Acetylation as a Chemical Ionization Technique in Medium-Pressure Chemical Ionization Mass Spectrometry J. Ronald Hass" National Institute of Environmental Health Sciences, P.O. Box 42233, Research Triangle Park, North Carolina 27709
Willard B. Nixon and Maurice M. Bursey" Venable and Kenan Chemical Laboratories, The University of North Carolina, Chapel Hill, North Carolina 27514
Mixtures of helium and biacetyl are shown to be effectlve lonlring reagents for a variety of organlc functional groups. Trends noted in ion cyclotron resonance (ICR) studies relating to absence of acetylated product Ions are reproduced at high pressure, and the ratio of acetylated to protonated product Ions correlates with observations in ICR as well. ?he stereoselectivlty of blacetyl noted In the ICR results are duplicated and found to be reproduced in both new and previously tested systems.
Correlation of ion reactivities between chemical ionization (CI) and ion cyclotron resonance (ICR) data is typically not straightforward. The inherent problem is that the number of collisions suffered by an ion under CI conditions is greater than in ICR, especially when the latter is operated in the normal drift mode. Hence, a study designed to find the conditions for obtaining acylation reactions by chemical ionization similar to the analogous reactions observed under ICR conditions is indicated. As an example, self-acylation for acetone, Equation 1, is a prominent reaction under the low pressure conditions of ICR Torr, approximately 10 ms reaction time) ( I ) . CH,COCH,'. + CH,COCH, + CH,CO(CH,COCH,)' + CH,. (1 1 However, under CI conditions (1 Torr, 10-ps source residence time), the concentration of the product ion above or any type formed by acetone clustering upon the acetyl ion, CH3CO(CH3COCH3),+,is not significant (less than 5%) ( I ) . Rather, the bulk of the ion current (ca. 7 5 % ) consists of protonated clusters, H(CH3COCH3),' ( I , 2). Furthermore, it is indicated from a study of proton transfer reactions between ketones (3) that ketones in general would yield no better results as acyl-transfer reagents than acetone, reacting principally as proton transfer agents ( 4 , 5 ) . A priori, the acyl-transfer potential of biacetyl, a reagent extensively used for acylation reactions in ion cyclotron resonance studies, is not significantly more promising. The (CH3C0)3+ion was detected in the radiolysis of biacetyl and implicated in the formation of a host of products (6). Under ICR conditions (CH3C0)3+ is the predominant acylating species (7), but the radiolysis experiments indicated other important contributors to the total ion current. Hence, it may be inferred that, under CI conditions, the spectrum of biacetyl would be cluttered with a variety of product ions not encountered in ion cyclotron resonance. Indeed, the 2-Torr spectrum of biacetyl shows a large number of other peaks in addition to m / e 129, (CH3C0)3+. Also, the self-acylation reaction of biacetyl to produce (CH3C0)3+,Equation 2, CH,COCOCH,'. t CH,COCOCH, + (CH,CO),+ t CH,CO. (2) is only approximately four times faster than the self acylation reaction of acetone (Equation 1) ( I , 8). Therefore, it is unlikely
that CI conditions would promote the formation of sufficient acylation species (CH3C0)3+to compete with protonation. Thus, a selection of CI conditions for acylation by biacetyl reagent must eliminate a background of superfluous biacetyl reagent peaks and promote the generation of a significant quantity of the acyl-transfer species, (CH3C0)3+. Since these clustering reactions are generally of high order, reduction of biacetyl partial pressure while adding an inert diluent gas circumvents the problem of cluster ions. Specifically, a reduction in the biacetyl reagent pressure from standard chemical ionization conditions of 1 Torr to 300-600 pm significantly reduces the interfering background. Also, dilution of the biacetyl reagent with helium, typically 250 pm, increases the acyl ion transfer efficiency of samples tested. In the absence of helium dilution, a significant reduction in the relative intensity of mle 129 is observed with a corresponding increase in the relative intensity of m / e 101. Consequently, the procedure is to introduce sample via the gas chromatograph with helium carrier gas flow so that the desired He pressure is maintained and to admit biacetyl reagent separately to the mass spectrometer source. Independent control of helium and biacetyl pressures has produced successful results for acyl-transfer chemical ionization which were found not to be feasible using biacetyl alone at higher pressure. The helium thus serves as a diluent in terms of the sample spectra produced, and not as an obvious reagent; the intent is quite different from that of a mixed reagent gas ( 9 , I O ) .
