A ~ I Chem. . iee3, 65, 1281-1287
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Micro Flow Rate Particle Beam Interface for Capillary Liquid ChromatographyIMass Spectrometry Achille Cappiello' and Fabrizio Bruner Zstituto di Scienze Chimiche, Universith di Urbino, Piazza Rinascimento 6, 61029 Urbino, Italy
A laboratory-made aerosol generator has been developed to interface the effluent of a HPLC packed capillary column and a quadrupole mass spectrometer. The new couplingdevice is designed to be fully compatible with a commercially available desolvation chamber and the momentum separator of a Hewlett-Packard 59980B particle beam unit. The aerosol generated by minimal solvent input (1-5 bL/min) allows an improved signal response for high water content mobile phases, yielding improved chromatographic performance during gradient analysis. Sensitivity is also improved by the scaled-down flow rate. Reversed-phaseanalysis has been performed on a mixture of human hormones using the new interface. Unnoticeableinstrument contaminationdue to mobile-phaseintroduction was observed over a 6-month working period. INTRODUCTION Interfacing liquid chromatography and mass spectrometry has become a priority task for the analysis of nonvolatile and/or thermally unstable compounds. Over the past decade many attempts have been made to accommodate the liquid effluent into a preexisting ion source. Some of them have been succeeafullydeveloped, but none has shown the extensive application possibilities peculiar to a GC interface. This shortcoming is largely due to the radical difference between a liquid chromatographic environment (solute at room temperature) and the mass spectrometry electron impact sourcerequirement (gasphase a t a relatively high temperature and in a very high vacuum) plus the enormous difference in the chemical properties of the molecules suitable to liquid chromatography. Because of their softness, alternative ionization techniques may contribute to the detection of high molecular weight compounds but they often lack the important structural information normally supplied by an E1 source. Particle beam interface, developed by Browner and coworkers,lY2 allows the acquisition of library-searchable E1 spectra from the liquid effluent originating from an HPLC analytical column. On the chromatograph side of this interface, the liquid effluent is forced into a fused-silica capillary tubing surrounded by a coaxial helium gas flow. At the end of the tubing, the gas mixes with the liquid to produce an aerosol. A desolvation chamber allows solute enrichment of the aerosol drops, thus forming a mixture of solvent vapors, helium gas, and dry solute particles. A capillary nozzle, located at the end of the desolvation chamber, produces a high-velocity beam of the mixture aimed into the center of a multiple-stage momentum separator. The combined action (1) Willoughby, R. C.; Browner, R. F. Anal. Chem. 1984,545, 2626. (2) Winkler, P. C.; Perkins, D. D.; Williams, W. K.; Browner, R. F. Anal. Chem. 1988,60, 489. 0003-2700/93/0365-1281$04.00/0
of a powerful pumping system and a series of coaxial skimmere allows the elimination of the low-momentum portion of the mixture prior to the final introduction of dry particles into a normal E1 source. The sample is thus ionized, not as a gaseous distribution of single molecules, but as a homogeneous dispersion of molecular aggregates. This particular sample distribution may explain the consistent loss in terms of sensitivity when a particle beam interface is employed. The size of the particles is dependent on the formation of the initial aerosol drops. The processes governing the breakup of a cylindrical liquid jet, such as the one produced inside the capillary, were first investigated by SavartS3In the late 1970s,Rayleigh found a relationship between a quantity linked to drop size and the diameter of the liquid jet.4-5 Browner and co-workers, with their monodisperse aerosol-based interface (MAGIC-LC/ MS),' developed the first prototype of what is now currently known as a particle beam interface. In their interface and in subsequently available devices,6s7 the effluent of an analytical HPLC column (0.1-1.0 mL/min) is nebulized and finally introduced into the ion source as described above. The aerosol generator has been designed and optimized to better handle the flow rates from conventional columns. Optimum drop size distribution is strictly related to conventional effluents and rapidly worsens when lower flow rates are used. Even though mechanical pumps operate at relatively hot temperature and are fully gas ballasted, some contamination may be observed in the pump fluid, especially if a high water content mobile phase is used. Since part of the mobile phase passes through the momentum separator to the mass spectrometer, contamination of the ion source has been observed and a frequent maintenance schedule is required. The performance of the electron multiplier detector, final target for all the ion species produced by the mass spectrometer, rapidly decreases when the particle beam interface is employed intensively. A drastic reduction in the mobile-phase flow rate will certainly slow the contamination buildup, thus minimizing solvent influence in the analytical results. Micro-HPLC permits highly efficient separations with minimal solvent consumption. When packed capillary columns are employed, the flow rate required for an optimal chromatographic process is nearly 1pL/min.a10 Even with a limited sample capacity, micro-HPLC has found its primary role coupled with mass spectrometry. This high-sensitivity technique allows this limitation to be overcome by taking advantage of the very low effluent flow rates. (3)Savart, F. Ann. Chim. Phys. 1983, 53, 337. (4)Rayleigh, J. W. S.h o c . London Math. SOC.1978, 10, 4. ( 5 ) Rayleigh, J. W. S.Proc. R. SOC.London 1979, 29, 71. (6)Ligon, W. V.,Jr.; Dorn S.B. Anal. Chem. 1990, 62, 2573. (7)Baczynskyj, L.J. Chromatogr. 1991,562, 13.
