Anal. Chem. 1983, 55, 1531-1533
1531
Accurate Mass Measurement in Fast Atom Bombardment Mass Spectrometry James
M. Gilllam, Paul W.
bandis, and John L.
Occolowltz"
Lilly Research L,qboratories, Indianapolis, Indiana 46285
-
Two approaches to accurate ( 1 ppm) mass measurement of Ions generated by Bast atom bombardment are described. The first method Involves the use of a surfactant as an internal standard In the glycerol matrix and Is useful over a mass range of about m / z 280-780. The second method involves the use of a rotatable probe which alternately presents the separated sample and callbrant to the ion optics of the mass spectrometer. The mass range of the second method Is IlmHed only by thle ability of the mass spectrometer to generate slgnals sufficiently intense and well resolved for accurate mass measurement.
The advent of fast atom bombardment ionization (FAB) (1, 2) has resulted in the observation of molecular ions unobtainable in magnetic and quadrupole instruments using other ionization methods. Plasma desorption ionization (3) is generally capable of generating molecular ions from quite polar and labile compounds. However, the limited resolution of the time of flight instrument, so admirably suited to this method, has precluded accurate mass measurements. Accounts of accurate mass measurement of FAB generated ions have been reported. These have involved the use of glycerol as an internal standard with photoplate recording (4), the use of internal standards in the glycerol matrix (5), the use of split targets where standards and sample are separated (6), the use of' a multichannel signal averager in which the calibration peak is recorded during E1 and the sample during FAB (7), and the use of a data system (8). The mean mass measurement error reported for the more accurate of these methods is 3-5 ppm. Errors of this magnitude cause little trouble if the compound examined has a low molecular weight or if something is known about its elemental composition. However, there are instances where very little is known about the sample, e.g., it may be a relatively impure antibiotic in the early stages of separation. In these instances, especially for larger molecules, a mass measurement accuracy better than 3-5 ppm is necessary to limit the possible elemental compositions derived from the mass measurement to a reasonable number. Because the signals arising from FAB ionization are often as strong as those from E1 ionization, it appeared likely that mass measurement of FAl3 generated ions could be made with errors comparable to E1 measurements-about 1ppm. In the work presented here we report an investigation designed to establish the mass accuracy obtainable by peak matching FAl3 ions by using internal and external standards and to find which internal standards are the most useful.
]EXPERIMENTAL SECTION All measurernents were obtained on a Finnegan-MAT 731 mass spectrometer wing either the unmodified E1 or the unmodified FD/EI source olperating in the E1 mode with the filament switched off. Fast atoms were generated with an ion Tech BllNF gun placed opposite the ion source direct probe entrance port. The gun was modijfied by placing deflection plates, one at ground
Table I. Mass Measurements with Internal Standards "unknown"
"standard"
result
[alanine + HI' [leucine + HI' [arginine t HI' [adenosine t HI' [sucrose + Na]+ [vindoline + HI'
[Ga + H I ' [valine t 'Li]' [ZG + HI+ [HisHis + HI' [LeuTrp t HI' [Tobramycin t HI+
90.05560 132.102 34 175.119 32 268.104 59 365.10548 457.233 91
a
error, ppm 1.0 0.9 1.1
0.7 1.4 0.1
Glycerol.
potential and one at gun-anode potential, at the atom exit. Additionally, the size of the atom exit aperture was reduced and the aperture at the rear of the gun was closed. Argon atom energy was 6-10 keV. For experiments using internal standards or split target experiments not using the rotatable probe, samples were introduced via the FD probe lock on a 2.2 mm diameter circular copper target, or target of geometry described later, inclined at an angle of 70' to the ion optic axis. The rotatable probe was introduced through the E1 solids probe inlet, i.e., transverse to the ion optic axis and coaxial with the atom gun axis. The hole in the end of the solids probe inlet was bored out to accommodate the rotatable probe. Peak matching was performed at a resolution of 15000 using muliplier gains of 106-107and a detector band width of about 0.5 Hz. The position of the rotatable probe was optimized by moving the probe tip in a line transverse to the ion optic axis while observing a signal under FAB conditions. After the optimum position was found, the probe tip was locked in position by tightening the collet (Figure 3).
