Anal. Chem. 1981, 53, 421-427 M+Na
M
421
(3) Schuiunegger, U. P. Angew. Chem. 1075, 14, 679-688. (4) Levsen, K.; Wipf, H.-K.; Mclafferty,F. W. Org. Mess Spectrom.1074, 8 , 117-128. (5) Bruins, A. P.; Jennings, K. R.; Evans, S. Int. J. Mess Specfrom. Ion P h y ~1078, . 26, 395-404. (6) Matsuo, T.; Mats&, H.; Katakuse, I.; Wada, Y.; Fujita, T.; Hayashi, A. 6 b M . Mass Specfrom., in press.
v 760
770
780
790
Flgure 9. Raw CA linked-scan mass spectrum of a free peptide (H-Val-Tyr-Iie-His-Pro-Phe-OH). The precursor ion was the sodium cluster ion ( m l z = 797). An intense molecular peak was observed.
(7) Beckey, H. D. “Principle of Field Desorption Mass Spectrometry”; Pergamon Press: New Yo&, 1977. (8) Wachs. T.; Van de Sande, C. C.; Bente, P. F., III; Dymerski, P. P.; McLafferty. F. W. Int. J. Mess Spectrom. Ion Phys. 1077, 23, 21-27. (9) Matsuda, H. Inf. J . Mess Specfrom. Ion. Phys. 1074, 14, 219-233. (IO) Schuiten, H. R.; Beckey, H. D. Org. Mess Spectrom. 1072, 6 , 885-895. (11) Matsuo, T.; Matsuda, H.; Katakuse. I. Anal. Chem. 1070, 51, 69-72. (12) Bodanszky, M.; Klausner, Y. S.; Ondetti, M. A. “Peptide Synthesis”, 2nd ed.;Wiley: New York, 1976;pp 18-84. (13) Roilgen, F. W.; Giessmann, U.; Borchers, F.; Levsen, K. Org. Msss Specfrom. 1078, 13, 459-461. (14) Kambara, H.; Burlingame. A. L. Proceedings of the 27th Annual Conference on Mass Spectromeby and Allied Topics, Seattle, WA, 1979; on 897-69s. rr
(18-20). The FD-CA method using metastable ions and the
method introduced above complement each other and provide valuable information in the structural analysis of peptides.
ACKNOWLEDGMENT The authors acknowledge helpful discussions with Y. Izumi, Institute of Protein Research, Osaka University. LITERATURE CITED (1) Cooks, R. G.; Beynon, J. H.; Caprioii, R. M.; Lester, G. R. “Metastable Ions”, Elsevier: Amsterdam, 1973;Chapter 3. (2) Mclafferty, F. W.; Bente, P. F., 111; Kornfekl, R. T.; Sal, S . C . ; Howe, I. J . Am. Chem. SOC.1073, 95, 2120-2129.
(15) Biemann, K.; Cone. C.; Webster, B. R.; Arsenauk, 0. P. J . Am. Chem. Sm. 1068, 88, 5598-5606. (16) Monis, H. R.; Williams. D. H.; Ambler, R. P. Bbchem. J. 1071, 125, 189-201. (17) Wipf, H.-K.; Irving, P.; McCamish, M.; Venkataraghavan, R.; McLafferty, F. W. J. Am. Chem. Soc. 1073, 95, 3389-3375. (18) Shimonishi, Y.; Hong, Y. M.; Matsuo, T.; Katakuse, I.; Matsuda, H. Chem. Left. 1070, 1369-1372. (19) Matsuo, T.; Matsuda, H.; Katakuse, I. A&. Mess Spectrom. 1080, BA, 990-996. (20) Matsuo, T.; Matsuda, H.; Katakuse, I. Shltsutyo Bunseki 1080, 28, 169-174.
RECEIVED for review September 26,1980. Accepted December 10, 1980.
Scans for Preselected Neutral Fragment Loss in Double-Focusing Mass Spectrometry Bori Shushan and Robert K. Boyd’ Guelph-Waterloo Centre for Graduate Work in Chemistty, University of Guelph, Guelph, Ontario, Canada N7G 2W7
The theory of linked scans of the fields of a double-focusing mass spectrometer, for a preselected mass of the neutral fragment from decompositions of ions In the first field-free region, is presented. One of the scan laws thus derived has been described previously, but the other Is new. The problem of artifact peaks in such scans, and the theory and practice of the method of ac modulation in identifying them, is described with reference to a model system, 1,ldiphenylethane. Application of these techniques to identification of polychlorinated biphenyls, present at levels of order 1 % in compkx matrices of other compounds, illustrates both the power and llmltations of the method.