EXPERIMENTAL Chemical ionization mass spectra were obtained on a Finnigan 1015C Mass Spectrometer. Data acquisition and analysis were performed using a System Industries System/l50 data system. Pressure measurements were made with a Varian Model 801 thermocouplegauge equipped with a Varian NRC 531 gauge tube. Pressures are uncorrected for relative gauge response for biacetyl and helium. Except for those noted below, samples employed were commercial sampleswith no impurities detected by conventionalmass spectrometry. The exo,exo- and endo,endo-norbornane-2,3-diol were prepared stereospecificallyby published (11)methods and acetylated with acetic anhydride/pyridine. Samples were then introduced into the mass spectrometer by means of a CHDMS column (3% on Gas Chrom Q 100/120 mesh). Finally, a 3:l endo,endo:ezo,exomixture was prepared and an aliquot injected. All liquid sample solutions were prepared 0.1 mL/10 mL and solids 1 mg/l mL in n-hexane immediately before use. For mixtures of isomers, known mixtures of samples were prepared at ca. the 3 mg/lO mL level. The injection volume range was 0.5 to 3 ILLtypically. Biacetyl reagent was freshly distilled and refrigerated until use. Samples were introduced into the mass spectrometer source via a gas chromatograph with helium carrier gas with source pressure maintained constant at 250 wm. Biacetyl reagent was admitted to the source through the probe reagent gas line via a Series 203 Granville-Phillipsleak valve in a valving arrangement permitting initial vacuum roughing and biacetyl degassing. Spectra described are those representing the maximum response for the acetylated product determined by a limited mass search ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977
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of the reconstructed gas chromatogram (total ion current spectrum). The biacetyl-helium background has been subtracted. Consequently, the intensities of peaks from the reagent gas mixture appear much smaller in intensity than actually observed. Instrument sensitivity to y-decalactonewas measured by injection of varying amounts of material on the GC column and examining the mass spectrum of the peak which eluted. Sensitivity was determined as the minimum quantity of material which gave an interpretable mass spectrum. Most applications of CI suggest GC introduction as a realistic method for sensitivity determination. RESULTS General Reactivity Studies We briefly summarize preliminary results. Appropriate (0.40 Torr C4H602,0.25 Torr He) spectra are given in Table I. 1) The typical alkanes hexane and dodecane are not chemically ionized by the reagent. 2) The model haloalkane 1-bromohexane is not chemically ionized by the mixture. 3) Nitrogen-containing compounds expected by analogy with ICR data to react in fact do: 4-nitrobiphenyl and 2naphthonitrile are both acetylated and protonated. They also give molecular ion peaks presumably due to charge exchange and the latter has a few fragments of low intensity. 4) Examples of carbonyl compounds, three ketones and three esters, are found to be acetylated and protonated. The cyclic ketone l-carvone, in which the carbonyl group is less hindered by substituents than the acyclic ketones examined (4-methyl-3-octanoneand 3,5-dimethyl-4-octanone), gives a larger acetylation/protonation ratio than the acyclic compounds. The cyclic ester y-decalactone, in which the oxygen function is less hindered by substituents, likewise gives a larger acetylation/protonation ratio than methyl decanoate, or isobutyl isobutyrate. It also has a greater tendency to form cluster ions. 5) As a negative control, p-chloroanisole, which was not acetylated in ICR experiments, also is not acetylated under these conditions. These preliminary results indicate that the general trends observed for ICR reactivity towards acetylation (12) can be duplicated under the higher pressure conditions of C1. It should be pointed out, however, that computer subtraction of spectra is necessary in order to produce usable results, and that, because of the multiplicity of background peaks from biacetyl under these conditions, one cannot be sure that the mechanism in CI experiments is the same as in the lowpressure ICR results. This point will be the subject of further mechanistic studies. With this background information in hand, we now pose the question of greatest analytical interest: are the stereoselective characteristics of biacetyl noted under ICR conditions observable under CI conditions? Stereochemical Selectivity of Biacetyl at Higher Pressure. Under low pressure condtions of the ICR experiment, the ions from biacetyl have been repeatedly shown to react at different rates with members of stereoisomeric pairs (12-16). We can now report that this characteristic is also present in the high-pressure CI experiments on such pairs. We report three examples from a large number of esters studied with similar results. For example, analysis of the spectra (Table 11) of cis- and trans-4-tert-butylcyclohexylacetate injected separately and as a mixture on the gas chromatographic column shows a significant difference in the relative intensity of the acetylation peak. The relative intensity of acetylated molecular ion (m/e 241) is greater for the less hindered equatorial isomer (i.e., the trans compound). The intensity of this peak relative to the base peak (m/e 139) in a series of runs was about 3.3 times greater than that of the m/e 241 peak in the cis isomer. This is, of course, in the direction anticipated, and is presumably 1072
ANALYTICAL CHEMISTRY, VOL. 49,NO. 7,JUNE 1977
"; R
I1
9sl i9
R
R 5!
