2444
Anal. Chem. 7905, 57, 2444-2448
the surface by the laser is lo5per pulse. For sampling small deposits, this should allow both high sensitivity and reasonable statistical precision through repetitive sampling. Registry No. Ta, 7440-25-7. N
LITERATURE CITED (1) Bekov, G. I.; Letokhov, V. S . Appl. Phys. 8 1983, 30, 161-176. (2) Bekov, G. I.; Letokhov, V. S.;Radaev, V. N.; Baturin, G. N.; Egorov, A. S.;Kursky, A. N.; Narseyev, V. A. Nature (London) 1984, 372, 748-750. (3) Donohue, D. L.; Young, J. P.; Smith, D. H. Int. J . Mass Spectrom. Ion Phys . 1982, 43,293-307. (4) Donohue, D. L.; Young, J. P.; Smith, D. L. Int. J . Mass Spectrom. Ion Processes 1984, 56,307-319. (5) Donohue, D. L.; Smith, D. H.; Young, J. P.; McKown, H. S.;Pritchard, C. A. Anal. Chem. 1984, 56, 379-381. (6) Donohue, D. L.; Young, J. P.; Smith, D. H. Appl. Specfrosc. 1985, 39, 93-97. (7) Downey, S. W.; Nogar, N. S.;Miller, C. M. Int. J . Mass Spectrom. Ion Processes 1984, 61 337-345. (8) Downey, S. W.; Nogar, N. S.;Miller, C. M. Anal. Chem. 1984, 56, 827-828. (9) Fassett, J. D.; Travis, J. C.; Moore, L. J.; Lytle, F. E. Anal. Chem. 1983, 55, 785-770. (10) Fassett, J. D.; Moore, L. J.; Travis, J. C.; Lytle, F. E. Int. J . Mass Spectrom. Ion Processes 1983, 54,201-216. (1 1) Fassett, J. D.; Moore, L. J.; Shideler, R. W.; Travis, J. C. Anal. Chem. 1984, 56, 203-206. (12) Fassett, J. D.: Powell, L. J.; Moore, L. J. Anal. Chem. 1984, 56, 2228-2233. (13) Moore, L. J.; Fassett, J. D.; Travis, J. C. Anal. Chem. 1984, 56, 2770-2775. (14) kller, C . M.; Nogar, N. S. Anal. Chem. 1983, 55, 1606-1608. (15) Miller, C. M.; Nogar, N. S.;Gancarz, A. J.; Shields, W. R. Anal. Chem. 1982. 54. 2377-2378. (16) Moore, L.' J.; Fassett, J. D.; Travis, J. C. Anal. Chem. 1984, 56, 2770-2775. (17) Nogar, N. S.;Downey, S. W.; Mlller, C. M. Anal. Chem. 1985, 57, 1144-1147. (18) Young, J. P.; Donohue, D. L. Anal. Chem. 1983, 55,88-91. (19) Smith, D. H.; Walker, R. L.; Carter, J. A. Anal. Chem. 1982, 54, 827A-830A. ~
(20) Miller, C. M.; Nogar, N. S . Anal. Chem. 1983, 55,481-488. (21) Beekman, D. W.; Callcott, T. A.; Kramer, S.D.; Arakawa, E. T.; Hurst, G. S.;Nussbaum, E. Int. J . Mass. Spectrom. Ion. Phys. 1980, 34, 89-97. (22) Mayo, S.;Lucatorto, T. B.; Luther, G. G. Anal. Chem. 1982, 54, 553-556. (23) Williams, M. W.; Beckman, D. W.; Swan, J. B.; Arakawa, E. T. Anal. Chem. 1984, 56, 1348-1350. (24) Becker, C. H.; Gillen, K. T. Anal. Chem. W84, 56, 1671-1674. (25) Kimock, F. M.; Baxter, J. P.; Pappas, D. L.; Kobrin, P. H.; Winograd, N. Anal. Chem. 1984, 56, 2782-2791. (26) Kimock, F. M.; Baxter, J. P.; Winograd, N. Surf. Sci. 1983, 724, L41L48. (27) Parks, ?. E.; Schmitt, H. W.; Hurst, G. S.;Fairbank, W. M., Jr. Thin Solid Films 1983, 708,69-78. (28) Winograd, N.; Baxter, J. P.; Kimock, F. M. Chem. Phys. Lett. 1982, 68,581-584. (29) Burgess, D. R.; Hussla, I.; Stait, P. C.; Viswanathan, R.; Weitz, E. Rev. Sci. Instrum. 1984, 55, 1771-1776. (30) Burgess, D., Jr.; Viswanathan, R.; Hussla, I.; Stair, P. C.; Weitz, E. J . Chem. Phys. 1983, 79,5200-5202. (31) Hurst, G. S.;Payne, M. G.; Phillips, R. C.; Dabbs, J. W. T.; Lehmann, B. E. J . Appl. Phys. 1984, 55, 1278-1281. (32) Hall, R. 6.; DeSantolu, A. M. Surf. Sci. 1984, 737,421-441. (33) Cotter, R. J. Anal. Chem. 1984, 56,485A-504A. (34) Dittrich, K.; Wennrich, R. f r o g . Anal. At. Spectrosc. 