Anal. Chem. 1982,5 4 , 2362-2368
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Optimization of Experimental Procedures for Fast Atom Bombardment Mass Spectrometry S. A. Martin, C. E. Costeilo, and K. Biemann" Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02 139
A systematlc Investigation of the experlmental variables In fast atom bombardment mass spectrometry has been undertaken in order to optlmlre the experlmental technlque especially for exact mass measurements at hlgh resolution. Among the parameters studied have been the shape and materlal of the target, the nature of the ionlrlng gas, sample preparation, concentratlon, and matrlx. Measurements have been made in both posltlve and negatlve Ion modes. Interferences and artlfacts have been identlfled and methods have been developed to minimize thelr effects. A serles of recommendations for fast atom bombardment mass spectral experlments is presented as the result of these studles.
Since its introduction by Barber et al. in early 1981 (1,2) fast atom bombardment mass spectrometry (FABMS) has become a widely used soft ionization technique for the investigation of large and/or thermally labile compounds, i.e., peptides, cobalamines, carbohydrates, and antibiotics. The technique has its foundations in other areas such as the earlier work of Devienne with neutral beams (3) and the pioneering work of Benninghoven with secondary ion mass spectrometry (SIMS) (4-6). The relationship between FABMS and SIMS rests in the fact that in both techniques the sample is bombarded with particle beams moving with approximately the same kinetic energy and sample ions are produced as a result of the interaction of the beam with the sample. In FABMS the particle beam is neutral whereas in SIMS the beam is charged. The compound classes examined by FABMS range from simple peptides, such as the enkephalins (7-91, to very complex antibiotics such as Bleomycin A2 (10,II). Several authors (11-14) have demonstrated the usefulness of FABMS for the ionization of compounds which had previously been intractable by other forms of mass spectrometry or had produced molecular species of very low abundance or only fragment or cluster ions from which the molecular weight had to be deduced. Many compounds that require derivatization prior to analysis by other ionization modes can be ionized by FABMS without such chemical conversion. There has, as yet, been little detailed information published about FABMS operating conditions, despite the proliferation of publications on FABMS spectra. Here we describe modifications to a conventional ion source housing and FD sample probe to accept a commercially available neutral atom gun and the evaluation of various parameters which affect the ionizing efficiency and sample ion yield. These parameters were investigated in an effort to optimize the molecular ion yield and the production of structurally significant fragments without interference from impurities such as salts and buffers.
EXPERIMENTAL SECTION Instrumentation. A double focusing (Varian MAT 731) mass spectrometer of Mattauch-Herzog geometry with a mass range of 1-2000 amu at 8 kV accelerating potential was employed in this work. The EI/FI/FD ion source was used without modifications. The MAT 731 ion source housing is equipped with pork directly opposite each other and perpendicular to the ion optical system. The Ion Tech atom gun was attached to the ion source
Table I. Operating Parameters for FABMS (P,' torr, TSb= 32 OC, SEMCgain = 1 X lo6)
=
8X
gas
HT,d kV
LC,e mA
IC,ffiA
xenon
4.8
0.2
5.7 6.6 7.3
0.5
10.0 20.0
argon
4.8
5.2 5.8 6.2
0.8 1.1 0.2
0.6
30.0 40.0 10.0 20.0
1.0
30.0
1.2
40.0
a Pressure in ion source housing. Temperature in ion source housing, Secondary electron multiplier. High voltage of €550 power supply. e Limiting current of B50 power supply. f Equivalent ion current of neutral beam.
