Anal. Chem. 1986, 58, 1221-1225 (21) (22) (23) (24) (25)
Glish, G. L.; Todd, P.
J.; Busch, K. L.; Cooks, R. G. Int. J . Mass Spectrom. Ion Phys 1084, 56, 177. Keough, T. Anal. Chem. 1985, 57, 2027. Jennlngs, K. R.; Mason, R. S. In Tandem Mass SePctrometrK Mcbfferty, F. W., Ed.; Wiley-Interscience: New York, 1983. Levsen, K.; Schwarz, H. Mass Spectrom. Rev. lg83, 2 . 77. CRC Handbook of Chemistry and Phys/cs; Weast, R. C., Ed.; CRC
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Press: Cleveland, OH, 1979.
RECEIVED for review September 9, 1985. Accepted January 13, 1986. This work was supported in part by the National Science Foundation (Grant CHE84-08258).
Instrumental Conditions of Secondary Ion Mass Spectrometry That Affect Sensitivity for Observation of Very High Masses William Aberth Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, S a n Francisco, California 94143-0446
I t is demonstrated by comparlng the mass spectra of cesium iodlde cluster Ions obtalned under different Operating parameters that such factors as acceleration voltage, system pressure, and primary beam energy affect the high-mass signal current to a greater extent than that of the low mass. The measurements Imply that consideration should be given to these parameters for more effectlve analysis of highmass compounds.
Cesium iodide in many ways is an ideal sample material for investigating instrumental effects on the quality of mass spectra. The sample spectrum remains stable and produces constant-amplitude peaks for several hours thus allowing comparison of spectra obtained over a long period. Also, since the elements cesium and iodine occur as single isotopes and are relatively heavy, cluster peaks of (CsI),Cs+ or (CsI),I- can be obtained for very high mass. Positive cluster ions with n = 122 (m = 31853 daltons) ( I ) , and more recently n = 155 (m = 40433 daltons) (2),have been reported. These factors permit a sensitive analysis of the effects of various instrumental variables on the cluster ion spectrum as a €unction of mass (cluster size). The results of this investigation show the probable nature of factors that would limit observation of very high mass (>3000 daltons) bioorganic compounds and further suggest methods for overcoming these limitations.
EXPERIMENTAL SECTION Mass analysis was performed on a recently constructed single-stage Wien EXB mass spectrometer ( 3 , 4 ) .The Wein spectrometer is especially suited to the analyses of very high mass compounds because it is capable of analyzing these compounds at high acceleration voltage. The present instrument design (see Figure 1)is based partly on a previous machine design (5) and utilizes a commercial 63-cm-long EXB mass analyzer (Colutron Research Corp., Boulder, CO,Model 300-6).An acceleration voltage of up to 40 kV was used for this work. The high acceleration voltage yields high transmission efficiency (5, 6) and permits good mass resolution (7) with a single-stage analyzer. The ion source is an immersion lens type (8)that utilizes a cesium for the ion gun (Antek, Palo Alto, CA, Model Cs-160-250B) primary ion beam (9). An overall vacuum of about torr was maintained in the instrument. The ion signal was amplified by means of a postacceleration detector that utilized an aluminum collision electrode at -10 kV and a ceramic continuous dynode electron multiplier operating at -4 kV.
RESULTS AND DISCUSSION Figure 2 is a plot of (CSI)~CS+ cluster amplitudes obtained with a Wein acceleration voltage of 40 kV compared with similar spectra obtained by Campana et al. ( I O ) , Ens et al. ( 1 1 ) , and Baldwin et al. (12). All spectra are normalized to the n = 1 cluster peak. Differences in peak amplitudes for n > 1therefore reflect instrument conditions that affect the observation of the higher mass ions differently from the lower mass ions. All spectra from which the data of Figure 2 were derived were obtained by sputter ionization from a solid target of cesium iodide. The primary beams used were Cs' (Aberth and Ens et al.), Xe+ (Campana et al.), and Xeo (Baldwin et al.). Although Baldwin and co-workers had more complete data using an Aro primary beam, only the four data points published using Xeo were plotted in Figure 2. This is because the mass of the primary beam particles has been shown to have a strong influence on secondary ion efficiency (13). The similarity in mass xenon and cesium would tend to reduce the effects caused by primary particle mass difference and thus yielded a more meaningful comparison. A general observation about the data shown in Figure 2 is that the curves differ in amplitude from each other by increasing amounts as the cluster size increases. The cluster spectrum obtained with the Wein mass spectrometer lies significantly above the other curves a t the higher masses. Some possible instrumental factors that could yield such differences between the high- and low-mass relative signal amplitude are (1)pressure differences, (2) mass-dependent differences in detection efficiency, (3) differences in primary beam energy, (4) differences in flight time of analyzed ions (secondary ion acceleration voltage and drift distances), and (5) differences in ion source geometry. The relative importance of these instrumental factors will now be explored. Pressure Effects. Figure 3 shows the effect of increasing the source chamber pressure by introducing N2 gas. The source chamber (see Figure 1)includes the ion source as well as the main Einzel lens and encompasses an 80-cm path length of the secondary ion beam. The restricted pumping through a beam-defining irls, positioned between the Wein separator and the source chamber, reduces the pressure spillover into the separately pumped detector region to about 1/20 of the change in source chamber pressure. The acceleration voltage for the measurements shown in Figure 3 was 30 keV. The amplitude of the n = 1 cluster ion was unchanged for all source pressures except for the highest value of 3 X lo4 torr where