1984, 7 , 139-198. (35) Ready, J. F. "Effects of High Power Laser Radiation"; Academic: New York, 1971. (36) Peliin, M. J.; Wright, R. B.; Gruen, D. M. J . Chem. Phys. 1981, 7 4 , 6448-6457. (37) Berry, R. S.;Rice, S. A.; Ross, J. "Physical Chemistry"; Wiley: New York, 1980. (38) Olstad, R. A.; Olander, D. R. J . Appl. Phys. 1975, 4 6 , 1499-1508. (39) Comsa, G.; David, R.; Schumacher, 8.-J. Surf. Sci. 1980, 95,L210L216. (40) Husinsky, W.; Bruckmuller, R.; Blum, P.; Viehbock; Benes, E. J . Appl. Phys. 1977, 48, 4734-4740. (41) Levy, D. H. Sclence 1981, 274, 283-269. (42) Cowin, J. P.; Auerbach, D. J.; Becker, C.; Wharton, L. Surf. Scl. i o n , 78,545-564.
RECEIVED for review March 22,1985. Accepted June 24,1985.
Structural Determination of Unsaturated Mycolic Acids by Fast Atom Bombardment and Tandem Mass Spectrometry Analyses of Their Amino Alcohol Derivatives Michel Riviere, Monique Cervilla, and Germain Puzo*
Centre de Recherche de Biochimie et de GBngtique Cellulaires du C.N.R.S., 118, route de Narbonne, 31062 Toulouse Cedex, France The ethylenlc functlons of the mycollc acids Isolated from M. smegmails were transformed Into amlno alcohols. Thelr analyses by posltlve fast atom bombardment mass spectrom etry allows thelr molecular weight to be unamblguously established from their pseudomolecular Ions. Moreover their MIKE-CID (mass analyzed Ion kinetic energy colllslon induced dlssoclatlon) mass spectra permit the amlno groups borne by the aliphatic chaln and consequently the ethylenic functlons In the natlve molecule lnvestlgated to be located.
Mycolic acids are a-alkyl p-hydroxylated fatty acids of basic structure 1 which are present in the wall of mycobacteria, nocardia, and corynebacteria ( I ) . OH R~
I
- C H - CH- C O ~ H
I
Rl
1: R z , mer0 chain R, , O - ramified chain
Minnikin has shown that mycolic acid alkyl chain lengths, defined by the number of methylene units, permits the identification of some mycobacterial species (2). Daffe et al. recently noted that lipidic analysis allows identification of 22 species of mycobacteria among the 27 investigated (3). Knowledge of the exact structure is required to establish the metabolic pathway of the mycolic acids and also to resolve the problem of mycobacteria taxonomy. Their structures have been mainly elucidated using electron ionization (EI) mass spectrometry on mixtures of mycolic acids. By EI, methyl esters of mycolic acids (I) mainly given two rearranged fragment ions [R2CHO]+-and [R1CH2CO2CH3]+. allowing the identification of R2 and R1 and consequently the determination of the molecular weight. To establish their exact structures, Steck et al. proposed high-performance liquid chromatography (HPLC) for resolving mycolic acid mixtures isolated from M. smegmatis into molecular species (4). An a-mycolic fraction previously studied by Etemadi et al. (5) called B by Steck et al. (5) has been resolved by HPLC according to chain length. Electron ionization (EI) and chemical ionization (CI) mass spectrometry
0003-2700/85/0357-2444$01.50/00 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985
2445
-89.54
100-170 4 2 -I
. . ^
> t
598
Lo
z IY
z
50-
Y >
-15
*
+
5
685
-
454
I
I " " I " "
"
!,
141
I1
1 ' ' ' ' 1 ' ' ~ ' 1 ' ' ' ' ~ ' ' ' ' ~ 1 ' "
A ~ '
I,+.