housing via one of these ports through an adapter connecting a 69 mm 0.d. Conflat flange which fits the atom gun to a 75 mm 0.d. gold seal flange which, in turn, fib the port on the ion source housing. Several adapter lengths were tested, and the configuration shown in Figure 1,in which the atom gun is 90 mm from the probe tip, was adopted. The FAB gun is a B12N 100-kA neutral source with B50 constant current power supply purchased from Ion Tech Ltd., Teddington, England, and was used as supplied, except that the 1 mm i.d. hole through which the atom beam exits was narrowed t o 0.5 mm. The sample is introduced into the EI/FI/F? combination ion source on the tip of a probe which is positioned on the end of the FD push rod. A view of typical probe tips and the definition of angle of incidence (15)are shown in Figure 2. The standard probe tip is made of 303 stainless steel, SS, with 0 = 60' and has a sample surface area of 5.7 mm2. The position of the probe tip with respect to the FAB gun may be observed through the quartz window placed in the direct insertion probe port shown in Figure 1. The probe tip can be aligned visually by using the light emitted by the FAB gun (blue for xenon, purple for argon, and red for neon). Fine adjustments can be made until maximum signal is observed on the oscilliscope displaying any chosen ion beam. Table I lists a few standard sets of operating conditions for FABMS with varying neutral beams. The gases used for bombarding the sample are placed in buffer reservoirs at 1 atm pressure and fed through a metering valve to the FAB gun. With the modification to the Ion Tech gun mentioned above, gas consumption is low, only a few cubic centimeters of gas per hour. Chemicals. Glycerol was MCB ACS reagent grade redistilled at 1torr and 120 "C, methanol was nanograde from Mallinckrodt, and HC1 was Pierce constant boiling sequanol grade. Water was MCB Omnisolv glass distilled and glacial acetic acid was HPLC grade "Baker Analyzed" reagent. All other reagents were ACS grade and used without further purification. Leucine-enkephalin, bradykinin, and [Sarl,Alas]angiotensin I1 were purchased from Beckman. Fomblin oil was purchased from PCR. Hexakis(2,2,2-trifluoroethoxy)cyclotriphosphazene was synthesized according t o the method of Ratz et al. (16). The following reagent purity gases were purchased from Matheson: xenon, argon, neon, methane, carbon monoxide, carbon dioxide, and nitrogen. Sulfur hexafluoride was instrument purity and was also purchased from Matheson. Sample Preparation. The flow chart in Figure 3 describes the procedure routinely followed for FAB analysis. To minimize the problem of sodium and other interferences in FABMS, all solvents are nanograde purity and all glassware is cleaned in
0003-2700/82/0354-2362$01.25/00 1982 American Chemical Society
ANALYTICAL CHEMISTRY. VOL. 54, NO. 13. NOVEMBER 1982
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usually at a concentration of 1 pg/pL, is applied with a 10-pL syringe or 5-pL graduated capillary to the sloped surface of the probe. The volume, 0.3 pL,is spread so that a thin, uniform film is visible to the unaided eye. The push rod on which the probe is mounted is then introduced into the vacuum lock of the EI/ FI/FD ion source. The probe is pushed into the ion source until the tip of the probe breaks the line of sight to the FAB gun when observed through the quartz window. The instrument is then tuned, the probe is moved slightly to verify the optimum probe position, and the mass spectra are recorded. After the analysis, the remaining sample can he recovered from the tip with a syringe or by washing it off with a suitable solvent. If the sample is expendable, the tip is simply washed off with water and then cleaned as described above. Under the conditions outlined in Figure 3, after 5 min of scanning, visual inspection of the probe tip indicates that it is still completely covered by a layer of matrix and the intensity of the ion beam due to the sample has dropped only slightly, depending somewhat on the concentration of the sample in the glycerol matrix. Figure 4 indicates the maior ions attributable to the glycerol matrix.
r
L__-
I
Flgure 1. Schematic diagram of the MAT 731 combinatinn EIlFIlFD ion s w c e in FAB configuration: (A) gas inlet: (E) FAB gun, Ion Tech Ltd.; (C) flight tube; (D) neutral beam: (E) sample probe tip; (F) ion focusing lenses; (G) ion beam; (H) analyzer: (I) polt for direct insertion probe, in which Is placed a quartz window (not shown); (J) FD push
rod. $,DE V I E W : C R O S S S E C T l O N
B A C K V I E W : CROSS S E C T I O N MkTERIALS:
F A B OUN
A N O L E OF “ClDENCE
Flgure 2. Dimensions of sample probe tips used in FABMS and def-