RESULTS AND DISCUSSION In order to determine the accuracy possible in measuring the mass of FAB ions, we chose to measure the mass of some arbitrarily selected compounds by using compounds present in the same glycerol matrix as mass standards. It was not always possible to choose a pair of compounds which were compatible without some experimentation, even though each alone in the glycerol matrix gave good FAB spectra. Often one of the compounds would suppress the FAB ionization of the other. In the case of the phosphazenes (9),which yield intense steady FAB spectra when used neat, introduction of glycerol resulted in wildly fluctuating signals which precluded their use. Table I lists mass measurements obtained for some compounds having molecular weights in the 90-500 range. The results in Table I show that when internal standards are used it is possible to determine the accurate mass of FAB generated ions with an accuracy equal to that obtained for E1 generated ions. Unfortunately, our experience indicated that the search for satisfactory internal standards could be difficult and quite time-consuming for unknowns of higher molecular weight or yielding smaller ion currents than those listed in Table I. A more general method of accurate measurement required finding an internal standard compatible with most unknowns or developing a target geometry which would give accurate results when standard and unknown were
0003-2700/83/0355-1531$01.50/00 1983 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 55, NO. 9, AUGUST 1983 bn=5
70 60
-
IM + HIn=s 710
300 350
400 450
500
550
600 650 700
MI2
750
Flgure 1. FAB spectrum of Zonyl FSB in glycerol (0.2%).
r
am@
Flgure 2. Split targets examined for use with external standards. For the rightmost target sample and standard were placed on alternate quadrants.
Table 11. Accurate Mass Measurements with Zonyl FSB as an Internal Standard “unknown” vindoline t HI’ tobramycin t HI+ moxalactam t HI+ apramycin t HI’
result
457.233 56 468.266 68 521.109 38 540.288 09 uridine-5’-diphosphoglucuronate 625.005 33 disodium salt t H1+
error, ppm 0.7 0.6 0.6 1.0 1.1
separated (external standard). Internal Standards. Without a more substantial theory describing the FAB process it is not possible to a priori designate a particular class of compounds as meeting the criteria for internal mass standards. Namely, the standard must be readily dispersed in glycerol, not suppress the FAB spectrum of the unknown, not be suppressed by the unknown, and yield steady signals over a wide mass range. The choice of the surfactant described here was based on observation and the simple reasoning that a dilution solution of the surfactant would provide a sufficient concentration at the surface of the glycerol without suppressing the unknown. Our interest was mainly focused on the acidic, basic, and amphoteric surfactants manufactured by Du Pont. Although the acidic surfactant Zonyl FSP provided a greater mass range than the amphoteric surfactant Zonyl FSB, we chose the latter because we found it more compatible with a range of unknowns. Figure 1 shows the FAB spectrum of Zonyl FSB at 0.2% concentration in glycerol. Although fragments appear at lower mass than shown we have not investigated their suitability as mass standard peaks. As a proprietry product the structure of Zonyl FSB is not readily available. The partial structure presented here, which is sufficient to define the composition of ions in the spectrum of Zonyl FSB, was deduced from accurate mass measurements and an examination of product literature. All of the prominent ions in the spectrum shown in Figure 1 arise as shown.
Flgure 3. Detalls of retractable, rotatable FAB target: (A) adjustable collet; (B) rotatable target position lndlcator; (C) fixed outer probe: (D) Vespel insulator: (E) rotatable, retractable inner probe; (F) dual target; (G) machinable glass insulator.