(8)Crescentini, G.;Bruner, F.; Mangani, F.; Yafeng, G. Anal. Chem. 1988,60, 1659. (9)Cappiello, A.; Palma, P.; Papayannopoulos, I. A.; Biemann, K. Chromatographia 1990, 30,477. (10)Crescentini, G.; Mastrogiacomo, A. R. J.Microcolumn. Sep. 1991, 3, 539. 0 1993 Amerlcan Chemical Soclely
ANALYTICAL CHEMISTRY, VOL. 65, NO. 9, MAY 1, 1993
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Caffelne slgnal Intensity versus H 2 0 concentratlon In the moblle phase for a mlcrolnterface (a,top)and a conventlonal Interface (b,bottom). Mobile-phase flow rate 1 pL/mln. Flgure 4.
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Table I. Response Linearity Data between 0.3 and 60 ng Obtained by Monitoring the Molecular Ion of Caffeine ( m / z 194)' caffeine
conc (ng) 0.3 0.6 1.2 6.0
signal int (SD% ) 36.0 (4.2) 104.4 (4.9) 262.2 (2.3) 1954 (3.5)
conc (ng)
signal int (SD% )
12.0 30.0 60.0
4144 (2.8) 9829 (2.2) 18240 (4.1)
caffeine
Average of five injections for each concentration.
A radically new laboratory-made aerosol generator, developed to interface the effluent from a HPLC packed capillary column to a quadrupole mass spectrometer, is described. The new coupling device is designed to be fully compatiblewith a commercially available desolvationchamber and momentum separator of a Hewlett-Packard 59980B particle beam unit.
Poly ether ether ketone (PEEK) 250-pm4.d. packed capillary columnshave been developed and are currently being made in our laboratory to satisfy micro-HPLC demand.I1 These standard l/la-in.-o.d. polymer columns allow greater chemical and mechanical resistance and easier connection capabilities. The new interface improves the overall performance of the particle beam interface thus showing several advantages: (1)drastic reduction in solvent consumption with negligible contamination by solvent vapors of the pumping system, the ion source, and the analyzer. This characteristic may widen the choice of buffers or mobile phases potentially harmful to the instrument; (2) light improvement in sensitivity, due either to sample detection with selected-ion monitoring or to the minimum amount of sample needed to generate an interpretable mass spectrum; (3) better signal response for high water content mobile phases with improved chromatographic performance during gradient analysis.
EXPERIMENTAL SECTION Instrumentation. A 59980B Hewlett-Packardparticle beam interface (Hewlett Packard, Palo Alto, CA) was used to host the new aerosol generator. Desolvation chamber, momentum separator, and pumping system were employed as supplied by the company without further modifications. The interface was coupled to a Hewlett-Packard 5989A quadrupole mass spectrometer. The E1 ion source was operated at 70-eV electron energy and the temperature used was 250 "C, unless otherwise stated. The analyzer temperature was set at 120 O C . The GC/ MS interfacewas capped with a blank nut and ferrule and heated up to 250 "C. Conventional liquid chromatography was carried out with a Kontron Instruments 420 dual-pump binary-gradient HPLC system (Kontron Instruments, Milano, Italy). Microliter flow rates were obtained with an Acurate 1:70 splitter (LC Packings, Zurich, Switzerland). Isocratic analyses were carried out with a (11) Cappiello,A.; Palma, P.; Mangani, F. Chromatogrophia 1991,7/8, 389.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 9, MAY 1, 1993
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TIC of caffe2.d Ab I!n d anc e
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Scan 282 (4.344 inin) of caffe2.d SUBTRACTED Abundance
1000
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Flguro 5. Caffelne (30 ng) Injected vla the
100
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140
160
180
mlcrolnterface and Its relative mass spectrum.