' ' ' ' ' ' '
W
r:
-
rn/z
Figure 1. FAB mass spectrum of amino alcohol derivatives of a synthesized mycolate methyl ester.
Scheme I CH,- (CH,)fi CH= CH-(CH&~CO~CHJ
Y CH,-
I
- epoxidation - HN - (CH3)z - methanolysis
( c H , ) ~CH- 'fH-(CH,kCOzCH3
X
x 6:x
A:
Y =-OH
=-N(CH,),, Z -OH
,
Y =-N (CH,),
and NMR allow the following basic structures for the dialkene series to be established (6). The double bonds have been located by oxidative ozonolysis. CH,-(CH;~-
CH=CH-(CHJ -CH=CH-(CH,)
rn
?H
-CH-CH
P
I
"
-COOCH,
c 2 2 H45
YH
CH,- ( c H , ) ~ C H ~ H - ( C H , ~ C H = C H - C H - ( C H ) -CH-f.H -COOCH, n C11 H,, CH3
'
'-'
Recently we proposed CI mass spectrometry using mass ion kinetic energy (MIKE/CID) to locate double bonds in unsaturated fatty acid mixtures (7). Among the possible double bond derivatizations (8, 9) we selected transformation into amino alcohol ( 7 , l O )via epoxidation as described in Scheme 1.
MIKE/CID of the pseudomolecular ions generated by CI shows two intense fragments arising from the cleavage of the CHX-CHY bond of both isomers (7). Both fragment ions possess an imminium structure, and their masses permit the amino group of each pseudomolecular ion investigated to be localized when mixtures are analyzed. Moreover, this method has been successfully used for direct isomeric analysis of unsaturated fatty acid mixtures isolated from mycobacteria (7'). A similar strategy has been used by Gross et al. for locating double bonds on underivatized fatty acids, using MIKE/CID analyses of their (M - H)+ or (M Fe)+ ions, respectively, generated by FAB (9) and by CI (11). In the present work, we investigated the potentialities of MS/MS analysis in the structural determination of unsaturated mycolic acids isolated from M . smegmatis.
+
EXPERIMENTAL SECTION Mass Spectrometry. Mass spectrometric measurements were performed on a double focusing instrument, type V.G. ZAB-2F (V.G. Analytical, Altrin chain, U.K.). The instrument was equipped with a FAB ion source. The atom gun (Ion Tech, Ltd.)
operated at 8 kV and currents (between 1and 1.5 mA). Xenon atoms were used to bombard the sample. Sample Preparation. Ten micrograms of the investigated sample was dissolved in 0.5 pL of glycerol previously deposited on the target probe. For MIKE/CID analysis higher quantities of sample were required. MIKE/CID were obtained by increasing the helium pressure in the collision cell until the precursor ion abundance was reduced to approximately 20% of its original value. Fragmentation was measured by scanning the ESA potential over the energy range 8-0.5 kV in 80 s. Chemical Syntheses. Palmitic and elaidic acids were purchased from Fluka (Chemische Fabrik, 9470 Buchs, Switzerland). Compounds I, 11,and I11 were synthesized by condensing methyl palmitate and methyl elaidate and then reducing with sodium borohydride (12). These compounds were derivatized into amino alcohols according to the protocol previously described (7). The saturated compounds resulting from the condensation of two molecules of palmitic acid were fractionated after the epoxidation reaction. Bacteria. M. smegmatis, ATCC 14468,was grown on Santon's medium. Five-day-old mycobacterial cells were saponified under reflux for 5 h in a suspension of KOH 5% in benzene/ethanol/water (11511, v/v/v). After acidification and extraction with diethyl ether, the lipidic fraction was methylated with ethereal diazomethane. The a-methyl mycolates were eluted, using a diethyl etherllight petroleum gradient (bp 50 "C), from a Silicar CC7 column (Mallinckrodt) when the proportion of ether was between 5 and 10%. The fractions were analyzed, by thin-layer chromatography (Silicagel G from Merck), using light petroleum-diethyl ether 8:2 (v/v) as solvent. The a-methyl mycolate fractions were purified on a silver nitrate impregnated silicic acid column. The separation was followed by argentation thin-layer chromatography. RESULTS AND DISCUSSION Mass Spectrometry Analysis of Synthetic Mycolic Acids. In the generation of ions containing the whole molecule from derivatized mycolic acids, it seems likely that the surface ionization mode would be more appropriate