Interpretation of the mass spectrum of an unknown substance is usually based upon reasonable assumptions concerning the nature of the fragments produced in ionic decompositions of the type ml+
-
m2++ m,
(1) A conventional electron impact (EI) mass spectrum reflects all those ions formed in the ion source and thus corresponds to a superpositionof several fragmentation reactions described 0003-2700/81/0353-0421$01.00/0
by eq 1 with varying ml+ and/or m2+. It is possible to study specific reactions of the general type (eq 1)by investigating only those fragmentationswhich occur in the field-free regions in a mass spectrometer. Such techniques available up to about 1973 have been fully discussed in a recent monograph (1).The well-establishedvalue of “metastable peaks”, which underlie the conventional mass spectrum, is limited (1) by ambiguity in the interpretation of the apparent mass mt = (mZ2/ml). Consequently, various unorthodox methods of scanning double-focusingmass spectrometers have been developed, in order to study specified reactions of the type (I). For many purposes, it is most convenient to specify the parent ion ml+ and to generate a spectrum of all its daughter ions m2+. Historically, this was first achieved by use of reversed-sector instruments in which the magnetic sector precedes the electric sector; the magnetic field strength B is set to preselect m,+, and the various ions m2+ produced by fragmentation (spontaneous or collision induced) of ml+, in the field-free region immediately following the magnet, are successively detected by scanning the electric sector field E downward (2,3)while the accelerating voltage V is unchanged. Until recently, such daughter-ion spectra could not be obtained on instruments designed in the more orthodox configuration in which the electric sector precedes the magnet. However, methods are now available whereby scanning of 0 1981 American Chemical Society
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various combinations of the three fields V, E, and B, in some appropriate linked fashion, can be used to provide information on specified reactions like eq 1. Two of these linked-scans, viz., that in which E and V are scanned at constant B such that @ / V is constant ( 4 , 5 ) and that in which B and E are scanned at constant V such that B/E is constant ( 6 , 7 ) ,yield daughter-ion spectra of a preselected parent ion ml+. Of the scans developed ( 6 )to generate spectra of precursor ions of a preselected fragment ion m2+,one turned out to correspond to the classic “metastable defocusing” technique (8-10) whereby V is scanned upward while holding both E and B fixed. Methods requiring that V be scanned have the disadvantage that the ion source is thereby detuned; this problem may be avoided for precursor ion s a n s by use of a linked-scan in which only B and E are scanned such that B2/E is held constant ( 6 ) . All of these techniques yield useful information. However, the interpretation of even the conventional E1 spectrum of an unkown substance often proceeds (11) via identification of the neutral fragments m,. It is found that the masses of these neutral fragments are often characteristic, or partly so, of functional groups within the unknown molecule. In this spirit, scanning methods have been developed (12-14) which permit generation of spectra of precursor ions ml+ giving rise to preselected neutral fragments m,. One of these methods (14) pertains only to instruments in which the magnet precedes the electric sector and will be mentioned only briefly here. The other method (12, 13), like all the other linked-scan techniques, is applicable to instruments of either configuration. The purpose of the present work is to explore both the power and the limitations of this new linked-scan method (12, 13). The limitations mainly arise from the possibility of artifact peaks. These have been discussed previously, for other linked-scans, in both conventional ( 7 , 1 5 17 ) and reversedsector instruments (18,19). Understanding of the origins of these artifacts and of the methods proposed to identify them as such, is made possible through investigation of the threedimensional maps proposed and exploited by Lacey and Macdonald (20,211.
DERIVATION OF THE SCAN LAWS The approach to be used in this section closely follows that used previously (6)in generalized derivations of linked-scan laws for preselected ml+ or m2+. We now wish to generate analogous relationships for the case where m, is fixed. For a reaction described by eq 1 occurring in the first field-free region of a double-focusingmass spectrometer, the velocity of the daughter-ion m2+(which is the fragment actually detected in all such experiments) is equal (I) to that of the parent ion ml+. Then, if ( Vo,Eo, B,) are the settings of the three fields required to transmit main-beam ions m,+ to the collector, in the normal double-focusing mode, the following relationships are valid: kinetic energy of ml+ (main beam) = ZVO
(2)
momentum of ml+ (main beam) = (2rnlzV0)1/2 (3) kinetic energy of m2+ (field-free region) = (rnp/rnl)zVo (4) momentum of m2+ (field-free region) = (rn2/rnl)(2rnlzVo)1/2 (5) momentum of m,+ (main beam) = (2rn,~VO)l/~(6) where z is the electrical charge upon the ions, here assumed for simplicity to have the magnitude of a single electronic charge. It is straightforward to generalize the treatment to cases where, e.g., the parent ion is doubly charged and the daughter ion singly charged.