inition of the angle of incidence. chromicsulfuric acid cleaning solution and then soaked snccessively in distilled water, glass distilled water, and nanograde methanol. FABMS Analysis. The standard procedure for FAB analysis is as followa The s t a b l e s steel tip is coated with 50% (v/v) nitric acid for a few seconds and then rinsed with distilled water followed by nanograde methanol and allowed to dry. The sample, having been prepared according to the flow chart shown in Figure 3,
RESULTS AND DISCUSSION Modifications to the Ion Source Housing and Neutral Atom Gun. The adapter was preferred over mounting the gun directly on a gold seal mating flange for the following reasons. First, without an adapter the tip of the FAB gun would be within the confines of the ion source and therefore i t would be necessary to remove the FAB gun whenever the ion source has to be removed and, secondly, if the gun is very close to the sample, it must be operated at very low ion current to avoid rapid evaporation of the matrix. More importantly, i t was found that the 190 LIS turbomolecular pump with which the ion source housing is fitted does not have adequate pumping speed to allow sufficient gas tbroughput for operation a t very low neutral fluxes. In the configuration shown in Figure 1,in which the tip of the FAB gun is 90 mm from the tip of the sample surface, neither heating of the probe tip nor the associated deerease in ion signal is observed. In an attempt to lower the operating pressure of the ion source housing without decreasing the efficiency of the FAB gun, the flight tube shown in Figure 1was replaced with one having an inside diameter of 0.5 mm. Since the beam has a Gaussian distribution around the center axis, this still allowed the major portion of the heam flux to leave the gun and also allowed operation a t lower pressures in the ion source housing. A second modification was also made to decrease the pressure of the ion source without any loas of FAB gun efficiency. Since B12N is a saddle field source (17). it generates diametrically opposed beams of equal intensity, one of which strikes a beam monitor plate to indicate the equivalent ion current. The other portion of the ion beam travels through a flight tube where it is neutralized by a dense cloud of secondary electrons produced when the ions strike the sides of the flight tube. The pathway of the monitoring ion beam was blocked by a solid piece of aluminum in order to force all the gas to flow into the front section of the gun, thus permitting still lower ion source operating pressures. These two modifications reduced the standard operating pressure, measured 15 cm from the torr to 8 X IOd torr (using Xe ionization block, from 5 X as the gas). Target Material. Copper, 303 SS, and polyimide coated SS were investigated as FABMS probe tip materials. Copper performed satisfactorily, except that occasionally it forms cluster ions with the sample or matrix (see Table 11) which may complicate the mass spectrum. In addition, these tips have to be replaced frequently because even the dilute nitric acid used for cleaning erodes them significantly. The 303 stainless steel is not etched with 50% nitric acid during removal of the previous sample and has no sample memory. Wettability of the 303 SS is not as good as that of copper, but i t is adequate. Finally, polyimide coated stainless steel tips
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Dissolve in Matrix Compatible Solvent (lor glycerol e g. H20, MeOH, DMSO)
Apply 0.3111 to Tip
Record (t/-) FABMS acid (I) FAB base [-) F A 0
c Evaluate Data
[ Spectrum Molecular Weight?
Dominated by Salts
FINAL RESULT
Figure 3. Sample preparation flow chart of procedures followed in FABMS to analyze samples.
W U z 0 U
75
..
571
~
I
51 1 1 1 1
I
Flgure 4. FAB mass spectrum of glycerol which represents the “matrix background” observed in FABMS; m/z 93 = MH+ of glycerol (G); m l r 185 = (G2
+ H)’
etc.
were prepared to study the effects of surface charging of the sample. In the case of the copper and 303 SS tips this charge could be dissipated through the push rod which makes contact with the ion source block. It was reasoned that if the sample layer built up a charge upon impact by the beam, then the polyimide tips which isolate the sample from the block would not be able to dissipate the charge and a decrease in ion signal would occur. However, the coated probe tips functioned as well as the uncoated 303 SS tips and no decrease in ion intensity was observed. Furthermore the likelihood that ions (remaining in the incompletely neutralized beam) could reach the sample is remote in this ion source since the probe tip is
at +8 kV and the atom beam’s kinetic energy is never greater than 7.4 kV. Angle of Incidence. To determine the optimum incident angle for FABMS we machined several probe tips with incident angles ranging from 8 = 30’ to 0 = 90’ in 10’ increments. In each case the same conditions of ion current, pressure, temperature, and sample concentration were used, the only variable being the angle of incidence of the particle beam. The amount of sample applied to the tip, 0.3 pL, sufficed to produce a thin, uniform layer on the 5.7 mm2probe surface (area = 5.7 mm2 only for 0 = 60O). The application of 1.0 p L or more of sample to the tip produces a hemispheric
