Anal. Chem. 1994,66, 3664-3675
Liquid Chromatography/Time-of-Flight Mass Spectrometry with a Pulsed Sample Introduction Interface Alan P. L. Wang,t Xijian Guo, and Liang Li’ Department of Chemistry, Universiw of Alberta, Edmonton, Alberta, Canada T6G 2G2
Conventional high-performance liquid chromatography (LC) has been combined with time-of-flight mass spectrometry (TOFMS) with the use of a pulsed sample introduction (PSI) interface. The ion chromatogram obtained by using LC/ TOFMS is similar to that obtained with a UV detector. No significant peak distortion is introduced by the PSI interface. Various experimental parameters affecting the performance of the LC/TOFMS system are investigated. It is shown that the PSI LC/TOFMS system can handle liquid flow rates ranging from 0.5 to 1.6 mL/min. Water and various organic solvents can be used as the mobile phase. Volatile buffers and solvent modifiers can also be used. Several other parameters affecting the system performance, including the temperature of the sample vaporizer and the capillary tube in the interface as well as the flow rate of the makeup gas, are also investigated. In addition, the effective sample transfer efficiency of the PSI interface is studied. It is shown that, by pulsing only 0.31%0.61%LC effluentinto the mass spectrometer with the interface, a chromatogram can be obtained with its peak area equivalent to that obtained by introducing 37%-72% sample into the system continuously. This represents an increase of sampling duty cycle by a factor of 118 using the pulsed technique, although the overall detection duty cycle of the present LC/TOFMS system is still low. Finally, several examples of separation and detection by the PSI LC/TOFMS system are given to illustrate its analytical capability. The advantages and limitations of the PSI interface in comparison with other interfacing techniques are briefly discussed. Mass spectrometry combined with chromatographic separation is one of the most powerful techniques for trace analysis. Techniques such as gas chromatography/mass spectrometry (GC/MS) have become a routine method for the detection of organic compounds in a variety of complex matrices. However, many polar, nonvolatile molecules and thermally labile chemicals, including many highly water-soluble organic pollutants and biochemicals, are not amenable to analysis by GC/MS. At present, the separation of these molecules is generally achieved with high-performance liquid chromatography (LC). Thus, to extend the MS technique for the detection of these molecules, a powerful on-lineLC/MS system should prove very useful. Indeed, the research on the development of LC/MS systems has been actively pursued in the past decade or s0.l The great challenge here is to develop suitable interfaces for introducing the liquid flow into the mass spectrometer without significant compromise in the operating conditions of either the LC or the mass spectrometer. + Current address: Department of Chemistry, Hong Kong Baptist College, Hong Kong.
3664
Analytical Chemistry, Vol. 66, No. 21, November 1, 1994
There are several on-line interfaces that have been developed for LC/MS with varying degrees of ~ u c c e s s . ~These - ~ ~ include continuous-flow fast atom bombardment,”8 thermospray?-’’ particle beam,12J3heated pneumatic nebulizer/atmospheric pressure chemical ionization (AFCI),1”18 and e 1 e c t r o ~ p r a y . l ~ ~ ~ Among them, the electrospray technique has been proven to be particularly versatile for combining LC with MS for the detection of both low and high molecular weight chemical species.19-30 In developing a LC interface, the type of mass analyzer used for ion detection also deserves careful consideration. Thus far, almost all commercial LC/MS systems utilize scanning mass analyzers (sectors or quadrupole). However, time-offlight mass spectrometry (TOFMS) has recently received considerable attention for further development as a tool for LC detection.3143 There are several advantages in using TOFMS as a LC detector. TOFMS can potentially provide much higher detection sensitivity with unlimited mass range due to its higher ion transmission. In addition, a complete spectrum, equivalent to a full scan spectrum of a scanning mass spectrometer, can be recorded within a single pulse. Furthermore, because a mass spectrum can be obtained in a short time period, the spectral acquisition rate of TOFMS can be very high. Using current data processing technology, over 10 spectra/s recording speed can be readily achieved. This unique feature should prove particularly useful in (1) For a recent comprehensive review, see the following for examples: (a) Liquid ChromatographylMass Spectrometry, Applications in Agricultural, Pharmaceutical, and Environmental Chemistry; Brown, M. A., Ed.; American Chemical Society: Washington, DC, 1990.(b) Liquid Chromotographypfass Spectrometry: Techniques and Applications; Yergey. A. L., Edmonds, C. G., Lewis, I. A. S., Vestal, M. L., Eds.; Plenum Press: New York, 1990. (c) Liquid Chromatography-Mass Spectrometry;Niessen, W. M. A,, van der Greef, J., Eds.; Marcel Dekker: New York, 1992. (2) Scott, R.; Scott, C.; Munroe, M.; Hess, J., Jr. J. Chromatogr. 1974, 99, 395. (3) Tal’Rose, V.; Karpov, G.; Gordoetshii, I.; Skurat, V. Russ. J . Phys. Chem. 1968, 42, 1658. (4) Baldwin, M.; McLafferty, F. Org. Mass Spectrom. 1973, 7 , 11 1. (5) Henion, J. D. Anal. Chem. 1978, 50, 1687. (6) Caprioli, R. M.; Fan, T.;Cottrell, J. S . Anal. Chem. 1986, 58, 2949. (7) Ito, Y.; Takeuchi, T.; Ishi, D.; Goto, M.J. Chromatogr. 1985, 346, 161. (8) Continuous-flow Fast Atom Bombardment Mass Spectrometry; Caprioli, R. M., Ed.; John Wiley: New York, 1990. (9) Blakley, C. R.;Vestal, M. L. Anal. Chem. 1983, 55, 750. (10) Vestal, M. L; Fergusson, G. J. Anal. Chem. 1985, 57, 2372. (11) Vestal, M. L. Methods Enzymol. 1991, 193, 107 (12) Willoughby, R. C.; Browner, R. F. Anal. Chem. 1984, 56, 2626. (13) Wider, P. C.; Perkins, D. B.; Williams, W. K.; Browner, R. F. Anal. Chem. 1988.60, 489. (14) Carroll, D. 1.;Dzidic, I.; Stillwell, R. N.; Haegele, K. D.; Homing, E. C. Anal. Chem. 1975, 47, 2369. (15) Henion, J. D.; Thomson, B. A.; Dawson, P. H. Anal. Chem. 1982, 54, 451. (16) Thomson, B. A.; Iribarne, J. V.; Dziedic, P. J. Anal. Chem. 1982, 54, 2219. (17) Eckerlin, R. H.; E M , J. G.; Henion, J. D.; Covey, T.R. Anal. Chem. 1989, 61, 53A. (18) Sakairi, M.; Kambara, H. Anal. Chem. 1988, 60, 774. (19) Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B. Anal. Chem. 1985, 57, 675. (20) Smith, R. D.; Loo,J. A.; Loo, R. R. 0.; Busman, M.;Udseth, H. R. Mass Spectrom. Rev. 1991, 10, 359.