since volatilization induces pyrolysis. To analyze their behavior, mycolic acids (I, 11,111)were synthesized (see Experimental Section) and derivatized according to Scheme I into amino alcohols, giving compounds IA, IB, IIA, IIB, IIIAA, IIIAB, IIIBA, and 1 1 1 ~the ~ ; structures are summarized in the Scheme 11. The unfractionated mixture of amino alcohol derivatives was analyzed by FAB-MS using glycerol as matrix; the mass spectrum obtained is shown in Figure 1. In the molecular mass range, we observe intense peaks a t m / z 598 and 685 attributed to the expected protonated molecular ions (M + H)+of the amino alcohol derivatives of compounds I and I1 and 111, respectively. Their abundance and their lifetime permitted their analysis by MIKE/CID. Moreover fragment ions characteristic of the location of the amino group were observed a t m / z 541, 454, and 170. They arise from the
ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985
2446
Scheme I1
MH'598 MH.H,OJ
II
454
1
E Fo
0:25
os
I
I
0,75
1
Figure 2. M I K E X I D spectrum of the precursor pseudomolecular ion (MH)' = 598 generated by FAB from compounds I,, I,, II,, and I I E .
n
170
m
.
EE.
oh5
0.5
1
0,75
1
Figure 3. MIKEICID spectrum of the precursor pseudomolecular ion (MH)' = 685 generated from compounds I I I A A , IIIAB, III!,, and II IBB*(MH- 63) corresponds to the expulsion of dimethylamine and water.
Scheme IV Scheme I11 %C,
/CH3
+
+N-
H
- C' H - C H -
*-,
C H p ( C H 2 k CH=CH-(CH,)yjj CHdH-(CH,
+ -CHzN(CH3)2
+ -CH2OH
I
OH
YH
b- CH-CH "
-COOCH,
I
n+m+p=42,44,46
I
c 2 2 H45
-
reduction
- epoxidation - HN(CH3))
protonated molecular species by the mechanism previously described (7) and summarized in Scheme 111. The fragment a t m / z 170 which possesses the alkylimminium structure CH3(CH2)7CH=N(CH3)2+arises from the protonated molecular ions of compounds IB, IIB, IIIAB, IIIBA, and IIIBB. The complementary fragment ion (m/z 454) of structure
PH
OH (cH,)~~ICH-(CH,S~H-~H-CO,CH, C,&CH- CH I
,
-
arises from IAand 11, while the mlz 541 fragment comes from the isomers (IIIAA,III,, IIIBA)by one cleavage. Similar mass spectra concerning the ions arising from the analyzed sample were obtained by desorption chemical ionization using ammonia as reactant gas; however, the lifetime of the protonated molecular ions is shorter. To examine the possibility of analyzing mixtures of mycolic acids and to confirm the fragmentation pathway of their protonated molecular ions, the latter ions were analyzed by MIKEICID. Figures 2 and 3 represent the MIKEICID spectra of the protonated molecular ions (M + H )' m / z 598 and 685. Both of these ions eliminate one molecule of water (MH - 18), while expulsion of one molecule of dimethylamine (MH - 45) or dimethylamine and water (MH - 63) only occurred with mlz 685. The presence of the fragment ions at mlz 170 and m/z 454 arising from mlz 598 (Figure 2) and mlz 170 and m/z 541 from mlz 685 is of great interest (Figure
3). These ions which are also present in the mass spectrum (Figure 1)allow the localization, as previously described, of the amino groups of each protonated molecular ion analyzed. Analysis of a Diunsaturated Ethylenic Mycolic Acids Isolated from M . smeghlatis. A lipidic mixture containing mycolic acids isolated from M . smegmatis was fractionated by liquid chromatography (see Experimental Section). E1 analysis of the different fractions obtained indicated that one of them mainly corresponds to three homologues of diunsaturated a-methyl mycolate of molecular formula C79H15403, C77H15003, and C7SH14603.The ramified R1chain consists of an n-C22H45alkyl chain, while the number of methylene units corresponds to 42, 44, or 46 (see Scheme IV). The methyl esters of the mycolic acids, before the epoxidation and dimethylamine reactions, were reduced to alcohol functions to avoid a methanolysis step since they are insoluble in methanol. As shown in Scheme IV these myco-
ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985 main beam
m/r:ll89
2447
I
1
,
I "
750
BOO
850
900
950
1000
m/z
1050
1100
1200
1
0,75
EO
1150
1250
mass spectrum of amino alcoholic derivatives of diunsaturated mycolols.