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a decrease of 26% was observed. It can be seen from the curves that the higher mass ions are attenuated to a greater extent than the lower ones and that pressures less than lo4 torr are sufficient to seriously limit the range of resolvable cluster peaks. Figure 4 shows the high-mass portion of the recorded four spectra used to obtain the curves of Figure 3. These plots dramatically demonstrate the deleterious effect that instrument operating pressure can have on the quality of mass spectra. Similar strong pressure-correlated attenuation effects on cesium iodide cluster ions have been alluded to from observations on an FTMS instrument (14). The pressure attenuation effect may be especially important when the gas load from a FAB primary beam saddle field type gun or similar gaseous discharge type gun is used. Although the specifics of pressure changes caused by xenon FAB gun
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operation are not given by Campana et al. (10,15) and Baldwin et al. (12, 16),it is likely to be between 5 X loW7 and 5 X torr. And depending on the extent of differential pumping designed into the ion source region as well as the ion beam path length in the different pressure regions of the instruments used by these investigators, the greater attenuation of the high-mass cluster ions may partly result from these pressure effects. The pressure-correlated attenuation of the cluster ions can be attributed primarily to collision induced dissociation (CID) and neutralization reactions. Due to the high velocity of the beam ions relative to the background gas molecules, these molecules may be considered stationary, and the pressure attenuation is roughly proportional to the integral of the pressure over the trajectory of the beam ions. The increase of the attenuation with cluster size is probably due to the
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
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corresponding increase in cross section and greater fragility of the cluster ions. A similarity in pressure-associated attenuation effects can be attributed to organic molecules. Campana and Green (17) have observed a greater pressure attenuation of the (csI)&s+ ion (4293 daltons) relative to the C&+ ion (77 daltons), (90% vs. 50% attenuation). However, in comparing similar highmass ions such as (CsI)13Cs+(3513 daltons) relative to methylglucose polysaccharide (MGP) (18) (3510 daltons), our laboratory has observed a greater relative attenuation for the MGP (90% vs. 50%). The effectiveness of the background pressure in attenuating the ion beam will depend on a variety of factors such as molecular shape, bond strengths, ionization potential, background gas composition, and molecular size. It appears, however, that cesium iodide cluster ions are quite stable and are probably less sensitive to pressure effects than the equivalent size of many organic ions. Detection Efficiency. The range of cluster ion masses detected in the present work is between 393 daltons ( n = 1) and 18333 daltons ( n = 70). A mass range this large might be expected to produce a wide variation in secondary electron emission efficiency and consequently affect the relative counting efficiencies between the high- and low-mass portions of the cluster spectrum. It was therefore decided to measure the net gain per ion to establish the extent that energy and mass affect secondry electron emission. This was accomplished by measuring the ion count rate and multiplier current as a function of mass and ion collision energy (obtained from the sum of the conversion dynode voltage and the mass spectrometer acceleration voltage). Care was taken to keep the count rate for each measurement less than 104/s to minimize current saturation effects in the multiplier. Since the multiplier gain can be considered constant for a fixed multiplier voltage (maintained at -4 kV for all measurements) and fixed postacceleration voltage, the change in net gain can be attributed to a change in secondary electron efficiency. Figure 5 is a plot of the net gain values measured for collision energies of 10 (5 kV acceleration + 5 kV postacceleration) and 50 keV