0003-2700/94/0366-3664$04.50/0
0 1994 American Chemical Society
capturing fast-rising peaks in some forms of LC experimental parameters that affect the performance of the ~ e p a r a t i o n s This . ~ ~ high recording speed should also provide interface. Second, we demonstrate that this interface can be an opportunity in recording daughter spectra on the chroused to combine conventional, high-flow LC with TOFMS. matographic elution time scale, if tandem TOFMS is to be Several examples of separation and detection by the PSI LC/ TOFMS system are given to illustrate its analytical capability. used. However, unlike scanning MS, TOFMS detects ions In addition, the advantages and limitations of the PSI interface in a pulsed form. This presents a challenge as well as a unique opportunity in developing LC interfaces. Here the sample in comparison with other interfacing techniques are briefly utilization efficiency is a major concern in designing an discussed. interface. EXPERIMENTAL SECTION Recently, several interfaces have been reported for comLiquid Chromatography. The LC solvent delivery system bining LC with TOFMS.3143 These include continuous-flow consists of two Shimadzu LC-600 programmable liquid pumps fast atom bombardment (CF-FAB)33and e l e c t r ~ s p r a y . ~ ~ ~ ~ capable of delivering solvent with variable flow rates ranging Interfacing techniques are also being developed for combining from 1 pL/min to 10 mL/min. For chromatographic work, LC with matrix-assisted laser desorption ionization (MALDI) a Waters Nova-Pak C- 18column (Millipore Ltd., Mississauga, in TOFMS.35-38In electrospray/TOFMS, in order toincrease Ontario, Canada) is used for separation. A Rheodyne Model the sampling efficiency, ion storage devices can be used for 7125 syringe loading injector with either a 5-pL or a 20-pL the accumulation of ions between pulses. In the continuoussample loop is used for sample injection. In between the flow experiments, a flow probe is used in which the LC effluents column and the PSI interface, a Shimadzu SPD-M6A flow to the probe surface. Between the desorption events, photodiode array detector is used for obtaining a UV sample and matrix are concentrated on the probe due to solvent chromatogram. All flow lines are connected with the use of evaporation in vacuum. An alternative strategy for increasing 1/ 16-in.-0.d. and 0.040-in.4.d. stainless steel tubing. sampling efficiency is to use a pulsed sample introduction Pulsed Sample Introduction Interface. The PSI interface (PSI) method to introduce the LC effluents into TOFMS in has been described previou~ly.~~ In brief, it consists of a heated a pulsed form.34 This will take full advantage of the pulsed capillary tube for aerosol generation and a high-temperature ion detection nature of the TOFMS. The idea here is to use pulsed nozzle for sample vaporization (see Figure 1). The a large orifice in the interface to introduce large amounts of nozzle consists of two components, i.e., the nozzle body and sample in each pulse. This will result in more intense signals the nozzle head, separated by a water cooling system. Besides within a given pulse. An ion chromatogram constructed with the sample vaporizer, the nozzle head also contains an aerosol a number of such pulses would give large peak areas. We inlet and a gas outlet to waste. In the experiment, the effluent have previously reported the design of such a PSI interface from the LC flows without splitting into a 5-cm-long, 150for introducing neutral molecules into a time-of-flight mass pm-i.d. capillary tube. A Nichrome 60 heating coil (Pelican ~pectrometer.~~ In the PSI interface, sample molecules carried Wire Co., Naples, FL) is wrapped around the capillary tube by a liquid carrier pass through a heated capillary tube to for heating. The temperature of the tube is not uniform across generate an aerosol and then through a sample vaporizer of the 5-cm total length. Only the temperature in the middle of a high-temperature pulsed nozzle. The resulting samplevapors this tube is monitored by a type-K thermocouple (TC) and are introduced into TOFMS in a pulsed form and ionized by is measured to be -200 'C for most work reported here. laser-induced multiphoton ionization (MPI). After passing through the tube, the liquid flow is converted We report here our further studies on the PSI interface. into aerosol droplets and/or supersaturated vapor. These The main objectives of this report are twofold. First, in aerosols are then expanded into the sample vaporizer for developing the PSI method, an important parameter is the complete desolvation and vaporization. sampling efficiency associated with the PSI interface. We A stainless steel plate with a 700-pm-diameter orifice is report here the study of the effective sample transfer efficiency sealed to the sample vaporizer of the nozzle head using a in this PSI interface, along with our studies on the other copper gasket and knife edge seal. The inner area of the (21) Bruins, A. P.; Covey, T. R.; Henion, J. D. Anal. Cfiem. 