Figure 4. FAB
m+nvalues
vi
25 27 29
509 537
loyls were transformed into amino alcohol derivatives leading to the isomers IVU, IVAB, IVBA,and IVBB. The FAB-MS spectrum of these different isomers is presented in Figure 4. In the high mass range intense peaks are observed at m / z 1161,1189, and 1217 and correspond to the expected protonated molecular ions. Their presence unambiguously allows the direct determination of the molecular weights of the mycolic acids. Fragment ions are also observed and can be explained by the fragmentation mechanism summarized by Scheme 111. A set of three peaks is observed (mlz 706,678,650). The parity, the high masses of these ions, and the values of n, m, and p (n + m + p = 42, 44, 46) suggest that the ions contained the R1 chain and arose from the fragmentation of the protonated molecular ions I V u and IVm Such an assumption allows the determination of the p values ( p = 15,17, 19). The complementary fragmentations arising from the isomers IVBB and IVBAare observed at m/z 537 and m / z 565; so the (n m) values are 25 and 27. At low mass range a set of three peaks a t m / z 254,282, and 310 permits the determination of the n values (n = 13,15,17). Fragment ions allowing a direct determination of the m values are missing. From the n and p values previously established we can retain all the m values which satisfied the relation n + m + p = 42, 44, 46. By MIKE/CID analysis of a selected protonated molecular ion we tried to define the n, m, and p values of a given homologue. The MIKE/CID spectrum of the protonated molecular ion m/z 1189 (n + m p = 44) is shown in Figure 5. The loss of one molecule of water (MH - 18) or dimethylamine (MH45) and consecutive expulsion of water and dimethylamine (MH - 63) are observed. The ion at m / z 835 could arise from the expulsion of the alkyl alcohol (C23H47CH20H); however, similar fragmentation is not observed either in the mass spectrum (Figure 4) or in the MIKE/CID of pseudomolecular ions arising from synthesized mycolic acids (Figures 2 and 3). The four sets of ions separated by 28 mass units which arise by the mechanism summarized in Scheme I11 are of greater interest. The series m / z 961, 933, and 905 arises from the isomers IVU and IVBA giving m + p values of 31,29, and 27; the series m/z 706,678, and 650 results from the fragmentation of the isomers IVU and IVAB giving 19, 17, and 15 for the p values; the series m / z 565, 537, and 509 corresponds to the fragmentation of the isomers IVBB and IVBA and leads to n + m values of 29, 27, and 25. The series m / z 310, 282, and 254 finally gives n values of 17,15, and 13. The m values can only be obtained by difference ( m = 8,10, 12, 14, and 16) since fragment ions allowing its direct determination are missing. From Figure 5 we observe two sets of peaks at m / z 933 and m / z 537. Both of these ions contained the methylene units defined by associated t o p ( m / z933) and to n (mlz 537), respectively. To determine the m values, we analyzed the
+
+
.
E
565
0.25
650 0!5
k
EO
Figure 5. M I K E K I D spectrum of 1189 ( n m p = 44).
+ +
the protonated molecular ion m l z
precursor ion, m/z 933, present in low abundance in the mass spectrum (Figure 4), by MIKE/CID. This fragment ion of structure V was selected since the expected masses of the ions containing of the m and p values resulting from its decomposition do not overlap.