Table I. Factors by Which the Accelerating Voltage, Electric Sector Voltage, and Magnetic Field Strength Must Be Changed in Order To Transmit mz+Formed from ml+ the First Field-Free Region, Where m, = (m, - m , ) is Fixedo x
0
(V‘IV,,)
1
(B’IBn1
(E‘IE,,)
(1- 5) m1
mn ( m l / m n ) 1 ’ 2-( l -1 m1
a The values (V,,,E,,, B,) are those which transmit m,+ ions formed in the source.
Now suppose that the accelerating voltage is changed from Voto a value V’ given by V’ = (rn,/rn,)*Vo (7) where x is a dummy variable used (6)to generate the various possibilities. It turns out that the only useful values of x are small integers. The particular choice of the relationship (7) was dictated by the requirement to preselect m, (via B,) and to search for all precursor ions ml+. Substitution of eq 7 into eq 4 and 5, with some simple algebra, yields the results given in eq 8 and 9. If m2+ions produced in the first field-free kinetic energy of mz+(field free region) if accelerating voltage is V ’
}-
i
kinetic energy of ions mn [(l - - ) ( m , / m n ) x ] transmitted by electric m1 sector when E = E , momentum of m2+(field-free region) if accelerating voltage is V ’
t
(8)
)-
m [(I - “ ~ ~ m , / m n ~ ~ ~ + l =~ ~ ~ ~ ~ ~ m n ~ m1 momentum of main beam mn [(l- - ) ( m , / m n ) ( x + 1 ) ~ 2mn+ ] transmitted by \(9) m1 magnetic field B , if V = V,
i
region are to be transmitted through both electric and magnetic sectors and thus detected, the two field settings E‘and B’must be such as to match the kinetic energy and momentum, respectively. Thus, the terms in brackets, on the right-hand sides of eq 8 and 9, give the required ratios (E’/Ed and (B’IB,,), respectively. Table I lists the various possibilities for reasonable values of x . For only one case, that of x = 0, do just two of the three fields require to be scanned. The required linked-scan law is the relationship between E‘ and B’, viz. (B’/B,) = (1 - E’/1?3o)-’/~(E’/EJ or, dropping the primes (B/E)[l - ( E / E 0 ) ] 1 /=2 (B,/Eo) = constant (10) Equation 10 is just the linked-scan law given previously (12, 13) for a constant neutral fragment scan. Of the other cases, all of which require linked-scanning of all three fields, the case x = +1 appears to be relatively straightforward. B / E = B,/Eo V/Vo = ( B + B,)/B, (11)
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
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Q
Figure 1. Circuit diagram used to generate scan law given as eq 10. nalii constants : Test-point voltages as follows (aand fl are pr ( 1 ) as; ( 2 ) OB2; (3) 2flBn2; (4) B2/2Bn2;(5) :%B (6)- B 2 / 2 8 ’.
+
( 7 ) [(B2/2B,,2)2 2(B2/2Bn2)]/1O;(8) [ ( 8 2 / 2 8 n 2 )i2 2(B2/2B,,2)]q’i ( B 2 / 2 B n 2 ) .The link-scan unit is finally used to set E to the value calculated from eq 10 when B2 = 2Sn2;this latter setting was chosen to k e e p the AD534K (Analog Devices) chips wlthln their linear range.
-
None of the other cases in Table I appears to hold much promise. For example, the case x = -1 implies that all three fields must be scanned downward; since the initial setting of the magnetic field strength is B,, and thus relatively low, there will not be much scan range available. The advantage of generating scan laws by this general method (6) is that it has some of the character of an existence theorem, since it demonstrates that no other simple scan laws, for first field-free region reaction of the type given by eq 1, remain to be discovered.
EXPERIMENTAL SECTION All experiments were performed on a VG Organic 7070F doublefocusingmass spectrometer,equipped with a closable @-slit, a collision cell in the first field-free region, and a temperaturecompensated Hall-effect probe for measurement and control of the magnetic field strength. Unless stated otherwise, the accelerating voltage was 3.86 kV, the nominal ionizing electron energy was 70 eV, and the ion source temperature was 200 “C. The linked-scansare best generated digitally by computer-based methods (22). However, not all laboratories have the necessary hardware, and the present approach was to use the signal from the Hall-effect probe as the input to an analogue device arranged to generate an appropriate programming input signal to the power supply for the electric sector. A sketch of the arrangement used to generate the scan law given by eq 10 is shown in Figure 1. By careful trimming of the various integrated circuits, a typical performance would yield an output which followed the required function with a maximum deviation of 0.1%, over a range of (E’/Eo)from 0.65 to 0.85. The method used to apply an ac modulation to the accelerating voltage, and to lock in to the various harmonics of the ion intensity output, was the same as that described previously (I7).