1985, 59, 2642. sample vaporizer is 25 mm in diameter and 8 mm in length. (22) Bruins, A. P.; Weidolf, L. 0. G.; Henion, J. D.; Budde, W. L. Anal. Cfiem. It is heated by a coil of thermocoax heating wire (Phillips 1985, 59, 2647. (23) Pleasance, S.;Quilliam, M. A.; de Freitas, A. S.W.; Marr, J. C.; Cembella, A. D. Rapid Commun. Mass Spectrom. 1990, 4, 206. (24) Hail, M.; Lewis, S.;Jardine, I.; Liu, J.; Novotny, M. J . Microcolumn Sep. 1990, 2, 285. (25) Moseley, M. A.; Deterding, L. J.; Tomer, K. B.; Jorgenson, J. W. Anal. Chem. 1991, 63, 1467. (26) Perkins, J. R.; Parker, C. E.; Tomer, K. B. J. Am. Soc. MassSpecfrom. 1992, 3, 139. (27) Hemling, M. E.; Roberts, G. D.; Johnson, W.; Carr, S. A,; Covey, T. R. Biomed. Enoiron. Mass Spectrom. 1990, 19, 677. (28) Ling, V.; Guzetta, A. W.; Canova-Davis, E.; Stults, J. T.; Hancock, W. S.; Covey, T. R.; Shushan, B. I. Anal. Chem. 1991.63, 2909. (29) Hopfgartner, G.;Wachs, T.; Bean, K.; Henion, J. Anal. Cfiem.1993,65,439. (30) Banks, J. F., Jr.; Shen, S.;Whitehouse, C. M.; Fenn, J. B. Anal. Cfiem.1994, 66, 406. (31) Pang, H. M.; Sin, C. H.; Lubman, D. M. Appl. Spectrosc. 1988, 42, 1200. (32) Lustig, D. A.; Lubman, D. M. Rev. Sci. Instrum. 1991, 62, 957. (33) 33. Emary, W. B.; Lys, I.; Cotter, R. J.; Simpson, R.; Hoffman, A. Anal. Cfiem. 1990, 62, 1319. (34) Wang, A. P. L.; Li, L. Anal. Cfiem. 1992, 64, 769. (35) Li, L.; Wang, A. P. L.; Coulson, L. D. Anal. Cfiem. 1993, 65, 493.
(36) Williams, E. R.; Jones, G. C., Jr.; Fang, L.; Nagata, N.; Zare, R. N. In Applied Spectroscopy in Materials Science I t Golden, W. G . , Ed.; Proc. SPIE-Int. SOC.Opt. Eng. 1992, 1636, p172. (37) Murray, K. K.; Russell, D. H. Anal. Cfiem. 1993, 65, 2534. (38) Murray, K. K.; Russell, D. H. J . Am. SOC.Mass Spectrom. 1994, 5, 1. (39) Dodonov, A. F.; Chernushevich, I. V.; Laiko, V. V. Book ofAibstracts, 12th; International Mass Spectrometry Conference, Amsterdam, August 1991; Abstract 153. (40) Boyle, J. G.; Whitehouse, C. M. Anal. Cfiem. 1992, 64, 2084. (41) Zhao, J.; Zhu, J.; Lubman, D. M. Anal. Cfiem. 1992, 64, 1426. (42) Mirgorcdskaya, 0.A.; Shevchenko, A. A.; Chernushevich, I. V.; Dodonov, A. F.; Miroshnikov, A. I. Anal. Cfiem. 1994, 66, 99. (43) Verentchikov, A. N.; Ens, W.; Standing, K.G.Anal. Cfiem. 1994, 66, 126. (44) Kalghatgi, K.; Horvath, C. J. J . Cfiromatogr.1987, 398. 335. (45) Nugent, K. D. In HPLC ofpeptides and Proteins; Mant, C. T . , Hodges, R. S.,Eds.; CRC: Boca Raton, FL, 1991; p 697. (46) Monnig, C. A.; Dohmeier, D. M.; Jorgenson, J. W. Anal. Chem. 1991.63.807. (47) Kassel, D. B.; Shushan, B.; Sakuma, T.; Salzmann, J. P. Proceedings of the 41st ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, May 3GJune 4, 1993; p 10.
Analytical Chemistry, Vol. 66,No. 21, November 1. 1994
3665
1 T
TO
Electrical
waste +--
I
Copper, S t r i p
Make
Solenoid
Liquid I n l e t
Flgure 1. Schematic of the design of the pulsed sample introduction interface. Drawing is not to scale. (Adapted from ref 34.)
Electronic Instruments, Norcross, GA). In most studies reported here, the temperature of the nozzle front is >480 OC. The temperature near the heating wire is about 600 OC. The actual temperature of the aerosol is unknown, since a TC cannot be placed inside the sample vaporizer with the current design. Judging from the temperature gradient we observe across the nozzle head, we believe that the temperatures of the inner wall of the sample vaporizer and the aerosol are much lower than 480 OC. A solenoid placed inside the cold nozzle body is used to drive a plunger for introducing the sample vapor into the mass spectrometer in a pulsed form. The plunger is made of a long stainlesssteel needle (1.5 mm in diameter and 55 mm in length) which screws into a steel base for magnetic attraction. The other end of the needle forms a metal-to-metal seal against the orifice. During the operation, a voltage is applied to the solenoid to generate a magnetic field that attracts the plunger away from the orifice to form a jet. When the power is off, a spring is used to push the plunger against the orifice to make a seal. The sample pulse width is measured to be about 1 ms fwhm for the current experimental setup. Thus, although sample vapors are continuously generated inside the sample vaporizer, only a small portion are pulsed into the mass spectrometer. The pulse repetition rate used for the solenoid is the same as that of the ionization laser beam.
