface must meet several requirements: allow for high transfer efficiency, preserve chromatographic resolution and efficiency, promote only controlled chemical interactions,17 prevent thermal decomposition of analytes, and ensure removal of solvents or carrier fluids if necessary. From a chromatographic point of view, the transfer line should maintain its temperature very close to the SFC oven temperature, in order to preserve the physical characteristics of the mobile phase (density and viscosity) and to not alter the chromatographic integrity. During the present study, the transfer line played the role of being an actual oven extension, since the capillary separation column extended until it reached the transfer line tip. (17) Niessen, W. M. A.; van der Greef, J. Liquid Chromatography-Mass Spectrometry. Principles and applications; Chromatographic Science Series 58; Marcel Dekker, Inc.: New York, 1992.

Temperature variations along the transfer line were monitored by introducing a Teflon-insulated J-thermocouple (36 gauge) into the stainless steel tubing, from the tip toward the oven, and then by slowly withdrawing the thermocouple and reading the temperature for each 1 in. of tubing length. A very uniform reading was displayed, except for the two line ends. Near the SFC oven, the transfer line crossed through an electronic module which controls the SFC system, and an increase of 5-10 °C relative to the oven was observed. A fan was mounted in the electronic module, and several experiments were performed to find the optimum fan speed for a given temperature range. The position in the SFC oven wall where transfer line heating was supposed to start also had to be optimized. At the other, outside end of the transfer line, a drop of 5-7 °C was often observed. This end was insulated using a 0.5 in. thick Teflon ring, in order to prevent heat loss to the support which held it in the micrometer stage. When the connecting mini-T-union was protected with heatinsulating material, and the transfer line tip, which was heated to a higher temperature than the transfer line, was mounted on the union, this temperature drop was avoided. Finally, by supplying oven preheated makeup gas (700-1000 mL min-1) along the transfer line, the following results were obtained. At 60 °C and 100 °C oven temperatures, a (1 °C temperature variation was obtained along the entire length. At 100 °C, +2 °C near the oven and -2 °C near the tip, in the micrometer support, were sometimes displayed for about 1-3 in. length of transfer line. At 150 °C oven temperature, variations of (1.5 °C were obtained. Since practical SFC separations are performed well below 200 °C, optimization studies were not performed for higher temperatures; however, the materials used in the transfer line design will withstand temperatures up to 260 °C. Preliminary Optimization under Gas Sampling Conditions. Optimization of the mass spectrometer voltages was achieved by supplying a continuous pyridine-enriched nitrogen flow to the ion source through a 50 µm i.d. fused silica capillary, at 1-2 mL min-1. Nitrogen gas was allowed to bubble through a vial containing pyridine. For an open-to-air ion source, nitrogen is the least expensive and obvious choice for the purpose of this study. Carbon dioxide could have been used as well, since interference of carbon dioxide in an open atmospheric pressure chemical ionization source was not observed. Since careful temperature control of the pyridine vial and of nitrogen flow was not provided, the concentration of pyridine in nitrogen was below the saturation concentration. The capillary was passed through the transfer line, and the transfer line tip was positioned at 28 mm from the nozzle. Atmospheric pressure chemical ionization of a compound occurs in two steps: first, an appropriate reagent gas (nitrogen, oxygen, methane, ammonia, nitric oxide, etc.) is ionized by the primary electrons produced by the corona discharge, and second, the ionized reagent gas ionizes the analyte molecules through ion-molecule reactions. Since our ion source was open, and no specific reagents were supplied, the primary ions produced, shortlived N2+ and O2+ ions, quickly interacted with atmospheric moisture to produce hydronium ion-water clusters, H+(H2O)n.18 These hydronium-water clusters are the main reagent ions in an open API source. Ionization of analytes with higher gas-phase basicities than water occurs through proton transfer reactions. (18) Sunner, J.; Nicol, G.; Kebarle, P. Anal. Chem. 1988, 60, 1300-1307.

Figure 4. Nozzle voltage, ion chamber temperature, and curtain gas flow influence on pyridine signal.