A MODEL SYSTEM: 1,l-DIPHENYLETHANE In order to understand the origins of artifact peaks in constant neutral-fragment linked-scans, we investigated the case of the pure substance 1,l-diphenylethane. The fragmentation mechanism of the ions produced from this substance is dominated by loss of CH3 radicals, though loss of CHI sometimes occurs. Figure 2 is a contour-map version of the three-dimensional ion intensity map, plotted according to the method of Lacey and Macdonald (20,21).(Figure 2 is a composite of smaller maps published previously (17).) On such a map the conventional mass spectrum lies on the line (EVo/EoV = 1). Lines parallel to the (EVo/EoV)axis correspond to scans either of V with both B and E fixed or else
Ion intensity map for metastable Ions formed from 1,ldlphenylethane. See text for explanation and discussion. Figure 2.
of B and E such that B 2 / E = constant (6). A straight line joining the origin (not shown in Figure 2), to main-beam ion ml+ on the line (EVoIEoV = l),represents a B / E or F / V linked-scan with ml+ as preselected parent ion. The ion kinetic energy (IKE) peaks on the surface, shown as sets of confocal ellipses in Figure 2, represent ion current from m2+ formed in the first field-free region. The broad ridges of negative slope in Figure 2 represent appropriate reactions described by eq 1, occurring a t various locations within the electric sector. The intersections of these ridges with the (EVo/EoV= 1)line yield broad distributions of ion intensity (m2+) representing the conventional “metastable peaks’’arising from fragmentations in the second field-free region. The narrow ridges parallel to the (EVo/EoV) axis are due to reactions occurring during acceleration out of the ion source. The widths of the f i s t field-free region IKE peaks, in the horizontal (EVo/EoV)dimension, are due mainly to kinetic energy release on fragmentation. Closing down slit widths thus serves only to better resolve the detailed shapes of such peaks in this dimension but cannot significantly narrow them. On the other hand, the breadths of such peaks in the (B2Eo/E) dimension are largely controlled by the settings of all three slits (entrance, collector and fl slits). The lines representing constant neutral fragment linked scans must be calculated for each individual value of m,. The curves for m, CH4, CH3, and CH2 are shown on Figure 2. For the scan law given by eq 10, if B = B,, (,TIEo) = 0.61803. Note that such curves do n o t intersect the line (EVo/EoV= 11,i.e., no main-beamions will be detected at any stage in such a scan. A genuine peak will be observed when the linked-scan line intersects the center of one of the IKE peaks. Three types
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
mi€-:
Constant NeutralFrogment
/
%an,m,,=l5
I
I ~
I1
1r-Q
I
HT Modulation
I
j/
I
Lock-in
I
Amplifier
I
Output
Lock-in
Amplifier
~
output
I11
Constant neutral-fragment linked scan, m, = 16 (CH,), for 1,ldiphenylethane. (a)Unmodulated spectrum; peaks marked A are artifacts of type iil arising from interference from Intense m, = 15 transitions (Figure 2). See text for discussion of peak marked A' and small insert. (b) First overtone (second derivative) version of Figure Flgure 4.
Constant neutral-fragment linked scan, m, = 15 (CH,.), for 1,ldiphenylethane. (a)Unmodulated spectrum. The molecular ion at m l z = 182 and its first and second I3C isoto e peaks are major precursors of m, = 15; similarly for (M - CH,) P ion at m l z = 167. Peak marked A is an artifact of type iii arising from transition 167' 151' with true m,, = 16 (see Figure 2). (b) First overtone (second derivative) version of Figure 3a (see text). Flgure 3.
4a.
-
of artifact peaks can be easily predicted (15-17) to occur, from inspection of Figure 2: (i) relatively broad peaks due to intersection of the linked-scan line with diagonal ridges representing transitions within the electric sector, (ii) relatively narrow peaks due to intersections with horizontal ridges corresponding to fragmentations which occur during acceleration out of the ion source, (iii) peaks, of widths similar to those of genuine peaks, due to intersections with the wings of neighboring IKEpeaks arising from loss of neutral fragment of mass (m, f 1). Occasionally, if an unusually large value of kinetic energy release causes severe broadening of the IKE peaks, it is possible to observe artifacts of this type due to expulsion of a neutral fragment two mass units removed from the value of m, actually preselected. Figures 3a, 4a, and 5a show the results of constant neutral scans in 1,l-diphenylethane, for m, CH3, CHI, and CH2, respectively; the scan law used was eq 10. Comparison with the corresponding scan curves shown in Figure 2 immediately permits identification of real and artifact peaks in the constant neutral fragment scans. For example, chemical intuition would predict that loss of a high-energy fragment like CH2should be improbable; although the m, = CH2scan (Figure 5a) shows many peaks, inspection of Figure 2 reveals that they are, indeed, all artifacts. Clearly, production of a map like Figure 2 provides the most unambiguous identification of artifact peaks. Figure 2 was produced by laborious manual reconstruction of a large number of accelerating voltage scans. Even with much more rapid computer-based methods (15, 16), some minimum quantity of sample is required to permit sufficient time to generate enough detail that the resulting map shall be useful. Sometimes artifacts of types i and ii can be identified as such via their characteristic widths or from the fact that they need
HT M b t b
!I
Constant neutral-fragment linked scan, m, = 14 (CH,), for 1,ldiphenylethane. (a)Unmodulated spectrum. All peaks observed are artifacts,of either type i (broad peaks) rx type I// (narrower peaks). (b) First overtone (second derivative) version of Flgure 5a. Flgure 5.