Table 111. Mass Measurements with an External Standard on a Rotatable Probe “unknown”
“standard”
m/z 407 Zonyl FSB
m/z 405 NaH,PO, m/z 61 0
[ metkephamidea
(LY127623) + HI’
result
error, ppm
407.014 18 0.6 601.279 79 1.7
Zonyl
FSB m/z 878 849.340 94 3.6 UM1621C m/z 1070 1021.552 88 1.7 UM1621 m/z 1370 1347.737 80 1.3 UM1621 m/z 1470 1520.962 11 1.4 UM1621 a H-Tyr-D-Ala-Gly-Phe-Me Met-”,. H-Trp-Ala-GlyGly-Asp-Ala-Ser-Gly-Glu-OH. Ultramark 1621, phos[ H-Tyr-D-Ala-Gly-Phe-NH(CH,),]2. phazene mixture. [Peptide LY13968gb + H1’ [Peptide LY146957d t HI+ [Peptide LY142261e + H I + m/z 1521 UM1621
e
H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH~.
Table I1 gives the results of accurate mass measurements made by using Zonyl FSB as an internal standard. External Standards. Figure 2 illustrates the stationary targets which were examined for suitability in mass measurement by using external standards. Each of the five targets behaved similarly. It was not possible to achieve an ion source focusing condition which resulted in simultaneous optimum focus for standard or unknown. When a compromise focus was achieved, the amplitude of each signal was considerably less than optimum. And worse, mass errors of about 10 ppm resulted for mass measurements obtained in the compromise focus condition. The good accuracy obtained with internal standards and the poor accuracy obtained with external standards suggested that to successfully use external standards it would be necessary to use a movable probe. (The probe should operate to preserve the same geometry relative to the ion-optical system when either standard or sample was examined.) The rotatable probe illustrated in Figure 3 is suitable for the geometry of our mass spectrometer. Although both the standard and unknown are simultaneously illuminated by the atom beam, only the target face facing the flight tube yields detectable ions. Rotation of the probe in 180’ increments at the same rate as the accelerating voltage alternation used in peak matching results in alternate views of standard and unknown. These can be matched with an oscilloscope of appropriate persistence or until both peaks coincide with a peak profiie drawn on the face of the oscilloscope. Observation
Anal. Chem. 1083, 55, 1533-1537
of the m/z 185 from glycerol at a resolution of 25 000 showed no peak shift wlhen the glycerol was observed from either target face. Table I11 lists isome accurate mass determinations using the rotatable probe. The results using the rotatable probe are almost as good as those using internal standards. We believe that the somewhat greater error is due to the longer interval between presentation of standard and unknown peaks which arises from the present need to manually rotate the probe. Although this aplit probe geometry was necessary to obtain good mass measurements on our 731 mass spectrometer, we do not know if it is necessary for mass spectrometers having other ion optical geometries. Generally we have found the rotatable probe t o be preferable for accurate mass measurements because of the wide choice of mass standards it allows and the steady nature of the ion currents which it provides.
ACKNOWLEDGMENT We thank K. Brunee of Finnegan-MAT, Bremen, for suggesting the use of a rotatable probe. We also thank D. Sed-
1533
gewick, formerly of UMIST, for advice on modifications to the fast atom gun. Registry No. Zonyl FSB, 57534-42-6.
LITERATURE CITED Barber, M.; Bordoli, R. S.; Sedgewlck, R. D.; Tyler, A. N. J. Chem. Sac., Chem Commun. 198l, 7 , 325-327. Surman, D. J.; Vlckerman, J. C. J. Chem. SOC.,Chem. Common. 1981, 7, 324-325. Torgerson, D. F.; Skowronskl, R. P.; Macfarlane, R. D. Blochem. Biophys. Res. Commun. 1974, 60,616. Heller, D.; Hansen, G.; Yergey, J.; Cotter, R. J.; Fenselau, C. "Abstracts"; 30th Annual Conference on Mass Spectrometry and Allied Topics, Honolulu, HI; Amerlcan Society for Mass Spectrometry: 1982; pp 560-561. Rinehart, K. L., Jr.; et al. J. Am. Chem. SOC.1981, 703,8517-8520. Van Langenkove, A.; Costello, C. E.; Chen, H. F.; Biller, J. E.; Blemann, K. "Abstracts"; 30th Annual Conference on Mass Spectrometry and Allied Topics, Honolulu, HI: American Society for Mass Spectrometry: lg82; pp 558-559. Rlnehart, K. L., Jr. Science 1982, 218, 254-260. Morgan, R. P.; Reed, M. L., Org. Mass Spectrom. 1982, 77, 537. Olson, K. L.; Rinehart, K. L., Jr.; Cook, J. C., Jr. Biomsd. Mass Spectrom. 1977, 4 , 284-290.