API is a soft ionization method, and mainly protonated analyte molecular ions are produced. Spectra of pyridine are illustrated in Figure 4. The protonated molecular ion, PyH+ (80 amu), and its pyridine cluster, Py‚PyH+ (159 amu), are observed. The intense peaks at low molecular weight, observed in all spectra, are due not to compounds but to electronic noise produced by the pulser. Most often the baseline varied between 5 and 20 mV. Slight variations in baseline are expected after each cryoregeneration and especially after change of electron multipliers. With careful tuning, the baseline can be set manually to any value. The high concentration of pyridine in the source was responsible for the occurrence of the dimer, Py‚PyH+, and also for the disappearence of background H+(H2O)2, the main reagent ion in the source. Three important parameters strongly interfered with the pyridine ion intensity, making the signal oscillate from pyridine to its dimer, and vice versa. These were the nozzle voltage, ion chamber temperature, and curtain gas flow. Figure 4A,B illustrates relatively optimized conditions for signal monitoring. Slightly higher curtain gas flows, as in Figure 4B, resulted in better dimer breakdown. An altered voltage on the nozzle (Figure 4C) diminished the pyridine signal, while a low ion source temperature strongly favored dimer formation (Figure 4D). The effect was compensated for by an increase in nozzle voltage (Figure 4E). The formation of pyridine dimers offered the opportunity to study a more complex situation and investigate the effects of the above-mentioned parameters, in order to control and eventually completely eliminate dimer formation. Conditions for SFC experiments have been chosen to avoid dimer formation. Elution of compounds with high gas-phase basicities in very narrow and concentrated peaks might result in dimer formation if conditions are not properly adjusted. An interesting observation was that the variation of signal with curtain gas flow was not symmetrical about the needle xn axis. The needle yn was set at 3.5 mm. For an on-axis nozzle-needle Analytical Chemistry, Vol. 68, No. 11, June 1, 1996

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Figure 5. Variation of pyridine signal intensity under gas sampling conditions. (A) Dependence of signal on curtain gas flow. Makeup gas flow, 0 mL min-1; xc ) 2 mm, yc ) 28 mm, xn ) 0.7 mm. (B) Dependence of signal on makeup gas flow. xc ) 2 mm, yc ) 28 mm, xn ) 0.7 mm, yn ) 3.5 mm.

arrangement, i.e., xn ) 0, the Py‚PyH+ cluster decreases with an increase in curtain gas flow from 100 to 500 mL min-1, resulting in an increase in the PyH+ signal. At higher curtain gas flow (1000 mL min-1), the analyte was flushed away, and the PyH+ signal decreased while the H+(H2O)2 signal increased in the spectrum. When the needle was positioned to the right of the nozzle (i.e., xn ) +0.7 mm), we could observe the PyH+ and the Py‚PyH+ signals only when the curtain gas flow was very small (i.e., 100 mL min-1). For higher curtain gas flows, the pyridine was completely flushed away in this region of the ion source, and we can see mainly the H+(H2O)2 signal. For 1000 mL min-1 curtain gas flow, a slightly increased PyH+ signal can be observed again, but this phenomenon might be due to increased turbulence in the source. For comparison, when the needle was positioned to the left of the nozzle (i.e., xn ) -0.7 mm), only a very small variation of the PyH+ and Py‚PyH+ signals with increased curtain gas flows was observed, and H+(H2O)2 could not be seen. This suggests that we had a relatively constant, high concentration of pyridine in this region of the source. The intensities are smaller than for the case where xn ) 0, but this is due to the fact that the xn and yn axes of the needle are not perpendicular, and when moving the needle from xn ) +0.7 mm to xn ) -0.7 mm, even though yn ) 3.5 mm is maintained constant, the needle is actually placed at larger distances from the nozzle. We believe that the unsymmetrical setup of the ion source is responsible for this behavior (Figure 3B). The placement of the two oblong-shaped orifices might allow for preferential removal of the gas flow through orifice 1, which was designated for column positioning. In this case, the gas flow dynamics would concentrate the analyte on the left side of the nozzle, which would account for our observations. We repeated the experiment for xn ) +0.7 mm, at different yn positions (Figure 5). Increased curtain gas flows resulted in lower analyte signals (Figure 5A). Very high curtain gas flows resulted in a slight increase of signal, possibly due to increased turbulence. At 1928 Analytical Chemistry, Vol. 68, No. 11, June 1, 1996