(40 kV acceleration 10 kV postacceleration) a t the postacceleration electrode. The data points for the two energies fall along roughly parallel straight lines with a poositive slope. The lower average gain of the 10-keV postacceleration energy data can be attributed, at least partly, to the lower energy of the secondary particles striking the electron multiplier (1keV vs. 6 keV) and may not be due to the lower cluster ion energy striking the postacceleration electrode. The positive slope to the net gain curves implies that there is a very low velocity threshold for secondary particle production. At the lowest collision velocity of 1.55 X lo6 cm/s (n = 30,lO-keV postaccelerator collision energy) the secondary efficiency appears to be increasing with mass. Measurements have been made in this laboratory at u = 1.38 X lo6 cm/s ( n = 155,40-keV postaccelerator collision energy) (2) and elsewhere at about u = 0.8 X lo6 cm/s (n = 122,10.68-keV postaccelerator collision energy) (1,19). It is interesting to note that these low velocity values are well below the threshold for kinetic electron emission of about 5 X lo6 cm/s measured for metal cluster ions (20-22). Beuhler and Friedman have reported a threshold of about 1.7 x lo6 cm/s for water cluster ions (23), and a similar threshold value was observed for bovine insulin ions (24). It has been postulated that this low threshold may arise from collisionally displaced lattice atoms near the detector surface, which upon relaxing can provide an electron with sufficient energy to escape (23). Another possibility that can explain the even lower threshold observed for cesium iodide cluster ions is the collisional ejection of an I- ion. An iodine negative ion originating from the colliding cesium iodide cluster ion can carry sufficient kinetic energy from its parent ion to break its molecular bonds and escape the surface after undergoing a knock-on type collision with substrate atoms. Experimental evidence indicates that a significant percentage of the negatively charged secondary emission from cesium iodide cluster ions is composed of ions originating from the colliding beam (25). Primary Beam Kinetic Energy. Increasing the energy of the primary particle can greatly increase the yield of secondary ions. Schueler and co-workers (26) have shown that yields of secondary alkali halide cluster ions can increase by a factor of 100 as the Cs+ primary energy is increased from 3 to 14 keV. An important question arises as to the effects of the primary beam energy on the higher order cluster ions. The work of Schueler and co-workers showed no change in the cluster ion ratio Y,/Yl for n = 2 and 3 over the primary energy range. However, preliminary work in this laboratory
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
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(27) on glycerol matrix sputtering has shown a strong correlation between primary energy and higher order glycerol ion cluster yield. Figure 6 shows a comparison of cesium iodide cluster spectra produced by using primary Cs+ energies of 4 and 10 keV. These spectra are normalized to the n = 1peak to better show differences in relative efficiencies of the higher cluster production. In addition to the greater relative yield of the higher mass cluster ions, an overall increase in secondary ion yield for n = 1of about a factor of 3 was observed for the spectrum produced by the 10-keV primary ions. This increase in secondary yield is consistent with similar increases in secondary yields of high-mass organic ions sputtered from liquid matrices that have been observed in this laboratory. Acceleration Voltage Effects. Increasing the acceleration voltage of a mass spectrometer has the general effect of improving transmission. However, an additional benefit can be provided by the accompanying reduced transit time for the mass-analyzed ions. Very high mass ions are often unstable to unimolecular dissociation, and the reduced transit time will increase the survival rate for these ions and thereby increases the detected signal. Figure 7 compares the cluster spectra of CsI obtained a t 40-keV and 5-kV acceleration voltages. The spectra are normalized to the n = 1 cluster peak to eliminate effects caused by ion optical differences in transmission. The transit time at 5 kV is 2.8 times greater than at 40 kV, and it is clera from Figure 6 that this increase in time significantly attenuates the spectrum at higher masses. Note also the greater value for the ratio of the cluster peaks Y13/Y14 for the 5-keV spectrum compared to the 40-kV spectrum (13 vs. 7). The increased value reflects the greater instability of the n = 14 peak relative to the 13 peak (17). This ratio is a good indicator of the total ion transit time. In the spectrum of Campana et al. (10) (see Figure 2) the Y13/Y14peak ratio is about 88 and corresponds to 11 times greater flight time than the Wein instrument a t 40-kV acceleration. This analysis, of course, cannot be applied to the work of Ens et al. (see Figure 2), since their time-of-flight instrument detects the parent ion and its decomposition products at the parent ion mass position. This
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is the reason given for the relative lack of structure in their cluster spectrum envelope (11).
CONCLUSION In an effort to mass analyze biological compounds of increasingly greater molecular weight, it becomes necessary to consider the effects of a wide range of instrumental parameters. Success or failure may depend on the degree to which these parameters can be optimized. Signal attenuation due to background pressure may become a major consideration if a gaseous discharge type FAB gun is used for the primary beam. The substitution of a cesium ion gun, which contributes virtually no gas load to the source chamber, would be one logical alternative (8,9,28). Other alternative low-gas-load primary beam sources include an indium ion gun (29) and a gold negative ion gun (30). Additional source chamber pumping, however, may still be necessary to reduce pressure contributions from evaporation when liquid sample matrices are used. ACKNOWLEDGMENT I thank Arnold Falick and A. L. Burlingame for a critical review of the manuscript and Charles Delwiche for assistance in taking data. Registry No. CsI, 7789-17-5.