+
V, (cH,),N-cH-
Y
( C H J ~ C H - F H -(cH,)
x mtp =29
VA
F
PH CH-CH-CH,OH , mlz933 i I CmHa
:X=N(CH,),,
vg:X=OH
Y=OH
,
Y:N(CH,),
This ion eliminates both a molecule of water and dimethylamine upon collisional activation. Furthermore two series of fragment ions are observed at m / z 706 and 678 and m/z 282 and 254. From the p values previously established (15,17,19) one can assume that ions m / z 706 and 678 arise by a cleavage of the CHY-CHX bond of the precursor ion VA by a mechanism not yet elucidated. By analogy we assumed that the fragment ions m/z 282 and m/z 254 arise from isomer VB giving 10 and 12 for the m values. These "mn values can be overestimated since the protonated molecular ions m / z 1161 and m / z 1217 could also participate in the formation of fragment precursor ion m / z 933. So for a diunsaturated homologous mycolic acid defined by n + m + p = 44 and m + p = 29, the proposed method allows the identification of two isomers defined by n = 15, m = 10, p = 19 and n = 15, m = 12, p = 17. These values are in agreement with those proposed by Gray et al. (6). The proposed method could also be applied to the detection and structural elucidation of epoxy mycolates (13). Registry No. I, 97613-94-0; IA, 97613-95-1; IB, 97613-96-2; 11, 97613-97-3; IIA, 97613-98-4; IIB, 97613-99-5; 111, 97633-43-7; IIIAA, 97614-00-1;IIIAB, 97614-01-2;IIIBB, 97614-02-3;IIIBA, 97614-03-4; CH,(CH,),,CH=CH(CH,)ioCH=CH(CH2)19CH(OH)CH(CO,H)(CH,)21CH3, 83504-44-3; CH3(CHZ)&H=CH(CH~,,CH=CH(CH,),,CH(OH)CH(CO,H)(CH,),,CH,, 83504-45-4; CH,(C~GHB,)CH(OH)CH(CO,H)(CH,),,CH3, 97613-83-7; CH3(C~,H,G)CH(OH)CH(C~~H)(CH,),,CH~, 97613-85-9.
LITERATURE CITED (1) Assellneau, J. "The Bacterial Lipids"; E. Lederer, Hermann: Paris, 1966. (2) Minnikin, D. E. "Lipids, Biology of Mycobacteria": Ratledge, C., Ed.: 1982; pp 95-184.
2448
Anal. Chem. lQ85,57,2448-2451
(3) Daffe, M.; Lanklle, M. A.; Asselineau, C.; Frebault, V. L.; David, H. Ann. Microbiol. (faris) 1983, 1348, 241-256. (4) Wong, M. Y. H.; Steck. P. A.; Gray, G. J . Bid. Chem. 1979, 2 5 4 . 5734-5740. (5) Etemadi, A. H.; Pinte, F.; Markovits, J. Bull. SOC.Chim. Fr. 1967, I , 195-198. (6) Danielson, J. J.; Gray, G. R. J . Bid. Chem. 1982, 257, 12196-12203. (7) Cervilla, M.; Puzo, G. Anal. Chem. 1983, 5 5 , 2100-2103. (8) Minninkin, D. E. Chem. f h y s . Lipids 1978, 2 1 , 313-347. (9) Tomer, K. B.; Crow, F. W.; Gross, M. L. J . Am. Chem. SOC. 1983, 105,5487-5488.
(10) Audier, H. E.; Bory, S.; Fetizon, M.; Longevialle, P.; Toubiana, R. Bull. Soc. Chim. Fr. 1974, 3034-3035. (11) Peake, D. A.; Groww, M. L. Anal. Chem. 1985, 5 7 , 115-120. (12) Lederer, E.; Portelance, V.; Hansen, S. K. Bull. SOC.Chim. Fr. 1952, 4 13-41 7. (13) Daffe, M.; LanOelie, M. A.; Puzo, G.; Asselineau. C. Tetrahedron Lett. 1981, 439, 7518-7519.
RECEIVED for review April 12,1985. Accepted June 10, 1985.