lower curtain gas flows, the signal increased when the discharge needle was positioned closer to the sampling orifice. At higher curtain gas flows, the signal was less dependent on the needle yn axis positioning. An appropriate makeup gas supply was able to bring back the lost signal (Figure 5B). The higher the curtain gas flow setting, the higher was the makeup gas flow needed to reach the initial signal intensity. Optimization under SFC Sampling Conditions. For the SFC-TOFMS experiments, data were collected by averaging the results of several separate injections of standard solutions. For flow optimization studies, only pyridine standards were used. For the effect of ion source temperature, 2-ethylhexanoic acid and phenanthrene were used as well. Background Stability. A stable and reproducible background is a basic requirement for an API source. The main ions observed in the source were water clusters, with H+(H2O)2 strongly predominating in the spectrum. Along with water clusters, H+(H2O)n, where n ) 1-4, other ions were observed at much lower intensities (NO+, O2+, H+(N2)(H2O)n, where n ) 1, 2, and H+(N2)H2O). The nature of the ions and their relative abundances in the spectrum were dependent on curtain gas flow, nozzle voltage, ion chamber temperature, the dryness of the source,19 and the nozzle-discharge needle distance. Several studies have shown the importance of this last parameter for achieving chemical equilibrium conditions in the ion source,20,21 but a detailed dicussion is beyond the scope of this paper. Once temperature equilibration of the source was achieved, a stable background was displayed for 6-7 days. Usually after a week, regeneration of the TOF cryogenic pump was necessary. Discharge Needle Positioning. With the SFC, a short study was performed to identify the effect of nozzle-needle distance on analyte signal intensity. This distance is one of the parameters which determines the residence times/reaction times for the ions in the source. A detailed study of this effect, with its kinetic and thermodynamic implications, is given by Kebarle and co-workers.18 Contrary to their results, in which the H+(H2O)2 ion intensity decreased and the PyH+ ion intensity increased with increasing nozzle-needle distance, in our case, both ion intensities decreased with increasing nozzle-needle distance (a result expected from our previous studies performed under gas sampling conditions). The experiment was repeated a couple of times, with the same results. Nevertheless, our setup did not allow for measurements under exactly the same conditions. Our determinations were performed between 3 and 6.5 mm nozzle-needle distance (with the signals decreasing to about 5% at 6.5 mm, compared to their original value at 3 mm), while theirs were between 10 and 22 mm.18 Flow dynamics in the two sources were also completely different. Ion transport in the ion source occurs through three major mechanisms: convective transport, where ions are carried along with the gas stream; diffusive transport from higher to lower concentrations; and electrostatic transport, which refers to ion drift in the direction of the existing electrical fields.22 Once the analyte molecules in the expanding supercritical fluid carrier are ionized (19) Dearth, M. A.; Korniski, T. J. J. Am. Soc. Mass Spectrom. 1994, 5, 11071114. (20) Dzidic, I.; Carroll, D. I.; Stillwell, R. N.; Horning, E. C. Anal. Chem. 1976, 48, 1763-1768. (21) Zook, D. R. Mass spectrometric sampling from the atmospheric pressure ion source. Ph.D. Dissertation, Montana State University, Bozeman, MT, 1990.

Figure 6. Dependence of signal intensity on xc/yc column tip position. Curtain gas flow 300 mL min-1; makeup gas flow, 80 mL min-1; xn ) 0 mm, yn ) 3 mm.

Figure 7. Dependence of signal intensity on makeup gas flow at different xc column tip positions. Curtain gas flow, 300 mL min-1; yc ) 7.5 mm, xn ) 0 mm, yn ) 3 mm.