LITERATURE CITED Katakuse, I.; Nakabushi, H.; Ichihara, T.; Sakurai, T.; Matsuo, T.; Matsuda, H. I n t . J . Mass Spectrom. Ion Processes 1984, 57, 239-843. Aberth. W. I n t . J . Mass Smcrrom. Ion Processes 1986, 68. 209-212. Wein, W. Ann. Phys. 1898, 65,440-452. Wein, W. Ann. Phys. 1902, 8 , 244-267. Aberth, W. Biomed. Mass Spectrom. 1980, 7,367-371. Aberth, W. Proceedings of the 10th International Mass Spectrometry Conference, Swansea, United Kingdom, Sep 9-13, 1985, in press. Aberth. W. Presented at the 33rd Annual Conference on Mass Spectrometry and Allied Topics, San Dlego, 1985. Aberfh, W.; Burlingame, A. L. Anal. Chem. 1984, 56, 2915-2916. Aberth, W.; Straub, K. M.; Burlingame, A. L. Anal. Chem. 1982, 5 4 , 2029-2034.
Anal. Chem. 1986, 58, 1225-1227 Campana, J. E.; Barlak, T. M.; Colton, R. J.; DeCorpo, J. J.; Wyatt, J. R.; Dunlap, B. 1. Phys. Rev. Len. 1981, 47, 1046-1049. Ens, W.; Beavis, R.; Standing, K. G. Phys. Rev. Lett. 1983, 50, 27-29. Baldwin, M. A,; Proctor, C. J.; Amster, 1. J.; McLafferty, F. W. I n t . J. Mass Spectrom. Ion Processes 1984, 54,97-107. Wlttmaack, K. Phys, Len. A 1979, 69A, 322-325. Castro, M. E.; Russel, D. H. Anal. Chem. 1985, 57,2290-2293. Colton, R . J.; Campana, J. E.; Barlak, T. M.; DeCorpo, J. J.; Wyatt, J. R. Rev. Sci. Instrum. 1980, 51, 1685-1689. McLafferty, F. W.; Todd, P. J.; McGllvery, D. C.; Baldwin, M. A. J. Am. Chem. SOC. 1980, 102,3360-3363. Campana, J. E.; Green, B. N. J. Am. Chem. SOC. 1984, 106, 531-535. Forsberg, L. S.; Dell, A,; Walton, D. J.; Ballou, C. E. J. B o / . Chem. 1982, 257,3555-3563. Katakuse, I.; Nakabushi, H.; Ichihara, T.; Fujita, Y.; Matsuo, T.; Sakurai, T.; Matsuda, H. Mass Spectrosc. 1985, 33, 145-147. Thum, F.; Hofer, W. 0. Surf. Sci. 1978, 90, 331-338. Beuhler, R. J.; Friedman, L. I n t . J. Mass Spectrom. I o n Phys. 197?, 23, 81-97. Staudenmaier, G.; Hofer, W. 0.; Llebl, H. Int. J. Mass Spectrom. I o n PhyS. 1976, 1 1 , 103-112.
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(23) Beuhler, R. J ; Friedman, L. Nucl. Instrum. Methods 1980, 170, 309-31 5. (24) Sundqvist, B.; Hedin, A.; Hakansson, P.; Kamensky, 1.; Saiehpour, M.; Sawe, G. I n t . J. Mass Spectrom Ion Processes 1985, 65,69-89. (25) Wang, G. H.; Aberth, W.; Falick, A. M. I n t . J. Mass Spectrom. Ion Processes, in press. (26) Schueler, 6.; Beavis, R.;Ens, W.; Main, 0. E.; Standing, K. G. Surf. Sci. 1985, 160, 571-586. (27) Morita, Y.; Aberth, W.; Burlingame, A. L. Presented at the 32nd Annual Conference on Mass Spectrometer and Allied Topics, San Antonio, TX, May 1984. (28) McEwen, C. N. Anal. Chem. 1983, 55,967-968. (29) Barofsky. D. F.; Giessmann, U.; Bell, A. E.; Swanson, L. W. Anal. Chem. 1983, 55, 1318-1323. (30) McEwen, C. N.; Hass, R. J. Anal. Chem. 1985, 57,892-894. '
RECEIVED for review August 5, 1985. Resubmitted January 21, 1986. Accepted January 21, 1986. This work was supported by the National Institute of General Medical Science under Grant GM 32315.
Use of Time Resolution To Eliminate Bilirubin Interference in the Determination of Fluorescein Frank V. Bright, George H. Vickers, and Gary M. Hieftje*
Department of Chemistry, Indiana University, Bloomington, Indiana 47405
Timeresolved fluorescence spectrometry Is used to eliminate blllrubln Interference In the fluorometrlc datermlnatlon of fluorescein. Desplte complete spectral overlap of the excltatlon and emlsslon of these two gpecles, a difference In fluorescence llfetlme provldes the selectivity parameter used In this method. Results for the determlnatlon of 0.300 nM fluoresceln In the presence of up to 25.00 FM blllrubln (1:8.3 X IO4) using a sampling oscilloscope and an optical delay llne show excellent accuracy (