Quantitative Fast Atom Bombardment Mass Spectrometry of Silylated Silica Surfaces Steven J. Simko, Mark L. Miller,' and Richard W. Linton* Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27514
An atom source for fast atom bombardment mass spectrometry (FABMS) of Insulating samples under static condltlons Is discussed. Low prlmary flux densities allow outer monolayer analysls of octadecyldlmethylchlorosllane (ODS) reacted silicas. Measurement of organlc secondary Ion intensities from the chromatographic supports as a function of ODS coverage shows a linear correlatlon after suitable normallration procedures. Ratlos of SIOH+/SIO+ decrease wlth Increasing ODS coverage, conflrmlng the removal of surface sllanol groups by reactlon wlth ODS. Results correlate with other surface analysls and chromatographic studies, suggestlng resldual surface silanols are largely Inaccessible at hlgh ODS coverages.
Secondary ion mass spectrometry (SIMS) is an important method for the analysis of metal and semiconductor surfaces. Not as widespread, however, is the study of insulators such as glass or chromatographic substrates by SIMS, since these samples charge under the influence of the primary beam, resulting in suppression of the secondary ion signal (1). Much effort has been exerted in developing procedures for alleviating this effect; these have been reviewed recently by Werner and Warmoltz (2). One method that has proven successful is substitution of energetic neutrals for the primary ion beam, often referred to as fast atom bombardment (FAB). Typical FAB experiments are performed on samples dissolved in a liquid matrix (usually glycerol) which allows diffusion of subsurface sample molecules to the vacuum-liquid interface where they are desorbed and ionized by the primary beam. This provides a renewable source of sample molecules and allows acquisition of stable, reproducible spectra for extended periods (3). This technique has helped to expand the range of samples amenable to mass analysis, including some compounds which are involatile, labile, and otherwise intractable by other ionization methods (such as high molecular weight compounds). FAB also can be used without the liquid matrix to probe the surface structure of nonconducting solid samples ( 4 ) . Glasses, catalysts ( 5 ) ,and polymers (6, 7) have been studied, Present address: Monsanto Polymer Products Co., Akron, OH
44313.
with researchers reporting higher signal intensity and more reproducible spectra than with other charge neutralization techniques. Briggs, for example, achieved a fivefold increase in sensitivity using FABMS, compared to SIMS with electron beam charge neutralization, for the study of polymer surfaces (7). The surface and molecular specificity of fast atom bombardment (FAB) make it a promising technique for the characterization of chromatographic support surfaces. Of interest is the measurement of both covalently-bound organic species (silane) and surface functionalities of the inorganic substrate (silanols) as a function of silane coverage. The potential of particle bombardment mass spectrometry (FABMS or SIMS) for the measurement of hydroxyl groups as a function of surface hydroxylation has been suggested for titanium dioxide, by monitoring the TiOH+/TiO+ion intensity ratio (8), and for glass, by monitoring the SiO+/SiOH+ ion intensity ratio (5). It has also been shown that FAB secondary ion intensity ratios (X+/Si+, where X = Al, B, Ti, Zr, Li) linearly correlate with the elemental concentration ratios of various glasses (5, 9). The present study concerns the evaluation of a FABMS system for application to surface analysis studies of bondedphase chromatographic materials. The use of particle bombardment mass spectrometry as a structural probe of organics physisorbed on inorganic substrates ( l o ) ,or for the identification of organics adsorbed on chromatographic substrates (TLC paper) has been demonstrated previously (11). This study addresses the capability of quantitating relative coverages of organics chemisorbed on inorganic substrates in submonolayer amounts. Of interest are the mass spectral characteristics of the organic and hydroxyl functionaliaties as they relate to the surface structure of the chromatographic surface and, ultimately, to chromatographic behavior.
EXPERIMENTAL SECTION Reagents. Indium foil was obtained from Alfa Products and octadecyldimethylchlorosilane from Petrarch Systems. Whatman Partisil 10 silica (RR-129-7A)with a specific surface area of 323 m2/g, average particle size of 7.51 pm, average pare diameter of 96.2 A, total pore volume of 0.777 cm3/g, and bulk density of 0.39 g/mL was used throughout the study. Preparation of Bonded Phases. Ten grams of Partisil 10 was dried in air at 150 "C for 1-2 days before refluxing in CC14, 2.5 mL of pyridine, and varying amounts (depending on coverage
0003-2700/85/0357-2448$01.50/00 1985 Amerlcan Chemical Society