RECEIVEDfor review February 7,1983. Accepted May 9,1983.
Determination of Reactive Components in Silicone Foams S. V. Dublel, 0. W. Girlfflth, C. L. Long, G. K. Baker, and Robert E. Smith" The Bendlx Corporation, Kansas City Division, D/816, SA-1, P.O. Box 1159, Kansas City, Mlssouri 64141
Analytical methods have been developed for the determlnation of reactive components used In the formulation of room temperature vulcanlzed ( R N ) sllicone foams. The reactive components Include total sllanol (SIOH), silane hydrogen (SIH), tetrapropoxysllane (TPS), and dlphenylmethylslianoI (DPMS). Total SiOH and SIH are determined by Fourler transform Infrarctd (FTIR) spectrometry. the SlOH peak at 3687 cm-' and the SIH peak at 2168 cm-' were used for quantitatlon. The TPS content was determined by gas chromatography (GC), using a solid capillary open tubular (SCOT) column and linear programmed temperature control. The DPMS content was determined by gel permeation chromatography (GPC) using THF solvent.
Recently, much interest has been shown in silicone polymers. Their use in industry is well documented (1-5). Studies on theories of rubber elasticity have used end-linked poly(dimethylsiloxanes) as models (6-8).Another class of silicones includes foams tlhat are cured at room temperature by mixing an organotin catalyst with a silicone prepolymer (9). They have thus been called room-temperature-vulcanized (RTV) silicone foams. Their use as encapsulants for integrated circuits has been described (IO,11). It would be expected that the concentration of reactive end groups and cross-linking agents would greatly affect the properties of the silicone polymer produced. A review of analytical methods for silicones described general methods without giving experimental details (12). However, specific methods for determining the reactive end groups SiH and SiOH have been developed. Hexafluoroacetune has been used to determine total -OH content (13),but hexafluoroacetone is hazardous andl not commercially available. Titration with
the Karl Fischer reagent can determine SiOH content (14,15) but is subject to interferences from water and SiH. The IR bands caused by SiOH and SiH have been characterized for some time (16) but are subject to interferences from water, solvent, and siloxane bands. The advent of Fourier transform infared (FTIR)spectrometry and computer software has made possible the technique of subtractive FTIR to remove these interferences. For the determination of cross-linking agents, chromatographic methods are preferred. Two possible cross-linking agents are tetrapropoxysilane (TPS) and diphenylmethylsilanol (DPMS). Gas chromatography of TPS, as well as a number of other silicon-containing compounds, has been described (17);however, no analytical methods for the determination of DPMS are available. In the Bendix laboratory, a RTV silicone consisting of TPS, DPMS, silanol terminated poly(dimethylsiloxane), and poly(methylhydrosi1oxane)was prepared. The following polymerization reactions involving TPS, SiOH, and SiH have been described (18). S" =SX-OH+ESI-H
_c
(I)
ESI-O-S!E+H,
HID
OCHXHKH, 4c
sb0H
Sn
+ CH,CH2CH.0 - Si - OCHICH~CH, OCHCH2CHI
0 E
Ha0
-
-
SI 0 - Si. 0 SI E
(2)
0 Si ill
Because of their importance in cross-linking, Bendix has developed methods for the determination of SiOH, SiH, DPMS, and TPS. These methods involve FTIR, gas chro-
0003-2700/83/0355-1533$01.50/0 0 1983 American Chemical Soclety