by the corona discharge needle, the resulting ions start to spread according to the above-mentioned mechanisms. Not all three mechanisms have the same importance in determining ion motion. Fluid flow interferences become strong in the vicinity of the ion sampling orifice, and the so called “vacuum suction” 23 effect results in fluid flow-induced velocities, which can competitively interfere with electric field-induced velocities. This effect contributes beneficially to sampling efficiency, since it increases the ion current sampled, relative to the case when sampling would not occur through a vacuum interface orifice. Ion beam divergence occurs mainly through radial diffusion, ion mobility in the externally applied fields, and space charge repulsion. An intense ion source, such as a corona discharge, produces a strong, concentrated ion beam, and mutual Coulombic repulsions have the tendency to spread the ions extremely fast, reducing the sampled ion current. As a result, ions should be sampled as fast as possible after they are produced, i.e., the distance between the discharge needle and the nozzle sampling orifice should be very small. On the other hand, we are dealing with ion-molecule reactions in the source, and consideration of kinetic and thermodynamic aspects of the involved reactions results in a compromise when positioning of the needle is considered. x/y-Axis Transfer Line Tip Positioning. A strong interdependence was found to exist in the alignement of sampling orifice, discharge needle, and transfer line/column tip. At the beginning, we presumed that the signal intensity would decrease upon placing the transfer line tip at a greater distance from the nozzle. The results are shown in Figure 6. As a first approximation, the experiment confirmed our supposition. The signal decreased as the transfer line tip was positioned farther from the sampling orifice on both xc and yc axes. For xc ) 0 mm, there was an onaxis, aligned nozzle-column tip arrangement. However, even though maximum signals resulted from the on-axis arrangement, the signal was very unstable and irreproducible under these conditions, which can be seen from the very large error bars for these positions. Further studies, as it will be demonstrated, showed that we were actually dealing with a much more complex situation. Most of our studies were performed at a small off-axis setting of the column tip, i.e., xc ) 0.5 mm. For low makeup gas flow settings, increased curtain gas flows resulted in progressively decreased signal.

We presumed that increased makeup gas flows would bring back the maximum signal when the column tip was positioned farther from the nozzle, either on the xc or the yc axis. The experiment was repeated for two yc positions (yc ) 7.5 and 12.5 mm). A slight increase in signal intensity with increased makeup gas flow was observed only at larger xc values, but the signal remained very low compared to its maximum value at xc ) 0 (Figure 7). Flow Interactions. Even though convective flows have a less pronounced effect on the motion of ions in the ion source compared to electrical fields or space charge effects, the influence of these flows cannot be neglected. It is especially the interaction between these flows, i.e., curtain gas and makeup gas, confronted with each other, which can result in complete signal loss if not properly adjusted. Even though dedicated studies were not pursued, it is our opinion that fluid flow interactions have a significant effect, especially on the motion of molecules prior to their ionization, rather than on the motion of the ions. The molecules can be easily flushed away by unfavorable gas flows, while ions might still be able to penetrate them. The existing electrical fields govern the energy and motion of the ions. Several experiments have been conducted to study these interactions. Nevertheless, absolute signal intensity values should not be compared from one figure to another. Factors like a slight misalignment, a new restrictor, or different stages of cryoregeneration all might have contributed to variations in signal intensity, but the experiments performed for drawing a single figure were always conducted on the same day, under exactly the same conditions. Some of the experiments have been repeated several times, and the trends were always the same. Dependence of Signal Intensity on Makeup Gas Flow at Different Curtain Gas Flow Settings. A complex consideration of fluid flow interaction effects revealed that an eventual “optimum” for the makeup gas flow might be found for a given curtain gas flow (Figure 8). At zero makeup gas flow, there was no signal, probably due to low heat transfer to the supercritical CO2 expansion region. As the makeup gas flow increased, a maximum was reached, and then the signal decreased again. In accordance with our experience from gas sampling conditions, with increased curtain-gas flows, increased makeup gas flows were necessary to focus the analyte in the ionization region to reach the maximum signal. The decrease of signal intensity at higher makeup gas flows can be due to several possibilities. Shorter residence times in the ion source10 might be one. Increased turbulence due to stronger interaction between two high flows could result in the spreading of analyte molecules or ions, resulting in reduced signal

(22) Busman, M. Space charge effects in high pressure mass spectrometry sources. Ph.D. Dissertation, Montana State University, Bozeman, MT, 1991. (23) Potjewyd, J. Focusing of ions in atmospheric pressure gases using electrostatic fields. Ph.D. Dissertation, University of Toronto, Canada 1983.

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Figure 8. Dependence of signal intensity on makeup gas flow at different curtain gas flow settings. xc ) 0.5 mm, yc ) 8.5 mm, xn ) 0 mm, yn ) 3 mm.

