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Anal. Chem. 1987, 59, 2642-2646
Ion Spray Interface for Combined Liquid Chromatography/Atmospheric Pressure Ionization Mass Spectrometry Andries P. Bruins,’ Thomas R. Covey, and Jack D. Henion* Cornell University, Drug Testing and Toxicology Program, Ithaca, New York 14850
An Interface for liquid chromatography/mass spectrometry (LC/MS) was constructed that combines the principles of lon evaporatlon and electrospray. This devlce Is called an “ion spray” LC/MS Interface. The total etfiuent from a mlcrobore LC Is fed through a pneumatlc nebullrer floatlng at f3 kV and dispersed Into charged droplets In dry nltrogen at atmoepherk pressure. Ions emitted by the charged droplets are mass analyzed by a triple quadrupole mass spectrometer capable of sampling Ions from a region at atmospheric pressure. Ions lndlcatlng the molecular welgM of a sample are observed wlth little or no fragmentatlon. Multiply charged Ions in solutlon show multiply charged lons In the corresponding mass spectra. Colilslon-Induced dlssoclatlon reactions generate fragment ions correspondlng to structural units. The full-scan detection limit appears to be about 10 ng for ion& or selected neutral compounds and about 10 pg under selected ion monitoring conditions.
The emission of ions from charged droplets offers a mild ionization technique for mass spectral determination of polar and ionic compounds ( I ) . This process, which has been called ion evaporation (Z), not only takes place a t atmospheric pressure but also is considered one of the ionization mechanisms in the low pressure thermospray ion source (3). Combined liquid chromatography/mass spectrometry (LC/MS) with a thermospray interface is now widely used and the interface can be purchased for nearly all commercially available mass spectrometers. The interface accepts the total flow of effluent from a standard HPLC column operated at 1-2 mL/min and has proven to be rugged and reliable. On the other hand, it requires careful control of temperature at the vaporizer and the ion source. The heat applied to the LC effluent stream may cause decomposition of thermally labile molecules and ion current instability is a frequent problem. The fundamental problem of LC/MS with conventional flow rates is the inability of a standard chemical ionization (CI) mass spectrometer vacuum system to pump the total solvent vapor of the evaporated eluent. The thermospray LC/MS combinationhas cleared this hurdle by the connection of an additional pumping line directly to the ion source. No vacuum at all in the ion source clearly obviates the problem altogether. Indeed, one of the earliest reports of on-line LC/MS described the use of an atmospheric pressure ion source ( 4 , 5 ) . Understandably, interest in atmospheric pressure ionization has been limited since the traditional manufacturers of analytical mass spectrometers do not offer an API source. However, atmospheric pressure ion sources can take advantage of the simple construction and reliable operation of LC/MS interfaces with pneumatic nebulizers for the dispersion of On temporary leave from State University, Department of Pharmacy,A. Deusinglaan 2,9713 AW Groningen, The Netherlands. 0003-2700/87/0359-2642$01.50/0
liquids such as the heated pneumatic nebulizer (6) and the liquid ion evaporation interface (7, 8). The API source is also required for the electrospray interface for LC/MS. The generation of a charged aerosol beam by electrospray in air at atmospheric pressure was first reported in the scientific literature in 1917 (9) but reference has been made to an experiment performed in as early as 1745 (10). When a liquid in a small capillary is held at several kilovolts, a fine mist or a thread of droplets is generated by electrical forces that draw the liquid out of the capillary even without the use of a pump (11). The liquid flow rate is a few microliters per minute or less but can be increased to liters per minute for spray painting (12). Electrospray takes place at a lower spray voltage if a grounded plate is positioned opposite the capillary (13). The natures of the solvent and the capillary-to-plate distance are among the parameters that affect the spray (9-11,13). An increased liquid flow rate is obtained by pumping the liquid into the capillary, but a stable fine spray is increasingly difficult to obtain above 10 FL/min. Electrospray as an ionization technique for mass spectrometry has the potential of producing ions even at very high mass, as was demonstrated by Dole and co-workers (14). However, mass analysis and detection of ions generated from polystyrene molecules with an average molecular weight of 51 000 is nearly an impossible task with modern instrumentation. Besides claims in patents (15-1 7) there has been no report of experimental data on smaller molecules. Two research groups have demonstrated that electrospray is feasible as an LC/MS interface for microbore LC with 5-10 FL/min flow rates (18-21). Since electrospray operates without the input of heat into the spray-ionization step, labile and polar samples are ionized without thermal degradation (20). The atmospheric pressure ion evaporation interface for LC/MS (7, 8), which also does not use heat, and differs from the electrospray interface only in the spray and charge process, has already produced a mass spectrum of the very labile compound adenosine triphosphate. A problem in atmospheric pressure ion sources is the sampling of ions into the vacuum region of the mass spectrometer without extensive clustering with water and other polar molecules during the free-jet expansion stage. A dry nitrogen gas curtain in front of the ion sampling orifice (2) prevents solvent vapor from entering the orifice. Ions are drawn toward the orifice by an electric field. However, many sample, solvent, and other background ions are formed as clusters with neutrals in the ionization region and are as such admitted into the ion sampling orifice. By acceleration of these cluster ions in the free-jet expansion stage, cluster ions collide with nitrogen curtain gas molecules that have carried ions into the sampling orifice. A mild collision suffices to break hydrogen bonds in cluster ions; vigorous collision, however, can cause fragmentation of sample ions (21, 22). The API source equipped with the curtain gas and declustering region as described above is well suited for the incorporation of electrosprayionization (Sciex Inc., Thornhill, Ontario, Canada). Furthermore, its large size (22 cm diameter, 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987 - 3 . 0 kV
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Flgure 1. Schematic diagram of the ion spray interface and atmospheric pressure ion source (not drawn to scale) with nitrogen gas curtain: (1) 50 pm i.d. fused-silica capillary; (2) 0.20 mm 1.d. stainless steel capillary; (3) 0.8 mm i.d. Teflon tube with narrow bore insert; (4) ion focusing lens, serving as counter electrode for ion spray; (5) orifice holding plate with 100 pm i.d. conical orifice. Drawing not to scale.