Time-of-FlightMass Spectrometer. A linear time-of-flight mass spectrometer is used for ion detection.34 The flight tube is mounted vertically in a six-port cross pumped by a 6-in. diffusion pump (Varian Associated, Inc., Lexington, MA). The 1-m-long flight tube is differentially pumped by a 4-in. diffusion pump (Varian). A liquid nitrogen (LN2) trap assembly is used to cool part of the flight tube and the entire acceleration lens assembly. The trap was manufactured by R. M. Jordan Co. (Grass Valley, CA) to our specifications. All other vacuum components were constructed in-house. The pressure of the system during operation is normally 5 cm) is not necessary to generate stable signals. Shorter tubes are less prone to the clogging problem. With a 5-cm-long tube, clogging rarely occurs. We have also studied the flow rate dependence of the makeup gas on the performance of the interface. Figure 6 shows the peak area of indole-3-acetic acid as a function of the C02 flow rate. The mobile phase is methanol at a flow rate of 1 mL/min. Similar results are obtained when the mobile phase flow rate is in the range of 0.5-1.6 mL/min. It is found that the increase of the flow rate reduces the sensitivity of the system. However, the peak width is independent of the makeup gas flow rate in the working range examined. This is also true for other compounds examined, including vanillic acid and chrysene. It appears that the main function of the makeup gas in the interface for LC/TOFMS is to prevent feedbackof the hot vapor into thecold solenoid. The minimum flow rate needed to prevent feedbackis dependent on themobile phase flow rate. It is our experience that when the C02 flow rate used is less than 100 mL/min and the mobile phase flow rate is 1 mL/min, hot vapor feedback can sometimes occur
Table 1. Sample Transfer Efflclency of the Pulsed Sample Introductlon Interface effective transfer transfer compound m p ("C) yield (96) efficiency (%) tryptophol pyrene indole-3-acetic acid acetaminophen triphen ylene vanillic acid chrysene
H I
I
0.5
inch
U
Flgure 7. Schematic of the experimental setup for collecting the samples introduced by the PSI interface for the study of the effective transfer efficiencies.
during the experiment. Thus, the flow rate of the makeup gas is always kept above 100 mL/min, although the sensitivity may not be at its optimum, as shown in Figure 6. Other makeup gases such as helium and argon are also tested, and similar results are observed. However, COS is chosen here since it can be readily trapped by the liquid nitrogen trap once it is expanded into the vacuum system, resulting in low operational vacuum pressure. Sample Transfer Efficiency in the PSI Interface. Now that the major parameters affecting the performance have been assessed, the next question to be addressed is concerned with the sample transfer efficiency associated with the PSI interface. An ideal interface would introduce all of the sample into the mass spectrometer. With a pulsed ion detectionsystem such as TOFMS, one of the major goals in designing the PSI interface is to introduce a large amount of sample in a number of pulses to allow us to construct a chromatogram with a large peak area. The peak area is ultimately used for the determination of the sample concentration. The following experiment was performed to determine the sample transfer efficiencyin the PSI interface. Figure 7 shows the schematic diagram of the setup for collecting samples pulsed into the system by the PSI interface. The boat-shaped sample collector shown in Figure 7 is made of copper, and it is attached to a solid insertion probe. In the experiment, the sample collector is first immersed in liquid nitrogen for about 1 min and then inserted into the TOFMS. The liquid nitrogencooled sample collector is placed about 2 cm away from the orifice of the PSI interface (see Figure 7). A known amount of sample is injected into the interface and vaporized. After the gas phase molecules exit the orifice, they condense onto
59 149-151 165-169 169- 172 197-200 210-213 252-254
0.52 0.38 0.3 1 0.6 1 0.53 0.44 0.38
61 45 37 72 62 52 45
the sample collector. The amount of sample collected is then determined by using HPLC with a UV detector. The results of this measurement for several compounds are listed in Table 1. In this table, the sample transfer yield (Y) is defined as the ratio of the amount of the sample collected (N,l) to the amount of the sample injected (Ivinj). It appears that the variation in transfer yield from compound to compound is relatively small. This small variation could be due to the differences in vaporization efficiencyand the ability to generate a fine aerosol among the compounds examined. In order to make a direct comparison of the sample transfer efficiency between a continuous sample introduction interface and a pulsed sample introduction interface, we use the term effective sample transfer efficiency (E) to characterize the interface. Since quantitation in LC is based on the peak area in the chromatogram, we can define E as APIAt,where A, is the sample peak area of the chromatogram generated from the actual sample introduced by the interface in a continuous or pulsed form to the mass spectrometer, and At is the peak area of the chromatogram obtained by assuming the total sample injected is being continuously transferred into the MS system. In a continuous sample introduction interface, the calculation of the effective sample transfer efficiency is quite straightforward. Here, the peak area ( A ) is directly proportional to the total amount of sample being detected (N). A is equal to kN, where k is a constant related to the properties of the detector and the recorder. The peak area (Ap) from the chromatogram with an injection of Ninj moles of sample is equal to kN,l, if only N,l moles of sample are transported by the interface to the mass spectrometer and collected in the sample collector. However, the peak area (At) would be kNinj if all of the sample injected were introduced into the mass spectrometer. Thus, the effective sample transfer efficiency is equal to N,l/Ninj, or the sample transfer yield. In the PSI interface, the sample introduced into the system is concentrated into a number of pulses. From each sample pulse, an ionization signal or a data point can be generated. In the end, a chromatogram is constructed from these data points. Thus, the size of the peak area in this chromatogram is dependent on the intensity of the individual data points. The intensity of a data point is in turn directly related to the amount of thesample contained in the individual sample pulse. Although the total amount of sample pulsed into the vacuum over the entire peak elution time (N,l) is small in the PSI interface, the amount of sample per pulse can be quite high, resulting in a large peak area. Clearly, in the calculation of the effective sample transfer efficiency for the PSI interface, A, is not equal to kN,I. However, At is still equal to kNinj, Ana&tkal Chemktry, Vol. 66, No. 21, November 7, 1994