ShimadzuLC-SA isocratic pump (Shimadzu,Tokyo,Japan). For micro-HPLC analyses a zero-volume Valco injector equipped with a 60-nL internal loop was employed (Valco, Houston, TX). A Hewlett-Packard 2.1 X 100 mm Hypersil ODS C18 reversedphase packed with 5-pm particles was used as the analytical column. Capillary columns were made in our laboratory from a 1/16-in,-o.d.,250-pm4.d. PEEK tubing (Alltech Associates Inc., Deerfield,IL) and packed with C18 reversed-phase5-pm particle size purchased from Phase Sep (Queensferry,UK). (Fora detailed discussion see ref 8). The 50-pm-i.d. 140-pm-0.d.fused-silica capillary tubing used in the nebulizer was purchased from Polymicro Technologies (Phoenix, AZ). Reagents. All solvents were HPLC grade from Farmitalia Carlo Erba (Milano,Italy) and were filtered and degassed before use. The human hormones were purchased from Sigma Scientific (St. Louis, MO).
RESULTS AND DISCUSSION Aerosol Generator. In a nebulizer random disturbances induce surface instability of the liquid jet, resulting in its breakup into a continuous series of aerosol droplets (Figure 1). In a conventional interface this process is optimized for mobile-phaseflow rates ranging from 0.1 to 1.0mL/min. When lower flow rates are introduced into the nebulizer capillary, an undesirable phenomenon takes place (Figure 2). After initial aerosol formation, a alow-growingliquid droplet sticks to the end of the capillary tubing (Figure 2a, top). Once the droplet is broad enough, it adsorbs into the internal gas coaxial tubing surface (Figure 2b, middle). Surface-active properties of the liquid force the capillary to flex sidewards while the liquid effluent goes upstream into the helium tubing. Once
the process is stabilized, the helium flows over the liquid stream and produces no aerosol (Figure 2c, bottom). A reduction of the nebulizer capillary diameter was tried in order to increase the linear velocity of the liquid, but only a minimal improvement was noticed. Very narrow tubings (diameter less than 25 pm) were also easily clogged by mobilephase impurities and worked for a very short time. To overcome these limitations, a laboratory-made aerosol generator for microliter flow rates has been designed (Figure 3). Several attempts have been made to optimize aerosol production, but the guideline was still to avoid liquid interactions with the surrounding tubing and to force it to form a spray. In the new interface the end portion of the helium coaxial tubing has been widened around the fusedsilica capillary, thus keeping the slow-growingliquid droplet away from the internal gas tubing wall. The enlargement of the tubing sharply restricts ita diameter around the capillary tip, thus keeping the contact surface between the capillary and the gas restrictor to a minimum. This sharp restriction avoids any active surface interaction of the liquid with the tubings and also boosts the gas stream. The peculiarity of the new interface consists in forcing an infinitesimal liquid output to form a fine and homogeneous aerosol. In the absence of helium flow, the liquid effluent coming out of the capillary a t 1pL/min does not tend to be forced out by the incomingsolvent, but remains in this position as an expanding droplet for continuous minimal liquid intake. When the helium is allowed into the new nebulizer, as soon as the droplet size exceeds the outside diameter of the capillary, the gas pressure over the exposed drop surface exceeds the surface-active forces, thus wrenching it from the
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 9, MAY 1, 1993 Scan 62 (1.402min) of HlSO6a.d SUBTRACTED
Abundance I29
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140
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280
Flguro 6. Comparlson between mass spectra of testosterone obtained at low concentratlon (60 ng) (a, top) and at high concentratlon (600 ng) (b, bottom).
nebulizer to form an aerosol. With the solution adopted in this case, the mechanism for aerosol formation is optimized when the enlarged tubing has an internal diameter of l/lG-in., with a length of 2 mm. As shown in Figure 3, the exit of the gas is limited by a circular restriction with a diameter of 0.4 mm. The capillary tip protrudes outside the gap to generate
the aerosol. Since the outside diameter of the capillary tubing is 140 Ccm, the circular crown open to the helium flow has a thickness of 260 pm with a surface of 0.11 mm*. The linear velocity of the gas through the circular crown is close to 200 m/s. This gas jet acta aa a cutting edge on the exposed drop surface. As soon as the drop protrudes from the capillary
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 9, MAY 1, 1993 Scan 1048 (23.273min) of H0403b.d SUBTRACTED SCALED Ahtindance 8000
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efficient introduction of the effluent from a capillary column into the ion source via a particle beam interface. The modifications in the process governing aeroaolformation may alter some of the parameters specificto this kind of interface. Some of these changes were found to be very positive for the LC/MS requirementa and may increase the application possibilitiesof this technique. The best approach is probably to compare the new interface to a conventional one by performing specific experiments. Of course, in our laboratory the nebulizer supplied with the 69980B Hewlett-Packard particle beam unit was available. Ligon and DornG and Baczynskyj' modified their particle beam interfaces to make them compatible with a double-sector mass spectrometer. In their work they outlined several interesting strategies for investigatinginterface performance. Although caffeine is far from being considered a reference compound for a particle beam interface evaluation, it is still widely used for interface optimization and it often represents the first step in the use