12 cm depth) allows great flexibility in the construction and positioning of an LC/MS interface. The instruments used thus far for electrospray ionization were home-made or extensively modified commercial mass spectrometers (18-21). We describe the construction of an improved ion evaporation/electrospray LC/MS interface for a standard, commercially available API mass spectrometer.
EXPERIMENTAL SECTION A Sciex TAGA 6O0OE triple quadrupole mass spectrometer data system combination equipped with an atmospheric pressure ion source was used. Ions are sampled into the vacuum system through a 100 pm i.d. orifice in the tip of a cone pointing toward the source region. The area in front of the orifice is flushed with high-purity dry nitrogen gas which acts as a gas curtain to keep solvent vapor and contaminants away from the orifice. High vacuum in the mass analyzer is maintained by cylindrical cryosurfaces maintained at 16-20 K located around the quadrupoles. While mass spectra are recorded, the indicated pressure is 9 x lo4 Torr. When argon is introduced for collision induced dissociation (CID), the pressure rises to 2 X Torr at a target gas thickness of 300 X 10l2atoms/cm2 in the second quadrupole (8). The collision energy was 120 eV in the laboratory frame. Figure 1 is a schematic drawing of the nebulizer-assisted electrospray interface. Standard fittings, unions, and tee pieces were used for connecting the concentric tubes. For negative ion operation, the stainless steel capillary no. 2 is kept at -3 kV and for positive ions, at +3 kV. These voltages were obtained from a reversible polarity, 0-10 kV power supply (RHR 10PN30, Spellman, Plainview, NY). Sharp edges were removed from the spray tip of the stainless steel capillary by electropolishing. Nitrogen at 2.5 bar pressure was used for pneumatic nebulization to provide a measured linear gas velocity at the exit orifice of 216 m/s. This high gas velocity facilitates “centering” the stainless steel capillary (2) at the tip of the Teflon tube (3). The position of each of the three concentric tubes was adjusted to give a fine symmetrical spray plume. This is achieved when tube 1protrudes from 2, which in turn protrudes from the Teflon nebulizing tube. We assume that a conducting layer of liquid acts as a connector between capillary 2 and the liquid meniscus. The protective polyimide coating should not be removed from the tip of fused silica tubing 1. The length of the interface is not critical and can be adapted to the dimensions of a particular ion source. Mass calibration of the quadrupole mass filters was done on the cluster ion series CHSC00--(H20),,produced when the interface passes 0.001 M ammonium acetate and is directed on-axis toward the ion samplingorifice. During normal LC/MS operation, the interface was positioned 5-10 mm off-axis to prevent the sampling of large cluster ions. Nitrogen gas boiled off from liquid nitrogen was used as the nebulizing gas. Liquid chromatographywas done with a Waters 510 pump, and a Model 680 flow and gradient controller (Milford, MA). A Rheodyne 7520 injector (0.2-pL or 1.0-pL sample loop) (Cotati, CA) and a Shandon Hypersil3-pm CIS,1 mm X 10 cm column (Scwickley, PA) completed the micro HPLC system. A 1m length
of 50 pm i.d. fused-silica tubing (no. 1 in Figure 1)was used to transfer the effluent from the column to the ion source. Most samples were separated on the column,but a few were introduced into the source by flow injection without a column. A W detector was not used in micro LC/MS experiments to avoid chromatographic band broadening resulting from the UV detector cell and associated connecting tubing. Sampleswere eluted under isocratic conditions.