3669
as defined above. Thus, E = Ap/(kNinj). In reality, A, (in arbitrary units) can be easily calculated from the chromatogram, and Ninjis known. To find out the k value, we need to examine carefully how we construct the chromatogram. From the experiment, we know that the individual sample pulse profile is close to a rectangular shape with a width of about 1 ms. Now, if we assume that a chromatogram was constructed using a continuous ion detection technique, then the peak in the chromatogram would consist of a series of rectangles with equal widths but different heights. The area of an individual rectangle ( A i ) can be readily estimated by multiplying the height (Hi) by 1 ms, and it should be equal to kNi, where Ni is the amount of sample contained in the sample pulse. Thus, Ni = Ai/k = (Hi X l)/k. Since the sum of the amount of sample under all individual sample pulses should be the total amount of sample introduced into the mass spectrometer, we have Ncol = EN/ = CAi/k or k = CA~/N,,I.Thus, E = AP/ (Winj) = Ap/[(CAi/Nmi)Ninj] = (Ap/CAi)(NmI/Ninj) = YAP/ CAi. To calculate the peak area, a curve fitting method is used. Figure 8 shows a typical flow injection peak (dotted line) obtained by injecting indole-3-aceticacid into the LC/TOFMS system without theuse of a column. The solid line is generated from a curve fitting program, Matlab (The Mathworks, Inc., Natick, MA). The experimental data fit well to an exponentially modified Gaussian f u n ~ t i o n . This ~ ~ , ~function ~ is used to determine the peak area as well as the peak height. The peak height is then used to calculate the area of an individual sample pulse at a given elution time according to the equation, Ai = Hi X 1. The ratio between the peak area (Ap) in Figure 8 obtained by the fitting function and the area sum of all individual sample pulses (CAi) is determined to be 118. Thus, for indole-3-acetic acid, the effective sample transfer efficiency is 0.3 1% X 118 or 37%. This simply means that although only 0.31% of the sample or LC effluent is introduced into the vacuum system with the PSI interface, it is equivalent to transferring 37% of the sample into the mass spectrometer with a continuous sample introduction interface. However, without a sample enrichment device, it is difficult to introduce 37% of the LC effluent into the vacuum without overloading the pumping system. Since other compounds examined give peak profiles similar to those shown in Figure 8, the E values listed in Table 1 are simply calculated by multiplying Y by 118. Table 1 shows that the effective sample transfer efficiency ranges from 37% to 72% for the compounds examined. This demonstrates that the PSI interface provides an efficient means of introducing LC effluent into the mass spectrometer without overloading the vacuum pumping system. Combined with a pulsed ion detection system, it should provide high detection sensitivity. However, the overall sensitivity of the LC/TOFMS system is related not only to the sample transfer efficiency but also to other factors such as the background noise level, the ionization volume, the ionization efficiency, and the ion detection efficiency. Note that although the sampling efficiency has been increased by a factor of 118 using the pulsed method, the (51) Grushka, E.Anal. Chem. 1972, 44, 1733. ( 5 2 ) Folcy, J. P.J . Chromafogr.Sci. 1984, 22, 40.
3670
AnalyticalChemistry, Vol. 66,No. 21, November 1. 1994
000
experimental data
Time ( s e c )
Flgure 8. A typical peak obtained by the LWTOFMS system. The dotted line is the experlmental result from the injection of lndole-8 acetic acM. The solM line is generated from the curve fitting program.
overall detection duty cycleof the present LC/TOFMS system is still quite low. In this MPI technique, the pulse width of the ionization laser beam is very short, Le., 7 ns. The laser beam is operated at 10 Hz. Thus, the maximum ionization duty cycle that can be obtained with the laser ionization technique is 10 X 7 x = 7 X lo-* for a continuous sample beam. With the PSI interface, the effective duty cycle improves to be 7 X lo4 X 118 = 8.26 X 106. It should be noted that the pulse width of the sample beam produced with the present PSI interface is 1 ms, as indicated above. Clearly, the overall detection duty cycle of the LC/TOFMS system can be further improved by using longer ionization pulse width. Currently the detection limit of the system is compound dependent, ranging from picograms to nanograms. For example, the detection limits for aniline and tryptamine are about 10 and 100 pg, respectively. Figure 9 shows the mass spectrum of aniline obtained by using the PSI interface with a total injection of 20 pg of the sample (Le., 1 pg/ML). In Figure 9, the peak at m / z 93 is from the molecular ion of aniline. The peakat m / z 45 is from the background molecules. Performance of LC/TOFMS. Figure 10 shows the chromatograms of a mixture of seven polyaromatic hydrocarbons (PAHs). These seven PAHs (in the order of elution) are naphthalene, 1-methylnaphalene, acenaphthene, fluorene, phenanthrene, 1-methylphenanthrene, and 9-bromophenanthrene. The mixture of these chemicals at an amount of 10 ng each is separated under a solvent gradient condition. Initially, the mobile phase containing 70% CH3OH and 30% H 2 0 is kept at a flow rate of 1 mL/min for 6 min. The composition of the mobile phase is then gradually changed from 70% CH30H to 100%CH3OH in 20 min. Figure 10A is the LC/UV chromatogram recorded at 280 nm from the UV detector. Figure 10B shows the ion chromatogram of these seven PAHs obtained by using LC/TOFMS with the PSI interface. The GPIB-based data system is used for recording and storing all the mass spectra during the experiment, followed by mass spectral data processing for generating the ion chromatogram. As Figure 10 illustrates, the LC/TOFMS ion chromatogram is almost the same as the UVchromatogramin termsof peakshapes and retention times. No significant peak broadening or other chromatographic distortions are found.
1 .o
10
Mt
'
93
.-
45 6
:A) LC/UV
0.8
3
I
5
c
7
3
A
6 0.6
.-C
v) c
a
3
8
46
2 0.4
5
0.2
0.0 , 0
5
10
15
1 20
Time (min) 1 .o
(B) LCROFMS 4
0.8 0
50
100
150
MI2 Flgure 9. Multiphoton ionization mass spectrum of anlline obtained by using the PSI interface with a total Injection of 20 pg of the sample. MethanolIs used as the mobile phase at a flow rate of 1 mllmin. Laser ionization is performed at 266 nm with a laser power density of -5 X lo8 W/cm2.