attributable to steric interference. It is interesting that protonation also is affected by the stereochemistry of the functional group, as can be expected from examination of the literature (17). Here, however, the ratio of relative intensities of the M + 1 (m/e 199) peaks in the two spectra differ by only about 1.5. Thus, in this case, the acetylation process is more stereoselective than the protonation. We have shown before that sensitivity to steric environment of functional groups is a function of the complexity of the ionizing species under ICR conditions, so this analogy is not surprising. It is a bit disappointing, however, that under our conditions, the selectivity is not as great as in the ICR spectrometer. There, by careful control of relative pressure of reagent and sample, it was possible to produce intense signals for one isomer under conditions such that no signal above noise level was obtained for the other, in a virtually stereospecific test (15). The rationalization of the order of the rates of acetylation of cyclohexane systems has been detailed before (15). However, the wide variation in conditions encountered in CI-GC/MS apparently precludes such stereospecificity. For example, as a component elutes from the GC, detectable signal is normally recorded while source partial pressure of that component varies two to four orders of magnitude. On the other hand, the lower pressure limit (below which acetylation reactions are not observed) differs from the upper limit (above which analyzer pressure becomes excessive) by only a factor of two. Our data are in fact optimized within this stringent restriction. Data for the cis and trans isomers of 4-tert-butylcyclohexyl propionate as a second example were similar to those obtained for the corresponding acetates. In a series of experiments, the ratio of relative intensities of m/e 255 in the trans and cis isomers was nearly constant at 3.2; that of m/e 213 (M + 1) was 1.3. These results therefore support the previous conclusion: acetylation is more stereoselective than protonation in this system as well. In typical protonation gases, the selectivity between these isomers is not great either. In 1 Torr of methane, the trans/cis ratio is 1.3; in 1 Torr isobutane, 1.8. Apparently some stereoisomers do have sufficiently different reactivities that the high-pressure acetylation results are truly striking. Consider the endo,endoand exo,exo isomers of 2,3-diacetoxynorbornane,chosen as models for derivatives of possible metabolites of dieldrin. Figures 1and 2 demonstrate the results we obtained Figure 1is a plot of the total ion current resulting from the injection of a 3 1 mole ratio of endo,endo-and exo,exo-diacetate injected onto the column. A single-ion plot of M + 43 (m/e 241) shows that the endo isomer, which should produce less steric interference to the approach of the acetylating ion, is acetylated;
Table I. Representative Spectra of Typical Compounds Using 0.40 Torr Biacetyl and 0.25 Torr Helium. Isotope Peaks Are Not Tabulated %
Compound
m le
4-Nitrobiphenyl 2-Naphthonitrile
4-Methyl-3-heptanone
199 200 242 126 153 154 168 196 129 171
3,5-Dimethyl-4-octanone I-Carvone
Isobutyl isobutyrate
Methyl decanoate
7-Decalactone, 100 ng yDecalactone, 1 0 nga a
Estimated
as sensitivity
157 199 151 193 237 301 145 187 231 289 187 229 273 373
total sample Rel. ion int. current 58 100
18 7
68 100 4 8 100 23 100 71 6 13 100 19 2
2
6
5 68
14 100
100
40 3 4 100
213 257
85 30 100 60
27 2
3 46 40 14 63 37
limit.