intensity. However, increased turbulence might have positive effects also; in some of our experiments, it was possible to increase or bring back a small or lost signal, even if the signal did not reach its maximum value. Another noticeable aspect in Figure 8 is the fact that the maximum absolute signal was obtained for the highest curtain gas flow. The data presented further in Figure 9 show that this maximum is actually very much dependent on the yc axis positioning of the column tip. Dependence of Signal Intensity on yc Column Tip Position at Different Makeup Gas and Curtain Gas Flow Setttings. Different positions of the column tip on the yc axis resulted in beneficial or deleterious effects, depending on the curtain gas and makeup gas flow settings. High makeup gas flows (300 mL min-1) resulted in a very unstable signal, the maximum being strongly dependent on tip positioning and curtain gas flows (Figure 9A). The signal decreased progressively with increased curtain gas flow only at larger nozzle-column tip distance (yc ) 15.5 mm), where the interaction between the two flows became insignificant and, presumably, the curtain gas flow dictated the amount of analyte which was able to reach the sampling orifice. When the column tip was positioned in the nozzle proximity (yc ≈ 6.5-8 mm), we observed the same trend as in Figure 8, where the maximum absolute signal results for the maximum curtain gas flow. For this rather unexpected result, a reasonable explanation would take into account the alignment between sampling orifice, discharge needle, and column tip. Even though precise positioning was possible using the two micrometer stages, the alignment of transfer line tip in the micrometer itself and the parallelism or perpendicularity among different elements were not exact. As a consequence, when the column end was very close to the discharge needle tip, a slight misalignment would have allowed for a strong makeup gas flow to flush the analyte from the ionization region. Under these circumstances, a high curtain gas flow would break the directional effect of the makeup gas, increase the turbulence, and consequently increase the signal intensity. A similar effect might be another answer for the decrease in signal at high makeup gas flows in Figure 8, but obviously the complexity of the interactions allows for much speculation. At lower makeup gas flows (150 mL min-1), where a less powerful interaction between the two gas flows is expected, a much more uniform and smooth variation of signal was observed, with a maximum between yc ) 9.5-12.5 mm (Figure 9B). The signal was less strongly dependent on curtain gas flows and column tip positioning. At even lower makeup gas flows (80 mL min-1), the signal progressively decreased with increased curtain1930 Analytical Chemistry, Vol. 68, No. 11, June 1, 1996

Figure 9. Dependence of signal intensity on yc column tip position at different makeup gas and curtain gas flow settings. xc ) 0.5 mm, xn ) 0 mm, yn ) 3 mm. (A) Makeup gas flow, 300 mL min-1; (B) makeup gas flow, 150 mL min-1; (C) makeup gas flow, 80 mL min-1.

gas flow over the entire yc region taken into study (Figure 9C), a situation that we already encountered in Figure 8 at very small makeup gas flows and in Figure 9A at low fluid flow interactions. Obviously, in order to make a satisfactory choice of operational parameters, a reasonable compromise must be made. Probably the setup illustrated in Figure 9B, which provides the most stable and independent signal, would be the most convenient. Dependence of Signal Intensity on Elution Pressure from the SFC Column. The observation that, under given circumstances, at higher curtain gas flows, higher makeup gas flows were necessary to reach the maximum signal led to the idea that the same effect might be obtained if the analyte is eluted at higher pressures from the SFC column (Figure 10). Surprisingly, as we increased the elution pressure from 150 to 250 atm, the beneficial effect of the curtain gas was lost. The intense multiple water cluster pattern invaded the spectrum, and the analyte signal decreased accordingly (as we mentioned earlier, during the experiments, the H+(H2O)2 cluster strongly predominated over the other clusters). An increased curtain gas flow was necessary to reequilibrate the situation and bring back the maximum signal. Further increase in curtain gas flow resulted, as already experienced, in a decreased analyte signal.

Figure 10. Dependence of signal intensity on elution pressure from the SFC column. xc ) 1 mm, yc ) 6.5 mm, xn ) 0 mm, yn ) 3 mm; makeup gas flow, 80 mL min-1.