not correspond to integral masses for the apparent precursor ions ml+. In our experience these criteria are of doubtful value in practice, and in any m e are inapplicable to artifacts of type iii. An alternative method of distinguishing real from artifact peaks, which is not as foolproof as methods (15,16)based on generating a map like Figure 2 but is applicable to a single scan, has been developed (17) for constant-precursor linkedscans ( B / E or E 2 / V constant). The same principle should apply to a constant-neutral fragment scan and may be understood qualitatively in t e r n of Figure 2; the full theory has
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
m
(A.
-
425
from the transition 180+ 165+,with m, = 15. The second derivative of the composite IKE peak is practically zero for a range including the point of intersection with the m, 16 linked scan line. Thus, even though it is known that the transition 181+ 165+does occur, there is no corresponding positive peak in the second derivative spectrum for m, = 16. Such effects would be very difficult to detect by any means in these linked scans and are probably detectable only via construction of maps like Figure 2. In Figure 5, all of the artifact peaks of type iii are negative in the second derivative spectrum (Figure 5b), as anticipated. However, there are also typically broad artifacta of type i, centered at apparent precursor m / z values of about 165.5 and 180.5. These survive as weak, broad but positive responses in Figure 5b, reflecting the complicated topology of the corresponding ridges in Figure 2. Effects of this type are unlikely to create confusion, however. +
Eve/ L V
Flgurs 6. (a) Schematic representation of an kn kinetic energy peak, Le., a cross-section through a map like Figure 2, parallel to the (€VdE,,V) axis. Arrows mark points of inflection. In regions I and 111 the sign of the second derivative is opposite to that in region 11. @) Same as (a), but for a case where severe rdiscrimination causes dishing of the peak. Regions I, 111, and V have positive second derivatives, while those in regions I1 and I V are negative.
been given previously (17). If the entire ion intensity map is oscillated in a direction parallel to the (EVo/EoV)axis, most conveniently (17) by applying a small ac component to the programming input of the accelerating voltage power supply, the detector response will contain an ac component. For sufficiently small ac amplitudes, it can be shown (17) that the magnitude of the response a t the first overtone (twice the modulation frequency) is proportional to the magnitude of the second derivative of the ion intensity with respect to V. Moreover, the phase of this response is directly related to the sign of the second derivative. Thus,a phase-sensitive detector locked in to the first overtone of the ac response can be used (17) to distinguish between intersections of a linked-scan line with portions of the ion intensity surface which differ in the signs of their second derivatives. In particular, this point is illustrated in Figure 6a for the case of so-called “Gaussian” IKE peaks; a first overtone response arising from detection of an intersection near the center of such a peak (a “true” response) will have a phase opposite to that arising from intersection of a linked-scan line with the wings of the peak (an “artifact” of type iii). Even in such an ideal case the method will work only if the artifacts correspond to intersections outside the central region bounded by the points of inflection (Figure sa). In practice, we have not thus far been faced with problems arising from this cause. Much more serious are problems associated with dished ion kinetic energy peaks (Figure 6b); the “horns” of such peaks are indistinguishable, on the present experimental basis (17), from the maxima of “Gaussian” peaks (Figure 6a). Artifacts of type i are readily identified, if necessary, by ac modulation of E rather than of V while type ii artifacts are reduced to practically zero intensity (17) by modulation of V. All of these features were explored previously for B / E linked-scans (constant parent ion scans) (17). The same qualitative expectations are valid for the present concern of constant neutral fragment scans. Figures 3b, 4b, and 5b are the first overtone spectra, with ac modulation of V, corresponding to Figures 3a, 4a, and 5a, respectively; the phasesensitive detector was set to make the true peaks positive and thus the artifacts negative. The method can be seen to work very well in this test case, as could be anticipated from the “Gaussian” forms of the IKE peaks (Figure 2). In all three cases, only peaks known to be genuine gave strong positive signals a t the first overtone; others were negative or were reduced to practically zero intensity. However, in Figure 4 (m, = 16) the peak marked A*,which is reduced to zero intensity in the second derivative spectrum (Figure 4b), is an ambiguous case. The insert on Figure 4a is the result of an accelerating voltage scan for mz+= 165 and shows that there is a genuine transition 181’ 165’ with m, = 16. However, the IKE peak for this transition appears as a poorly resolved shoulder on a more intense IKE peak arising
-
