Anal. Chem. 1988, 60, 1426-1428
1426
Effect of Primary Beam Energy on the Secondary Ion Sputtering Efficiency of Liquid Secondary Ionization Mass Spectrometry in the 5-30-keV Range William H. Aberth* a n d A. L. Burlingame University of California, Department of Pharmaceutical Chemistry, Mass Spectrometry Facility, San Francisco, California 94143-0446
A comparison Is made of the Integrated Ion count signals from equal sample amounts of methyl glucose polysaccharlde (3514 daltons) and bovine insulln (5733 daltons) obtained by using a llquld secondary Ionization mass spectrometry source at different Cs' prlmary beam energies ranglng from 5 to 30 keV. Results show that In both posltlve and negative ion analysis modes wlth glycerol and thioglycerol as the solvent matrix, secondary Ion efflclency Increases rapidly above 5 keV primary energy, reaches a maximum at 15-20 keV, and decreases at higher energies.
The sensitivity of a mass spectrometer is often of crucial importance when sample quantity is severely limited as is the case in many analyses of high mass biochemical compounds. It is generally observed with primary ion beam liquid secondary ion mass spectrometry (LSIMS) type ion sources (analogous neutral atom primary beam LSIMS sources are commonly referred to as FAB sources) that the production of secondary ions tends to increase with increasing primary beam energy up to 8-10 keV (the maximum range of most instruments). Similar effects have also been reported in solid SIMS type experiments (1-3). An important question arises as to whether a primary beam with energy above 10 keV will yield greater secondary ion ejection efficiency. Recent analyses using 30 and 35 keV Cs+ primary beams have yielded spectra for the first time of lysozyme (14 701 daltons) (4) and trypsinogen (23978 daltons) (5, 6), respectively, from samples in a liquid matrix. These results suggest that greater signal efficiencies could be obtained a t higher primary energies. On the basis of these observations, we have attempted to establish if there is a primary beam energy, within the range of 5-30 keV, at which secondary ion production efficiency is at a maximum ( 7 ) . We chose as our samples methyl glucose polymer (MGP) (3516 daltons), an acidic polysaccharide, and bovine insulin (5733 daltons) because they are representative of the species and mass range of interest to many biochemists in this laboratory and elsewhere.
EXPERIMENTAL SECTION The LSIMS ion source (8) utilizes a cesium ion gun (Antek, Palo Alto, CA, Model Cs-160-250B) for the primary beam. The gun is capable of producing a 10 keV Cs+ beam; however, it can be electrically floated an additional 20 kV above the source potential yielding a maximum beam energy of 30 keV. The primary beam is directed at the sample liquid matrix target at an incident angle of 70". It strikes the matrix after first passing through a 0.15 mm wide slit aperture positioned 1.5 mm above the target surface (see Figure 1). A potential difference of 40 V between aperture and target assists in accelerating the secondary ions back through the slit for mass analysis. Ten picomoles of bovine insulin and 2.5 pmol of MGP were used as samples. These were dissolved in 0.5 fiL of thioglycerol or glycerol located on a 2.5 mm diameter copper surface. During mass analysis, the sample target was maintained close to 0 "C by Freon cooling through the sample
probe (9). Analysis runs lasted until the signal dropped to a few percent of its maximum, which was typically 15-20 min. Some liquid matrix material was always present on the probe tip at the conclusion of a run indicating that the sample was depleted rather than that the probe tip had gone dry. Mass analysis was performed on a Wien mass spectrometer at 40 kV accelerating voltage (IO,11j. The signal from a portion of the mass spectrum, centered on the sample molecular mass, was registered on a 40 mm diameter microchannel plate detector. The sizes of the detected mass segment were 300 and 700 mass units for MGP and bovine insulin, respectively. The spectra were processed by use of position computing electronics (Surface Sciences Laboratories, Mountain View, CA, Model 2401) and pulse integrated on a 512-channel MCA. Total ion signal count rates were maintained at less than 50 000 counts/s to prevent excessive signal and resolution loss caused by the overall detection dead-time of about 3 ps per ion pulse. This limitation of counting rate also necessitated the use of small picomolar amounts of sample. The analysis of bovine insulin was performed on the positive ion while that of MGP was performed on both the positive and negative ions.