Table 11. Spectra of Isomers Using 0.40 Torr Biacetyl and 0.25 Torr Helium. Isotope Peaks Are Not Tabulated %
Compound
m le cis-4-tert-Butylcyclohexyl 139 acetate 199 241 trans-4-tert-Butylcyclohexyl 139 acetate 199 241 cis-4-tert-Butylcyclohexyl 139 propionate 143 195 213 255 trans-4-tert-Butylcyclohexyl 139 propionate 143 195 2 13 255
Rel. int. 100 2.8 6.0 100
5.9 15.7 100 8.8 12.3 28 5.6 100 9.2 14.3 57
18.1
61-
P8!CL
isRR0-
2 4
81 19 88 12 53 37 3 4 78 15
171
171 2 13
33 57 10 4 36 54
61-
total sample ion current 91.9 2.6 5.5 82.2 4.9 12.9 59.9 5.3 7.4 16.8 3.4 50.4 4.6 7.2
28.7 9.1
but under the same conditions more hindered exo isomer is not acetylated to a detectable degree, even though it is present in larger amount. The explanation of the order of rates of acetylation in norbornane systems has been made before (11, 14). We estimate that there is a difference of at least 100 in the detected amount of acetylated product of these two compounds. The M + 1 peaks of these isomers in this experiment differed in relative intensity by a factor of only 3. We note in passing that evidence against control of ion intensity by decomposition of M + 1 or M + 43 to tertbutylcyclohexyl ion or norbornyl ion was found by ICR ex-
CONCLUSIONS The following conclusions can be drawn. It is feasible to obtain acetylation spectra using biacetyl provided the biacetyl pressure is reduced to 300-600 microns and the reagent is mixed with helium. Second, ion cyclotron resonance studies can serve as models for predicting reactivity under medium pressure chemical ionization conditions for biacetyl. Third, the range of functional groups capable of being ionized by acetylation is comparable to those noted in earlier ion cyclotron resonance studies. Fourth, the abundance of acetylation and other ion-molecule products in chemical ionization spectra using biacetyl-helium reagent reflects the steric environment of the substituent being acetylated. Fifth, each case examined points to more selectivity by acetylation than by protonation. Sixth, relative reactivity toward stereoselective reagent gases can provide useful information which can be used in conjunction with data such as elution order on a judiciously chosen GC column to give information relating to the stereochemical environment of a functional group. Seventh, it is sometimes possible to optimize conditions to give not only stereoselective, but stereospecific acetylation, The importance of this observation for the simplification of natural product and metabolite analysis should not be overlooked.
LITERATURE CITED K. A. G. MacNeil and J. H. Futrell, J . Phys. Chem., 7 8 , 409 (1972). M. S. B. Munson, J . Am. Chem. Soc., 87, 5313 (1965). 6. L. Jelus, Ph.D. Dissertation, University of Clncinnatl, 1972. C. A. Kuether, personal communication. M. S. B. Munson, personal cornmunlcatlon. G. J. Mains, A. S. Newton, and A. F. Sclamanna, J . Phys. Chem., 85, 1286 (19611. M:K: k & n , T. A. Elwood. T. A. Lehman, and M. M. Bursey, Tetraheclron Lett.. 4021 (1970). R. C. Dunbar; D. A: Chatfield, and M. M. Bursey, Int. J. h k s s Spectrom. Ion Phys., 13, 195 (1971). D. F. Hunt and J. F. Ryan IIK, Anal. Chem., 44, 1306 (1972). D. F. Hunt, G. C. Stafford, Jr., F. W. Crow, and J. W. Russell, Am/. Chem., 48, 2098 (1976). H. Kwart and W. G. Vosburgh, J . Am. Chem. SOC..7 8 , 5400 (1954). M. M. Bursey, T. A. Elwood, M. K. Hoffman, T. A. Lehman, and J. M. Tesarek, Anal. Chem., 42, 1370 (1970). M. M. Bursey and M. K. Hoffman, Can. J . Chem., 49, 3995 (1971). M. M. Bursey, J. L. Kao, J. D. Henlon, C. E. Parker, and T. S. Huang, Anal. Chem., 48, 1709 (1974). J. R. Hass,M. M. Bursey, and R. L. Stern, Anal. Chem., 47, 1452 (1975). M. M. Bursey, J. L. Kao, and C. A. Sirnonton 111, Org. Mass Spectrorn., 11, 149 (1976). Cf. J. Wlnkler and F. W. McLafferty, Tetrahedron,30, 2941 (1974).
RECEIVED for review October 16, 1975. Accepted February 24, 1977. This work was supported in part by National Institutes of Health Grant GM15994. ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977
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