A REAL CASE: SCREENING FOR CHLORINATED BIPHENYLS Practical applications of constant neutral fragment scans are likely to be fast screening procedures for compounds containing specified functional groups, within a complex matrix of other substances (12-14). As an example of such a procedure, we inveatigated the possibility of screening crude biological samples for the presence of chlorinated biphenyls and other chlorinated residues. Metastable molecular ions of chlorinated biphenyls show intense peaks corresponding to loss of one chlorine atom as neutral fragment; collisioninduced decompositionsof the same ions, on the other hand, are dominated by loss of two chlorine atoms. It appeared that constant neutral scans for m, = 70 and m, = 35, with and without collisional activation, respectively, might provide a convenient method of screening for such chlorinated residues. In Figure 7 are shown three E1 spectra. Figure 7b is a spectrum of Aroclor 1232, a commercial chlorinated biphenyl preparation. Figure 7c is an E1 spectrum of 0.05 p L of Aroclor 1232 plus 5 p L of perfluorokerosene (PFK), the latter added as a convenient source of background ions to swamp out the chlorinated biphenyl peaks. (Thmtwo liquids are immiscible, and were mixed as the vapours in the batch inlet.) Figure 7a is an E1 spectrum of the crude hexane extract of Escherichia coli fed with 3,4-dichloroanilineunder anaerobic conditions, as described previously (23). It is extremely difficult to ascertain the presence of chlorinated biphenyls from Figure 74c. Figure 8 shows the results of five constant neutral-fragment scans with m, = 70. Figure 8b is for unadulterated Aroclor 1232;the spectrum shown was obtained without V modulation and corresponds reasonably closely to that predicted on purely statistical grounds. For example, molecular ions of tetrachlorobiphenylshave a natural isotopic abundance ratios for (35~1,):( 3 5 ~ 1 ~ 3 7 ~(36Ci237Cl2): 1): (35C13’cl3): ( 9 7 ~ 1 ~ ) of 0.32320.42180.2064:0.0449:0.0037, respectively. In order to predict relative intensities for (M - 236Cl)+-fragments, the statistical probabilities for choosing two W 1 fragments to the exclusion of 37Clmust also be taken into account. The numben of ways in which the molecular ions listed above can select two %C1atoms are 6,3, 1, 0, 0, respectively. Thus, ignoring kinetic isotope effects, the predicted intensities for the (M - 2%C1)+*fragments as observed in the constant neutralfragment scan with m, = 70 are given by the products of the two sets of ratios, viz., 100:65:11:0:0, for m l= 290, 292, 294, 296, and 298, respectively. Similarly, the relative intensities in the m, = 70 scan for the molecular ions of trichlorobiphenyls should be 100:33:0:0 for ml = 256,258,260, and 262, respectively. Corresponding calculations for dichlorobiphenyls yield predicted intensities of 1OO:O:O for ml = 222, 226, and 226, respectively. The intensities observed in the secondderivative spectra ( f i t overtone of ac modulation frequency)
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
70eV El
SPECTRA:
V35.MI
256 /
I
m
DI
I
PRECLRSOR
Flgure 7. Electron impact mass spectra: (a) crude hexane extract from E. coli, fed 2,4dichioroanilineunder anaerobic conditions; (b) Aroclor 1232; (c) 1% of Arocior 1232 in perfluorokerosene.
depend also on additional factors (17) and are not expected to follow these predictions. Figure 8b also shows the corresponding spectrum for Aroclor 1232 for a constant neutral fragment m, = 35. In this case, the statistically predicted ratios do not agree with those observed. In particular, more peaks are observed than expected, and these extra peaks survive in the second derivative spectrum. The extra peaks corresponding to odd values for the precursor mass ml probably arise from loss in the field-free region of a 36Clneutral fragment from (M - C1)+. fragment ions formed in the ion source from more fully chlorinated biphenyls. In addition, (M - HCl)+ fragment ions, formed in the ion source, can lose one %C1fragment in the field-free region, thus giving rise to intensity at euen values for precursor masses in addition to that predicted from molecular ions. Figure 8c shows constant neutral fragment scans, for m, = 70 and 35, respectively, for 1% of Aroclor 1232 plus 99% of perfluorokerosene (PFK). Without ac modulation, the m, = 70 scan shows large interferences from the PFK, and indeed this spectrum is of dubious value. However, the second derivative spectrum clearly distinguishes between peaks arising from the Aroclor 1232 and those due to the PFK. The latter behave as artifacts of type iii and correspond to collison-induced losses of CF3 (m, = 69) from various ions formed from PFK. The ac modulation technique is remarkably successful, in this example, in distinguishing between true and artifact peaks. The corresponding case for m, = 35 (Figure c) is not as subject to “chemical noise” from the PFK background, so that the ac modulation does not produce as dramatic an improvement. Figure 8a shows similar results for the real biological sample. Particularly for the m, = 35 scan, considerable chemical noise is observed,which is reduced in the second derivative spectra. The precursor ions observed at ml = 305,307,309, and 311
m/z
Flgure 8. Constant neutral-fragment linked-scans for m, = 70 (236Cl), all spectra coilisionally activated, and for m, = 35, no collisional activation: (a) crude hexane extract from E . coll, with and without modulation; (b) Aroclor 1232 no modulation of accelerating voltage; (c) 2% of Aroclor 1232 in perfluorokerosene,with and without mod-
ulation.