.-
1
.-
Y)
s4 0.6 9 .-*
7
L
Y)
-5 0.4 C
Another example is shown in Figure 11 for the UV and ion chromatograms of four indole derivatives, namely, 5-hydroxytrypamine (serotonin), tryptamine, melatonin, and tryptophol, with an injection of a mixture of 10 ng each. The separation is carried out under isocratic conditions with a mobile phase containing 40% methanol and 60% 0.3 M acetic acid aqueous solution at a flow rate of 0.8 mL/min. Representative mass spectra (single shot) at each chromatographic peak are shown in Figure 12. All peaks shown in Figure 12 are from the molecular ions and their fragments. This figure illustrates the unique ability of the MPI technique for generating not only molecular ions but also fragment ions for structural analysis. It should also be noted that with an injection of 10 ng each, the signal to noise ratio in the single shot spectrum as shown in Figure 12 appears to be good. The mass resolution is determined to be about 300 (tl(2At)). With this resolution, the isotope ratios can be readily revealed by expanding the peaks (figures not shown). We have also applied this LC/TOFMS technique to a variety of nonvolatile and relatively polar compounds. As an example, Figure 13 shows the ion chromatogram for the separation of four dipeptides. The UV chromatogram is also shown for comparison. The mixture injected into the C-18 column contains 400 ng of Tyr-Gly (MW=238) and 200 ng eachofGly-Trp (MW=261), Leu-Trp (MW=317), andTrpLeu (MW=317). Theseparation isperformedunderisocratic conditions with a mobile phase containing 48% methanol and 52% trifluoroacetic acid (0.1%) aqueous solution at a flow rate of 0.8 mL/min. The mass spectra of these dipeptides at the ion chromatographic peaks are shown in Figure 14. They
6
0.2
0.0 0
5
10
1
1 51 20
Flgure 10. UV absorbance chromatogram at 280 nm (A) and total ion chromatogram(B) of a mixture of PAHs (10 ng each): (1) naphthalene; (2) 1-methylnaphalene;(3)acenaphthene; (4)fluorene;(5) phenanthrene; (6) 1-methylphenanthrene; and (7) 9-bromophenanthrene.
are obtained by using the 266-nm laser beam with a power density of about 5 X lo6 W/cm2. At this high power density, fragment ions are generated as shown in Figure 14. In all cases, an intense molecular ion peak is observed. The fragments are quite extensive and informative, and they can be used for positive identification or structural confirmation bf the molecule of interest. For example, the isomeric species Leu-Trp and Trp-Leu can be distinguished on the basis of the difference in fragmentation patterns as shown in Figures 14C and 14D. To further illustrate the capability of this PSI interface for introducing nonvolatile species, the MPI mass spectra of Leuenkephalin (Tyr-Gly-Gly-Phe-Leu, MW=S55) and pentagastrin (N-t-Boc-P- Ala-Trp-Met-Asp-Phe amide, M W =767) are shown in Figure 15. These spectra are obtained by using the PSI interface for sample introduction with methanol as the liquid carrier at a flow rate of 1 mL/min. No separation column is used in this experiment. In each case, an intense molecular ion peak along with extensive fragment ion peaks is obtained. Most fragments can be assigned to the peptide AnalyticalChemistry, Vol. 66, No. 21, November 1, 1994
3671
1 .o
(A) LClUV
Serotonin 2
0.8
I
-
5Y -ae
B
3
i
3
MW
176
I
4 146
0.6
li8
\
0.4 0
50
150
100
250
200
300
MI2 0.2
Tryptamine
160
0.0 0
I
I
1
2
I
I
I
I
I
3
4
5
6
7
I
Time (min) 1 .o
M W = 160
130
( 8 )LCflOFMS
4
0.8
50
100
150
200
250
300
250
300
MI2
'"1 (C)
I
5B
e4 .2 -
0.6
Melatonin
232
0
0.4
C C
L
0.2
!2 .cn v) 0
0.0
0
0
I
I
1
2
I
I
I
I
I
3
4
5
6
7
structure. It appears that no thermal decomposition products are detected. Other small peptides (MW < 1000) such as morphiceptin, Met-enkephalin, and pentaphenylalanine can also be detected. However, the sensitivity of the technique for these small peptides is generally not very good. For example, about 5 pg of sample is used for generating each spectrum shown in Figure 15. This low sensitivity may be attributed to the low ionization efficiency of the MPI process for peptides. Note that MPI with other sample introduction techniques such as conventional laser desorption and matrixassisted laser desorption for neutral generation do not produce strong signals for peptides either.48.53-s6 Nevertheless, this 3672 Analytical Chemistry, Vol. 00, No. 21, November 1, 1994
100
150
200
MI2 1"-
Time (min)
Flgure 11. UV absorbance chromatogram at 280 nm (A) and total ion chromatogram (B) of a mixture of indoles (10 ng each): (1) 5-hydroxytrypamine(serotonin); (2) tryptamine; (3) melatonin; and (4) tryptophoi.
50
"
--I
.&C?
I (D)
161
Tryptoph o1
8
3
130
I 0
0
50
100
150
200
250
300
MI2
Figure 12. Multiphoton ionizationmass spectra of indoles at the peaks corresponding to the ion chromatogram shown in Figure 11B. Laser ionization is performed at 266 nm with a laser power density of - 5 x 100 W/cm2.
Tyr-Gly
""1
1
I
1
3
MW-238
0.6
ili
0.4
200
100
0
300
500
400
MI2
Gly-Trp
0.2 0
P
I1
HzN-CHz-C-NH-~-C-OH
fi s
0.0 0 1 .o
1
2
3 4 5 Time (min)
6
7
-m
(B) LCflOFMS
II
156
c 2 0)
187
5
Mw = 261
243
L
0
0.6
ofz
261
3
L MI2
.-B
5
s b
'Z
-2
1
1
0.6
0.4
s
0.2
0.0 0
3 I
I
1
2
I
130 I
4
117
86 2U J
2
w 0
0
I
I
I
3 4 5 Time (min)
6
7
I
Leu-Trp
200
100
300
Trp-Leu
I130 0
Figure 13. UV absorbancechromatogram at 280 nm (A) and total ion chromatogram (6)of a mixture of dipeptides: (1) Tyr-Oly, 400 ng; (2) Gly-Trp, 200 ng; (3) Leu-Trp, 200 ng; and (4) Trp-Leu, 200 ng.