RESULTS AND DISCUSSION Construction. In our first experiments on electrospray ionization, a stainless steel capillary with a fused-silica transfer line inside was placed 10-20 mm from its counter electrode in the ion source (14,18). The capillary was placed 5-10 mm off-axis which increased sample ion current and prevented droplets from hitting the ion sampling orifice. A fine smoke (11) or fog (23) could only be generated if the flow rate was below 10 rL/min and the precentage of water in methanol or acetonitrile was low. The position of the sprayer relative to the ion sampling cone had to be carefully adjusted for optimum performance but was easily reproduced once located. At higher flow rates and/or higher percentages of water, the electrosprayer produced a stream of larger droplets together with the desired smoke or fog. An increased voltage applied to the spray capillary dispersed the large droplets, but at the same time initiated a corona discharge (18-20) that substantially lowered the sensitivity for samples ionized in solution. A corona discharge is most easily formed during negative ion operation. Flushing the area around the spray capillary with pure oxygen suppresses the discharge by the capture of electrons emitted from the spray capillary (19). We found that a gentle flow of zero air or nitrogen doped with Freon was equally effective for preventing a discharge. Experience with pneumatic nebulizers incorporated in the heated pneumatic nebulizer and ion evaporation interfaces delivered with our instrument prompted us to construct an interface as shown in Figure 1. The electron scavenging gas blowing coaxially around the spray capillary also serves the purpose of nebulizing the liquid stream. The combination of pneumatic nebulization and an electric field tolerates higher eluent flow rates and a higher percentage of water in the formation of a spray of charged droplets. In pure electrospray a high electric field is required for both nebulization and charging. Experiments with the ion evaporation system (7) have already shown that a moderate voltage of *3 kV is sufficient for charging droplets when a liquid is pneumatically nebulized even at flow rates as high as 1 mL/min. The nebulizer-assisted electrospray interface shown in Figure 1 has been tested a t flow rates up to 0.2 mL/min, but it was found that better sensitivity is obtained at 40 pL/min, a flow rate compatible with 1 mm i.d. packed microbore columns.
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987
TOTAL
ION CURRENT
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Figure 2. Total ion current profile for ion spray LC/MS analysis of a mixture of five monosulfonated azo dyes and mass spectrum of the last component: (1) acM orange 7, (2) acid red 337, (3, 4) acM red 88 (2 isomers), (5) acid red 151; sample size approximately 20 ng per component; column 1 mm X 100 mm Hypersil 3-pm C,8 (Shandon); flow rate, 40 hL/min; isocratic elution, 30% acetonitrile, 70% 0.001 M ammonium acetate in water.
A second improvement over pure electrospray is that nebulizer assistance allows a larger distance between the spray capillary and its counter electrode, which, together with f3 kV spray voltage, reduces the electric field at the tip and prevents a corona discharge even with nitrogen as the nebulizing gas. Pneumatic nebulization has made the spray process independent from the position of the interface inside the ion source although best sensitivity is obtained at 3-4 cm from the counter electrode which houses the ion sampling orifice and 5-10 mm off-axis from the orifice. Operation on-axis suppressed the signals of samples and showed signals for cluster ions that were useful for mass calibration up to m/z 900. There are similarities and differences between nebulizerassisted electrospray described here and ion evaporation (2, 7). Both methods operate at room temperature and use the same spray and ionization process. The differences are in the liquid flow rate, the arrangement of electric fields, and the direction of the spray toward the ion sampling orifice for electrospray and across the orifice for ion evaporation. It appears this device has some of the merits of ion evaporation and electrospray and as such we propose to call it “ion spray”. Applications. The emission of ions from charged droplets is most effective if a sample is present as an ion in solution as was demonstrated for ion evaporation (2, 7 , B ) . The ion spray interface produces [M - HI- ions of the free acid form of monosulfonated azo dyes, steroid glucuronides,and steroid sulfates that were injected into the LC as sodium or potassium salts. Figure 2 gives an example of a total ion current (TIC) profile for the on-line micro LC/MS determination for a synthetic mixture of five organic sulfonated azo dyes. In the case of positive ions, dyes that contain positively charged quaternary nitrogen atoms show the corresponding positive ion as the base peak in the spectrum. Reduction of these dyestuffs, as observed in SIMS by Gale et al. (% did not I)take , place in our hands by ion spray LC/MS. Sample molecules with more than one ionizable functionality gave multiply charged ions. For example, disulfonated azo dyes and estradiol glucuronide sulfate gave [M - 2HI2ions of the corresponding free acids, while estriol trisulfate gave the [M - 3H]%ion as the base peak as shown in Figure 3. Sample molecules that are not ionized in solution can be lifted out of charged droplets by association with other ions (2, 7,8). Amines introduced in neutral solution were observed
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Flgure 3. Ion spray mass spectrum of estriol trisulfate. Column and flow rate see Figure 2, 20% acetonitrile, 80% 0.001 M ammonium acetate. Sample size was 200 ng. +
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