arise from the tetrachlorodiphenylamine known (16) to be present in the sample. There is also evidence for trichlorodiphenylamines. Characteristically broad artifact peaks of type i are present, which in the present example happen fortuitously to yield second derivative responses out-of-phase with the true peaks. However, the scan for m, = 70 serves to illustrate the limitations of the present technique. A very intense artifact, corresponding to a precursor mass of ml = 255, is seen in both unmodulated and second derivative spectra. That this peak is not due to some other nitrogenous metabolite is most easily seen by a comparison with the E1 spectrum (Figure 7c) of the same sample. Here, the intense interference is at m / z = 256, the lowest mass molecular ion of the isotopic molecular ions of trichlorobiphenyl. In fact, a B / E linked-scan showed that the background ion of m / z = 256 undergoes an intense collision-inducedtransition with m, = 71. It appears that the kinetic-energy release must be sufficiently large to significantly broaden the IKE peak for this transition that the artifact (m, = 71) cannot be distinguished as such (see discussion of Figure 6). Unfortunately, there was not sufficient sample available to completely characterize this JKE peak via a precursor ion scan for the background fragment ion at mlz = 185. However, all the available evidence supports this interpretation of this artifact; the apparent precursor mass is thus 255 instead of 256, since the artifact appears as a peak in the constanbneutral scan with m, = 70 whereas the neutral fragment actually lost has a mass of 71. This identification of the intense peak at ml = 255 in Figure 8a, as an artifact of type iii which cannot be distinguished as such in the second derivative spectrum, is further confirmed by Figure 8a. The background ion with m/z = 256 apparently
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
4 I’
-. .
--
.m
m
%
Flgwe 9. Constant neutral-fragment linked scans, m, = 35 (collision induced), for hexachlorobutadiene: (a) obtained with eq 10 as scan law; (b) obtained with eq 11 as scan law.
does not lose a neutral fragment with m, = 35, since the intensity ratios observed are very close to those found for Aroclor 1232. This background is thus unlikely to be due to a chlorinated compound at all. It is noticeable that the chemical noise is much worse in the m, = 35 scan than in that for m, = 70. We speculate that this will probably turn out to be an example of a more general trend, viz.,scans for neutral fragments of higher mass will turn out to be more discriminatory than those tuned to neutral fragments of lower mass. This is simply a reflection of the fact (11) that neutral fragments m, tend to be of fairly low mass. (Conversely, the ionic fragments m2+are usually of higher mass than m,. In turn, this trend can be rationalized on the basis of Stephenson’s rule, since larger fragments will in general tend to have lower ionization potentials.) Thus, ions produced from a complex mixture will tend to undergo many more reactions with m, = 35 than with m, = 70, for example. CONCLUSIONS The present work has been concerned with the theory and practice of linked-scans for preselected neutral fragments in ionic fragmentations described by eq 1, using double-focusing mass spectrometers of conventional geometry. All of such linked-scan spectra discussed thus far were obtained on the basis of eq 10 as the scan law. However, the present work also derived eq 11as a possible scan law, and Figure 9 compares scans obtained by using these two scan laws. Although all three fields must be scanned simultaneously in the case of eq 11, the required functional relationships between them are relatively simple. However, we found it relatively difficult, using our analogue circuitry, to accurately generate the required constant neutral scan (for m,, = 35) using eq 11as the scan law. Although not immediately apparent from the spectra reproduced in Figure 9 it is clear from the original charts that the two spectra do not coincide exactly and that the scan based on eq 11is in error while that based on eq 10 does not deviate significantly from the correct result. (The errors referred to are in the precursor masses; the differences in relative intensities, between the two scans, are due to ion-source detuning and to variations in detector response when the accelerating voltage V is scanned. Hexachlorobutadiene was used in these experiments as a convenient test
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compound in checking, relatively unambiguously, the performance of our control circuitry. Partially chlorinated cornpounds can also lose HC1 (m, = 36), and in our initial experiments it was possible to confuse the linked-scan for m, = 35 with that for m, = 36.) Artifact peaks in linked-scans, which are a major concern (15-17) in instruments of conventional geometry (electric sector preceding magnet), present a less severe problem in instruments of reverse geometry though even here care is necessary (18, 19). The present work has shown that ac modulation of the accelerating v tage can increase the discrimination of the constant neutr -fragment scan, as can choice of as high a mass as possible for the neutral fragment. When possible, useful cross-checks are provided through use of more than one neutral fragment. As pointed out previously (14,chemical ionization provides an additional stage of selectivity in the overall procedure. The present work may be regarded as a contribution to an approach (24) to mixture analysis which has been called “mass spectrometry without chromatography”, or MS/MS (mass spectrometry/mass spectrometry). Since the majority of mass spectrometer installations do not have specialized equipment, it is important to develop techniques which are applicable to more conventional instruments even if other designs (24) are better suited to MS/MS.