ANALYTICAL CHEMISTRY, VOL. 54, NO. 13, NOVEMBER 1982
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Table 11. Composition Assignments for Mass Deficient Ion Series (Glycerol/Fomblin/Cu Target, 6 min Exposures, M / A M = 10000)
m / z (obsd)
m/z (calcd) A:
144.857 59 146.855 78 149.859 1 8 151.857 38 161.859 18 163.857 38 165.855 58 165.854 1 8 173.859 1 8 173.860 14 173.860 41 175.857 38 175.857 40 175.858 61 188.787 12 188.788 77 190.786 55 190.786 97 192.784 05 192.785 17 200.785 04 200.788 77 202.783 53 202.786 97 204.785 17 204.779 65 204.783 77 206.778 62 206.783 37 206.781 97 208.775 28 208.780 17 212.782 71 212.786 37 214.781 02 214.784 57 216.779 29 216.782 77 216.783 77 218.780 97 218.778 88 218.7131 97 226.784 32 226.786 97 228.782 20 228.785 1 7 228.783 77 230.779 21 230.781 97 238.784 88 238.706 97 a Difference. millimass units. 144.855 94 146.854 68 149.859 24 151.856 23 161.858 63 163.858 85 165.854 85
mmu -1.6 -1.1 0.1 -1.1 -0.5 1.5 -0.7 0.7 1.0 -0.3 0.0 -1.2 -1.6 -0.4 -1.1 -3.7 -3.4 -5.5 -4.1 -4.7 -3.3 -4.9 -3.7 -3.6 -3.5 -4.5 -2.1 -3.1 -2.6 -3.0 -1.6 -2.8 -2.1
composition 63C~,F 63C~65C~F C263CU, c ,6JCU65CU c,63cu2 c363cu65cu
L
IZSO)3'0
40
8'0 6'0 7'0 lnoidenl Angle ( d a g )
8'0
80
I
Flgure 5. Relationship of sample ion abundance and incident angle of the neutral beam for a 2.5 mm diameter tip at constant ion source housing pressure and temperature and constant neutral beam flux (soli line and error bars). Dotted line represents the relationship of the ratio of ion intensity at low resolution (1:lOOO)vs. high resolution (1:lOOOO)
and angle of Incidence.
NaHW;, C:063CU265CU c:,63cu,65cu C,63CU6~CU c:,06Jcu3
c:
2 0 6 3 c u ,"5 c u C,63CU,6~CU
shape which would alter the angle of incidence forcing it toward the limit of 0 = Oo, i.e., the particle beam would be perpendicular to the sa:mple surface. Figure 5 shows the results for five sample loadings of [Sarl,Alas]angiotensin I1 at each angle and shows that a plateau is reached at 0 = 60'. Figure 5 also indicates the variability (expressed by the error bars) of the signal intensity in successive sample loadings and insertions which we attribute chiefly to the error involved in loading the sample and which reflects the difficulty in exactly reproducing the postion of the probe tip within the ion source. Even when the probe position was made more reproducible by a retaining collar, the exact same ion intensity was not observed. This could be due to the fact that the production of sample ions decreases with time, although experience shows that it does not interfere with qualitative measurements. In this case, where quantitatively comparable measurements had to be made, the effect of variation of ion abundance with time was minimized by measuring it in each experiment at a specific time interval after the sample had been placed in the path of the neutral beam. Since earlier reports (13,18) had implied that 0 = 70' (i.e., 20' in the earlier, unconventional definition (13, 18, 19)) is the optim,al angle, this series of experiments was repeated a number of times and with different samples. In all cases the pattern shown in Figure 5 was observed and the maximum signal was always obtained a t 0 = 60' for our FAB instrument configuration (Figure 1). Next it was investigated whether the angle of incidence has an effect on the extent of decrease in ion current when changing from low resolution (1:lOOO) to high resolution (1:lOOOO). The circled data points in Figure 5 represent this ion current ratio of various angles for the 2.5 mm probe tip. There is no significant change in this ratio, a result which is not unexpected because the diameter of the probe tip and
therefore the area over which the sample is spread is much larger than the 1mm slit in the extraction electrode, the first ion lens in the combination source. Furthermore, the ions do not form a collimated beam, as can be deduced from the ion burn on the extraction electrode in a pattern which indicates that there is a some angular spread of the beam. This seems reasonable considering the nature and geometry of the particle beam/sample interaction. This experiment was repeated with probe tips of 1mm and 5 mm diameter, respectively, the first equal to the extraction lens slit and the other much larger. The 1 mm diameter tips showed characteristics similar to those exhibited by the 2.5 mm tips (Figure 5 ) . The relative intensity ratio of low vs. high resolution was not constant and direct comparison of these values with the ratios determined for the 2.5 mm tips indicate that the ratios for the 1 mm tip are somewhat smaller. The drawback of the 1mm tips, with a sample capacity of