RESULTS AND DISCUSSION Figures 2 and 3 show the integrated signal vs primary beam energy for MGP positive and negative secondary ion mode, respectively. The negative mode yielded about twice the maximum signal efficiency as the positive mode and additionally proved to be superior in molecular signal quality. This may be due to the fact that the negative ions are not subject to potassium and sodium cationization and yield a dominant (M - H)- primary peak. A similar analysis of MGP in the positive mode using glycerol as the solvent matrix yielded a signal vs primary beam energy result that is qualitatively similar to that shown in Figure 2 for thioglycerol, except that a maximum of 37 K counts/channel occurred at 20 keV primary energy and the drop-off at energies above 20 keV is greater (see Figure 4). It appears from Figures 2-4 that the maximum of MGP ion production occurs a t 15-20 keV primary energy in both the negative and positive modes. Operating a t 15 keV primary energy beam, we have obtained a spectrum of MGP with a signal/noise value of about 10 using only 10 fmol of sample (see Figure 5). Similar high sensitivities have been observed for lower-mass (1300daltons) biocompounds by using a SIMS time-of-flight (TOF) mass spectrometer (12). The relatively high sensitivities for these instruments can be largely attributed to the near 100% duty cycle of the collected portion of the spectrum. This results from the nature of TOF ion detection (12) and from the use of a position-sensitive microchannel plate detector (13) in our laboratory. A plot of signal counts vs sample quantity yields a linear relationship covering about 3 orders of magnitude (see Figure 6). The primary source of background signal at low sample levels appears to arise from the solvent matrix alone since a blank run yields the same level of background as is shown in Figure 5 . A plot of signal vs primary energy for bovine insulin is shown in Figure 7. The shape of this plot is qualitatively similar to those for MGP but with an overall sensitivity re-
0003-2700/88/0360-1426$01.50/06 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 14, JULY 15, 1988
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Figure 4. Plot of the integrated secondary positive ion signal of 2.5 pmol of MGP in glycerol vs Cs' primary ion beam energy.
Flgure 2. Plot of the integrated secondary posklve ion signal of 2.5 pmoi of MOP in thioglycerol vs Cs' primary ion beam energy.
duction of about an order of magnitude. Again, the maximum signal appears to be in the range of 15-20 keV primary energy. A negative mode analysis of bovine insulin was not performed because it failed to produce a signal of practical intensity. It is interesting to note that a maximum in integrated signal occurs at about 15 keV. Increasing primary energy from the commonly used value of about 8 keV to this value can more than double the total signal. However, increasing the primary energy beyond 15 keV does not greatly enhance the signal or can even reduce it. The fact that lysozyme and trypsin were first observed by LSIMS at primary energies of 30 keV and higher ( 4 , 5 ) may be due in part to the higher secondary ion flux (resulting from the higher primary energy) which can improve detection in the scanning mode in which these measurements were made (even though secondary ion efficiency may not have been maximized). One possible reason for the signal decline a t the higher primary energies is that the Cs+ energy is deposited too deep in the matrix for optimum ionization of the molecules on the matrix surface (14).
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Flgure 5. Integrated spectrum of negative MOP using 10 fmol of sample in thioglycerol. The mass scale was calibrated by using cesium iodide and cesium fluoride cluster ion peaks.
The penetration depth can be reduced by increasing the angle of incidence. One could also reduce this depth by using a primary beam composed of molecular ions instead of atomic ions (15-17). The penetration depth of the atomic constituents of the molecular ions, separated upon surface impact, would be less than the depth of an equivalent mass single particle. By these methods, it should be possible to obtain increasing
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of 3000 daltons and higher, the improvement in molecular ion signal with primary energy outweighs the increase in chemical noise. However, for sample molecular ion masses below 3000 daltons, this may not be the case. For example, in the range of 700-1500 daltons we obtain a better signal/background value by using only 5 keV primary beam energy. The lower secondary ion intensity resulting from the reduced efficiency of ion production at the lower primary energy can be readily compensated for by increasing the primary Cs+ beam flux.
CONCLUSIONS
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Figure 8. log-log plot of the integrated negative MGP signal vs sample
amount. 20k
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Based on the preliminary results presented here, a maximum of high mass secondary ion efficiency is obtained at about 15 keV primary Cs+ energy. Although there may be a shift in the maximum position of the signal vs primary energies for secondary masses >6000 daltons, this seems unlikely since no shift in maximum is observed while going from 3514 to 5733 daltons. We have shown that increasing the primary beam energy to 15 keV can substantially improve higher mass secondary ion ejection efficiency. However, whether increasing the primary beam energy beyond 15-20 keV would be useful under analytical circumstances remains to be investigated. It is interesting to note that the spectrum obtained at maximum efficiency represents a detection of only 1molecule out of 2 X lo6 for MGP (see Figure 5) and 1 in 3 X lo7 for bovine insulin. It thus appears that signal detection in general is inefficient and substantial further improvements in highmass sensitivity may still be realizable.
LITERATURE CITED
5
10 15 20 25 PRIMARY BEAM ENERGY KeV
30
Flgure 7. Plot of the integrated secondary positive ion signal of bovine insulin vs Cs' primary ion beam energy.
secondary ion efficiencies with increasing primary energy over the range of 30 keV and beyond. A second issue to be considered concerns the effect of primary beam energy on chemical noise arising from the liquid matrix. This chemical noise increases with increasing primary beam energy and decreases with increasing mass. At masses
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RECEIVED for review November 23,1987. Accepted February 18,1988. This work was supported by the National Institute of General Medical Science under Grant GM32315.