of particle beam. We thought that caffeine could be considered a valid approach to the problem, while leaving open the possibility of extending the study to other compounds in subsequent experimentation. A calibration curve and the evaluation of result reproducibility using the new interface were performed. A 60-nL loop of a solutioncontaininga given amount of caffeine was injected five times for each concentration. The sample was introduced directly into the mass spectrometer via the new particle beam interface, without the column and with a mobile phase composed of equal concentrations of water and acetonitrile. The mass spectrometer was operating in SIM at m/z 194. The resulta are reported in Table I. The mean standard deviation, calculated by using the average of the peak area values for each concentration experiment, is f3.4 % A critical aspect of any LC/MS interface is ita response to a modification in the mobile-phase composition. Since most HPLC analyses are carried out using a proper gradient of
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ANALYTICAL CHEMISTRY, VOL. 85, NO. 9, MAY 1, 1993
mixable solvents, the LC/MS system response to variations in the effluent composition should be minimal. A comparison of a conventional PB-MS interface and the microversion is reported in Figure 4. An easily detectable amount of caffeine was injected with both interfaces, using different relative Concentrations of water and acetonitrile in the mobile phase. The m/z 194 ion signal intensity versus an increasing concentration of water was plotted, and it is reported in Figure 4a,b. Because of difficulties in correct mixing of solvents using micro flow rates with a binary HPLC system, relative concentrations higher than 90% or lower than 10% were not considered in this experiment. The difference between the two interfaces is clearly shown: The interface for microcolumns (Figure 4a) gives a flat response in a wide range of concentrations while the conventional interface (Figure 4b) shows a decrease in performance for mobile phases with increasing water content. Consequently it can be pointed out that the modified particle beam interface allows greater LC/MS flexibility when gradient analyses are performed. These results may be explained by the differencesin the nature of aerosol droplet production: In the microinterface the droplets are mechanically generated by the violent impact of the gas on the growing effluent droplet rather than by a spontaneous random liquid breakup. With the new interface, the influence of surface-active properties and polarity of liquidsmay have a minor role in aeroeol generation. Moreover, the evaporation process, in the direction of the momentum separator, is certainly accelerated by a consistent reduction in droplet size which offers an increased surface area. An indicative evaluation of the dimension of the droplets has been obtained through photographic images, with the assumption that the maximum diameter of the droplets is 5 pm. The improved efficiency in aerosol production may be observed through a better signal to noise response. The minimum detectable amount of caffeine which gives an interpretable mass spectrum is lowered by a factor of 10. Figure 6 shows 30 ng of caffeine injected with the new interface and its relative spectrum. The analysis was carried out with an 1:l ratio of water and acetonitrile in the mobile phase. To check the integrity of the spectrum at low and high concentrations, two different amounts of testosterone were injected into the system. Figure 6a shows the mass spectrum obtained
1287
by injecting 60 ng of testosterone while Figure 6b shows the results when 600 ng is used to collect the spectrum. Other than the different threshold used during the acquisition, no appreciable differences are evident in the two spectra. In isocratic conditions, an increase in solvent polarity usually results in a decrease in sensitivity. The effects of modifying solvent composition while recording the molecular ion of caffeine are shown in Figure 7. An iaocratic pump was employed and flow rate was kept at 1 pL/min during the experiment. A reversed-phase gradient micro PB-MS analysis of six human hormones is reported as an electron impact total ion profile in Figure 8a. A poly ether ether ketone (PEEK) C18 packed capillary column was used for this separation. The mobile-phase flow rate was kept at 3 pL/min. To evaluate chromatographic band broadening caused by possible sample interactions in the modified particle beam interface, an UV on-line codetection of the hormone mixture was performed. The 50-pm-i.d. fused-silica tubing used in the aerosol generation was converted in a UV detector microcell placed between the chromatographic column and the nebulizer. The UV profile obtained on-line prior MS analysis is shown in Figure 8b. No appreciable differences in the peak shape or width can be discerned by comparing the two profiles. Micro PB-MS analysis supplies high-quality, librarysearchable E1 spectra. Figure 9 shows a comparison between a representative micro particle beam mass spectrum (testosterone) and the corresponding NBS/ Wiley reference library mass spectrum. The two spectra are very similar visually and fully overlapping. The lowest detectable amount of a test sample (caffeine) injected via micro particle beam interface was 0.1 ng with a signal to noise ratio of 3:l for the m/z 194 ion current profile (Figure 10).
ACKNOWLEDGMENT We gratefully acknowledge Giorgio Famiglini for his invaluable assistance.
RECEIVEDfor review August 10, 1992. Accepted December 3, 1992.