H2N-CH-C-
0,
P
NH-CH-C-OH
I
FH2 CH \CHI Mw-317
317
I
156
z
li
ofz
11'
study indicates that nonvolatile species such as small peptides can be introduced into the mass spectrometer without significant thermal decomposition. The present LC/TOFMS technique can also be used to provide quantitative information. As an example, we have performed quantitation of caffeine in instant coffee. A brand
500
400
MI2
77
2-
5
01 ~ " ' ~ ' ~ ' ' I ~ " ' ~ " ' ' l ' ' ' ~ ~ ' I ' " '' , ' I " " I
(53) Grotcmcycr, J.; Boesl, U.; Walter, K.; Schlag, E. W. Org. Mass Spectrom. 1986, 21, 595. (54) Kinscl, G. R.; Lindncr, J.; Grotemeyer, J. Org. Mass Spectrom. 1991, 26,
1052. (55) Bccker, C. H.; Jusinski, L. E.;Moro, L. Inr. J. MassSpectrom. Ion Processes 1990, 95, R1. (56) Li, L.;Lubman, D. M. Anal. Chem. 1988,60, 1409.
0
100
200
300
400
, " ' I
,.+
500
MI2
Flgure 14. Multiphoton ionization mass spectra of dipeptides at the peaks correspondingto the ion chromatogramshown in Figure 136. Laser ionization is performed at 266 nm with a laser power density of - 5 X IOs W/cm*.
Analytical Chemistry, Vol. 66, No. 21, November 1, 1994
3673
Leu-Enkephalin
b2
M' Y3
I
JJj, ,
1
x 10
k Y 2
Pentagastrin
I Ij
II
I II
0
200
M-BocN
II II
600
400
M-Boc
800
MIZ
Flgure 15. Multiphoton ionization mass spectra of Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu, MW=555) (A) and pentagastrin (Kt-Boc-/%AlaTrp-Met-Asp-Phe amide, MW=767) (B). Methanol is used as the mobile phase at a flow rate of 1 mL/min. The amount of sample injected is about 5 pg in each case. Laser ionization is performed at 266 nm with a laser power density of - 5 X loe W/cm2.
51
0
10
I
I
PO
30
I 40
I
I
50
60
Concentration (ppm)
Figure 16. Calibration curve of caffeine obtained by using the PSI LC/TOFMS system. A C-18 column is used, and the mobile phase is 50% methanol and 50% water at a flow rate of 1 mL/min.
name of instant coffee purchased from a local store is dissolved in water and injected into a C-18 column for separation. The mobile phase used contains 50% methanol and 50% water with a flow rate of 1 mL/min. Figure 16 shows the LC/ TOFMS calibration curve of caffeine from standard solutions. The data of the caffeine peak area (in arbitrary units) are expressed as an average of five repetitive injections. Very good linearity (R2=0.993) is obtained from the calibration curve. From the curve, we find that the weight percentage of caffeine in the instant coffee is 2.37 f 0.14%. When this 3674
Analytical Chemistry, Vol. 66, No. 21, November 1, 1994
result is compared with the data obtained from LC with a UV detector, i.e., 2.31 f 0.02%, we find that there is an excellent agreement between the two methods. This study shows that the LC/TOFMS technique with the PSI interface is suitable for quantitative analysis. Strengths and Limitations of the Interface. Comparing the present LC/TOFMS system with existing LC/MS techniques, we note the following strengths associated with the PSI interface. The PSI interface, in principle, provides an alternative means for interfacing LC to any type of mass spectrometer that is capable of detecting ions in a pulsed form. The PSI interface described here is simple to operate and inexpensive to construct. Unlike other interfaces, the PSI interface does not require additional pumping systems in the interfacing region. The interface also provides high sampling efficiency, as illustrated above. The PSI interface bears some resemblance to the particle beam interface12J3 and the atmosphere-pressure spray with electron ionization (APEI).57 In all cases, samples are thermally vaporized, followed by ionization. In the case of the particle beam interface, a solute enrichment device is also used to reduce the solvent content prior to sample introduction into a mass spectrometer. In light of the fact that there are many different experimental parameters used, a direct comparison between these techniques is difficult. However, we note that there are reports of studies on sampling efficiency in the particle beam interface. The sampling efficiency is strongly dependent on the experimental conditions such as the solvent type and the amount of the sample i n j e ~ t e d . ~ ~ ~ ~ ~ Transfer efficiencies, determined by comparing the signal level obtained by using the interface with that obtained by an insertion probe, are reported to be between 2% and 15%5841 and as high as 70%efficiency for chrysene is reported for the universal interface, a technique closely related to the particle beam interface.62 We also note that the sampling efficiencies determined by a technique similar to that used in our study (Le., using a cold sample collector to condense the sample and measure the actual amount of the sample transported) are reported to be less than 2% in a commercial particle beam i n ~ t r u m e n t .In~ ~the case of APEI, we are not aware of any reports of studies on sampling efficiency. However, in terms of detection sensitivity, it has been reported that APEI provides sensitivity comparable to that of the particle beam interface for the detection of naphthalene and anthra~ene.~' One recent report of determination of PAHs by using particle beam LC/ MS with a ultrasonic nebulizer illustrated low nanogram detection l i m i t ~ . 6The ~ results obtained with our PSI interface (e.g., see Figure 10) appear to be better in detection sensitivity for the PAH molecules studied. Several factors including the (57) Sakairi, M.; Yergey, A. L. Rapid Commun. Mass Spectrom. 1991, 5, 354. (58) Bellar, T. A,; Behymer, T. D.;Budde, W. L. J. Am. SOC.Mass Specfrom.