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ACKNOWLEDGMENT We wish to thank W. F. Haddon for communication of ref 13 prior to publication and M. J. Lacey and C. G. Macdonald for bringing ref 15 to our attention. We are greatly indebted to Ian Renaud for advice and assistance with the electronics and to Stuart McKinnon for enduring our continual modifications to the mass spectrometer while continuing to provide the routine service. LITERATURE CITED Cooks, R. 0.; Beynon, J. H.:Caprloli, R. M.: Lester, 0.R. “Metastable Ions“;Elsevier: Amsterdam, 1973. Beynon. J. H.;Cooks, R. G. Res.lDev. 1071, 22, 26. Maurer, K. H.; Brunnee, C.; Kappus, 0.;Habfast, K.; Schroder, U.; Schulze, P. 19th Conference on Mass Spectrometry and Allied Topics; American Society for Mass Spectrometry: Atlanta, GA, 1971. Evans, S.; Oraham, R. Adv. Mass Specfrom. 1074, 6 , 429. Weston, A. F.; Jennings, K. R.; Evans, S.; Elliott, R. M. Inf. J. Mass Specfrom. Ion Phys. 1078, 20, 317. Boyd, R. K.; Beynon, J. H. Org. Mass Specfrom. 1977, 72, 183. Bruins, A. P.; Jennings, K. R.; Evans, S. rnf. J. Mass Specfrom. Ion Phys. 1078, 26, 395. Barber, M.; Eilbtt. R. M. 12th Annual Conference on Mass Spectrometry and Allied Topics; ASTM Commmee E-14: Montreal, 1964. Jennings, K. R. J. C h m . Phys. 1985, 43, 4176. Futreli, J. H.;Lancaster, K.; Ryan, R.; Sieck, L. W. J . Chem. Phys. 1065, 43, 1832. Mdafferty, F. W. “Interpretation of Mass Spectra”; 2nd ed.; W. A. Benjamin: Reading, MA, 1973. Lacey, M. J.; Macdonald, C. 0.Anal. Chem. 1070, 57, 891. Haddon, W. F. Org. Mass Spectrom., in press. Zakett. D.; Schoen. A. E.; Kondrat, R. W.; Cooks, R. G. J. Am. Chem. Soc. 1070, 707, 6781. Lacey, M. J.; Macdonald, C. G. Ausf. J. Chern. 1078, 37,2161. Lamy, M. J.; Macdonakl. C.G. Org. Mass. Specfrom. 1070, 74. 465. Shushan. 6.; Boyd, R. K. Int. J. Mass Spectrom. Ion Phys. 1080, 34, 37. Mwgan, R. P.; Porter, C. J.; Beynon, J. H. Org. Mass Spectrom. 1077, 72, 735. Ast, T.; Bozorgzaheh. M. H.; Webers, J. L.; Beynon, J. H.; Brenton, A. 0.Org. Mass Specfrom. 1070, 74, 313. Lacey, M. J.; Macdonald, C. G. Org. Mass Specfrom. 1077, 72. 587. Lacey, M. J.; Macdonald, C. G. Org. Mass Spectrom. 1078, 13, 285. Haddon. W. F. Anal. Chem. 1979, 57. 983. W e , C. T.; Bunce, N. J.; Beaumont, A. L.; Menick, R. L. J . Agrlc. Food Chem. 1070. 27, 644. McLafferty, F. W. ACG.Chem. Res. 1080, 73,33.
RECEIVED for review August 28,1980. Accepted November 24,1980. This work was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada, who also awarded a graduate scholarship to B. Shushan.