Ion Source Temperature. The effect of ion source temperature was evaluated only up to 180 °C for three analytes: pyridine, 2-ethylhexanoic acid, and phenanthrene. Higher temperatures would have interfered negatively with the cryogenic system of the TOF instrument. A pronounced increase in signal intensity with temperature was observed for pyridine, from 12 mV at 60 °C, to 43 mV at 180 °C. For the other two analytes taken under study, the signal intensity also increased with ion source temperature, but the effect was less obvious because of much lower intensity signals for these compounds. 2-Ethylhexanoic acid and phenanthrene have much lower gas-phase basicities than pyridine. The molecular ion of phenanthrene, which would have resulted from a charge transfer mechanism, was not observed. The choice of source temperature will depend on the nature of the analytes eluting from the SFC system. A high temperature may be desirable for desolvation purposes or cluster breakdown. An extensive discussion on the dependence of analyte signal intensity on ion source temperature is given by Kebarle et al.24 However, decomposition of certain analytes at higher temperatures and instrumental limitations have to be taken into account when deciding the optimum ion source temperature. Transfer Line Tip Temperature. Optimization of the transfer line tip temperature was found to be useful for each studied compound. The maximum temperature at the column tip was 360 °C. A maximum signal intensity for pyridine (MW ) 79) was observed at 150-160 °C tip temperature, and the signal remained relatively constant until the temperature reached 220-240 °C. At higher tip temperatures, 300-320 °C, a decrease of ∼40% in signal intensity was observed. The lowest tip temperature that gave maximum signal and the shortest elution time was chosen for analysis. Analytes such as 2-ethylhexanoic acid (MW ) 144), methyl benzoate (MW ) 136), and 1,4-naphthoquinone (MW ) 158) required 220-240 °C, while compounds such as phenanthrene (MW ) 178) and pyrene (MW ) 202) required 350-360 °C tip temperature. Generally, within the tip temperature range taken into study, from 120 to 360 °C, or from 200 to 360 °C for higher molecular weight compounds, the signal decreased by 4050% from its maximum value at the optimum tip temperature. SFC Column Positioning in the Transfer Line Tip. For a given temperature reading in the transfer line tip, the actual maximum temperature was found not to be at the very tip, but 6-8 mm inside the stainless steel tubing. The column tip was always positioned at the same level with the transfer line tip. No (24) Sunner, J.; Ikonomu, M. G.; Kebarle, P. Anal. Chem. 1988, 60, 1308-1313.

Figure 11. TOF spectra of selected compounds: (A) trans-1,2cyclohexanediol (MW ) 116), (B) methyl benzoate (MW ) 136), (C) 2-ethylhexanoic acid (MW ) 144), (D) 1,4-naphthoquinone (MW ) 158), (E) phenanthrene (MW ) 178), and (F) caffeine (MW ) 194).

significant variations in signal intensity occurred due to an eventual small misalignment between the two tips. Applications. Figure 11 gives some selected spectra of investigated compounds. The spectra are composed in most cases of the protonated molecular ion. In the case of trans-1,2cyclohexanediol, a water molecule is lost after protonation, and the (M + H - H2O)+ ion is prevalent. The quantities of analytes used to generate these spectra were in the low nanogram range. CONCLUSIONS The SFC-MS transfer line was meticulously tested and proven to meet the strict temperature requirements for preserving the integrity of the SFC separation. The importance of several factors, such as capillary column tip10 and discharge needle tip position in the ion source,12 makeup and curtain gas flow settings8,10-12 and their mutual interactions, and temperature effects, usually have been acknowledged and briefly described by most authors who have conducted experiments in the field, but quantitative Analytical Chemistry, Vol. 68, No. 11, June 1, 1996

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aspects about signal variations when these parameters are shifted from their optimal values have not been described. Our experiments reveal the importance and the complex nature of interactions between the above-described factors. In our work, we have found that a stable background is essential to obtain reproducible data. The main reagent ion in our source was H+(H2O). Its intensity, if it was present, or its absence in certain situations was always a good reference point in considering how well the system was performing. The analyte signal decreased with increasing distance between the sampling nozzle and the discharge needle tip. Maximum intensity signals were obtained for an on-axis nozzle-SFC column tip alignment, but the stability of the signal was poor. The best performance in terms of intensity and stability was obtained when the column tip was positioned 0.5-1 mm off-axis. The signal intensity was less strongly dependent on the positioning of the column tip on the y-axis if the makeup gas and curtain gas flows were properly set. Essentially, for a given curtain gas flow, an optimum for the

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makeup gas flow was found. Our experience shows that both flows should be set at a low value to give the best performance. At high flow settings, strong interactions between the two flows can lead to signal loss or instability. An interdependence between the elution pressure from the SFC column and the optimum for the curtain gas flow was also found. The signal intensity was also dependent on the transfer line tip and ion source temperature. ACKNOWLEDGMENT We thank Mingin Wu (Sensar Corp.) for his continuous help during this research project and Lon Que Adams from the BYU instrument shop for help in constructing the transfer line. Received for review September 18, 1995. Accepted March 12, 1996.X AC9509364 X

Abstract published in Advance ACS Abstracts, April 15, 1996.