1990, 1, 92. (59) Ho, J. S.; Behymer, T. D.;Budde, W. L.; Bellar, T.A. J. Am. SOC.Mass Spectrom. 1992, 3, 662. (60) Edman, K. R.; Kirk, J. D.;Browner, R. F. Proceedings of the 37th ASMS Conference on Mass Spectrometry and Allied Topics, Miami Beach, FL, May 21-26, 1989; p 130. (61) Ligon, W. V., Jr.; Dorn, S. B. Anal. Chem. 1990, 62, 2573. (62) Vestal, M. L. In Liquid Chromafography/MassSpectrometry, Applications in Agricultural, Pharmaceutical, and Environmental Chemistry; Brown, M. A,, Ed.; American Chemical Society: Washington, DC, 1990. (63) Tinke, A. P.; van der Hoeven, R. A. M.; Niessen, W. M. A,; Tjaden, U. R.; van der Greef, J. J. Chromatogr. 1991, 554, 119. (64) Singh,R. P.; Brindle, I. D.; Jones, T. R. B.; Miller, J . M.; Chiba, M. J. Am. SOC.Mass Spectrom. 1993, 4 , 898.
sampling efficiency and the ionization efficiency (laser MPI vs EI) may contribute to this difference. It should also be noted that the results obtained for quantitation of caffeine content in coffee shown in Figure 16 are quite comparable to those reported by using isotope dilution particle beam LC/ MS.65 However, in the PSI interface, a linear calibration curve can be readily obtained without the use of isotope compounds or other analogs, which can be useful and convenient in some quantitation applications. Compared to the particle beam and APE1 LC/MS system, one major limitation with the present PSI LC/TOFMS system is related to the ionization technique used. While MPI can be very selective and sensitive for the ionization of certain types of molecules such as aromatic compounds, it is not a universal technique for molecular i o n i z a t i ~ n .For ~ ~ future development, we plan to add an electron beam to this system for obtaining E1 spectra. This will enable us to generate classical E1 spectra for molecular identification and extend the applicability of the current system to a much broader range of biological and environmental chemicals. A direct comparison between these interfaces may then become possible. It should be noted that the sensitivity of the PSI interface could be further increased by combining a particle beam interface or a universal interface to it. In this case, the aerosol generation tube in the PSI interface would be replaced by a solute enrichment device. This would increase the amount of solute per pulse being introduced into the mass spectrometer. Another potential drawback of the present PSI interface is related to the use of thermal heating for samplevaporization. Due to possible thermal decomposition, the technique would have limited applicability for ionic and very polar chemicals such as sulfonated azo dyes. While molecules such as small peptides and a number of thermally labile aromatic acids that are not amenable to GC/MS can be studied as illustrated above and in our previous paper,34it appears that the present PSI interface is best suited for the detection of nonpolar compounds or polar compounds with some volatility. To increase its applicability, techniques currently being developed for the particle beam interface, such as the use of laser beam and fast atom beam for sample vaporization and/or ionizati0n,6~9~~could be adapted for the PSI interface. An alternative (65) Doerge, D. R.; Burger, M. W.; Bajic, S.Anal. Chem. 1992, 64, 1212. (66) Kirk, J. D.; Browner, R. F. Biomed. Enuiron. MassSpectrom. 1989,18,355. (67) (a) Lanyon, V.; Schilling, A. B. Proceedings of the 41st ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, May 36June. 4, 1993; p 214. (b) Richardson, J. C.; Browner, R. F. Proceedings of the 41st ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, May 3C-June 4, 1993; p 663. (c) Tinke, A. P.; van der Hoeven, R. A. M.; Niessen, W. M. A.; Tjaden, U. R.; van der Greef, J. Proceedings of the 41st ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, May 3C-June 4, 1993; p 761.
method of enhancing its general applicability is to generate ions directly from the LC effluent, followed by pulsing the ions into the mass spectrometer. This will take full advantage of several very powerful atmospheric ionization techniques such as APCI and electrospray. These techniques have been shown to be very effective for the detection of polar and ionic species. In these techniques, ions, not the neutrals, are generated at a high pressure and subsequently introduced into a mass spectrometer. Conceptually, ions generated at a high pressure can also be mechanically pulsed into the mass spectrometer. We are currently in the process of developing a nozzle to introduce the ions generated from APCI and electrospray into a time-of-flight mass spectrometer in a pulsed form. In summary, the pulsed sample introduction method provides an alternative means for interfacing LC with mass spectrometry. The PSI interface involving single stage pumping provides high sampling efficiency. The interface does not introduce significant peakdistortion in LC/TOFMS. Further improvement in instrumentation such as the addition of an E1 source and a solute enrichment device prior to sample introduction could enhance the utility of the PSI LC/TOFMS system. The present PSI system uses a heated capillary tube to generate aerosol, followed by thermal vaporization of the samples. Thus, the technique would be best suited for the analysis of nonpolar compounds or polar compounds with some volatility. To extend the pulsed sample introduction method for the analysis of ionic and very polar compounds, we are developing a technique based on the current work to mechanically pulse the ions generated at a high pressure using APCI and electrospray into a TOFMS for detection. It is our hope that the combination of several modes of operation (i.e., MPI, EI, CI, and electrospray) in a PSI interface would enable us to analyze a wide variety of compounds from nonpolar chemicals to ionic species in LC/TOFMS.
ACKNOWLEDGMENT Wearegratefulto Dr. F. F. Cantwell, University of Alberta, for many helpful discussions. We thank David Gowanlock for performing the computer curve fitting for Figure 8. This work was in part supported by the Natural Sciences and Engineering Research Council of Canada and Alberta Environmental Research Trust. Received for review March 11, 1994. Accepted July 7, 1994.' a Abstract
published in Aduance ACS Abstracts, September 1 , 1994.
Analytical Chemism, Vol. 66, No. 21, November 1, 1994
3675
