24Na. It was also noted that the background due to Bremsstrahlung from the 24Na did not significantly decrease the sensitivity. Based on this detector and set of counting and irradiation conditions, the sensitivity is on the order of a few tenths of a part per million Cu in aqueous solutions even in the presence of Na levels sufficient to prohibit measurement by y-spectrometry. In order to test the procedure for biological materials, samples of blood serum were doped with varying amounts of Cu before irradiation. With no treatment of the serum before or after irradiation the Ni K a X-ray intensity from the 64Cu gave a linear relationship with the amount of copper added (Figure 3). In this case, the high radioactivity of the serum sample did give more background (Bremsstrahlung and other characteristic X-ray peaks), decreasing the sensitivity for these conditions to the ppm level. I t should also be noted that the volume of sample from which the X-rays could be detected was smaller than in the case of water. A sample of freeze dried NBS standard bovine liver was also run, and its Cu concentration of 193 ppm was easily detected by this method. In the case of a sample of NBS orchard leaves, the background was lower and the activity level for the Cu concentration (12 ppm) suggested that, for plant material, the sensitivity would be similar to that for liquids (tenths of ppm). A series of USGS rock standards were analyzed by this technique. The very high activity samples gave background levels of 250-300 counts per minute under the Ni K a peak decreasing the sensitivity to on the order of several ppm Cu. The high Fe contents were, however, a more significant problem with the analysis of Cu in geological materials by this technique. The X-ray intensities for samples ranging from 13 to 52 ppm could be partially corrected by taking into account the absorption of Ni K n photons by Fe. The conclusion was that only when rocks of very similar or very low Fe concentrations are being considered is this technique really accurate. X-Ray counting can, however, be used for other elements in geological materials. The Cu K a X-rays from the decay of 65Zn were
0 grn CL addec to 1 r? Serum
Figure 3. Whole blood serum spiked with Cu prior to 30-minute irradiation at fluxof 2 X 1013n cm-* sec-' Count rates are corrected to a time four hours after end of irradiation The non-zero intercept reflects the concentration of Cu already in the serum
used to calculate the Zn concentrations using one of the USGS rocks as the standard. The results agreed well with the results from other techniques. Other X-rays observed in the geological materials were the Sm Kcu from 1 5 2 m E ~ and the Br Kcu from aomBr. In summary, the use of Si(Li) detectors to complenient y-ray spectrometry for instrumental neutron activation analysis is possible, even in complex matrices. Sensitivity is improved, and the analytical procedure is easier when infinitely thick samples are used in the holder described. However, there is the limitation that samples and standards must be similar, particularly with respect to the concentrations of elements having absorption edges just above the energy of the X-ray being detected. Thus. Cu levels in biological and plant material can be analyzed, but absorption by iron in geological materials presents a problem. Received for review February 14, 1974. Accepted April 17, 1974.
Mass Spectrometric Studies Using Compounds sotopically Labeled at Natural Abundance: A Development from Ion Kinetic Energy Spectrometry J. H. Beynon, D. F. Brothers, and R . G. Cooks Department of Chemistry, Purdue University. West Lafayette. Ind. 47907
In recent years, increased attention has focused upon the fragmentations of metastable ions that occur in the mass spectra of organic compounds. Measurements of the abundances, shapes, and apparent masses of the metastable peaks have found wide application in chemistry and so have measurements on the similar peaks that arise due to collision-induced dissociation (CID) of stable ions ( I ) . A field in which interest is particularly widespread is analytical chemistry where the properties of metastable and ( 1 ) R . G. Cooks, J. H . Beynon. R . M. Caprioll. and G . R. Lester, "Metastable Ions,'' Elsevier, Amsterdam, The Netherlands, 1973.
CID peaks are used to infer the individual steps in complex fragmentation sequences and ion structures (2). The development of ion kinetic energy spectrometry (IKES) has led to new techniques for carrying out even more refined measurements on both metastable and CID peaks. The ability to carry out measurements of both mass-to-charge ratio and translational energy on the product ions has enabled the entire reaction, including both the reactant and products, to be defined. Simultaneously, ( 2 ) J. H . Beynon and R . G . Cooks, Advan. Mass Spectrom., in press
A N A L Y T I C A L C H E M I S T R Y , V O L . 46, NO. 9, AUGUST 1974
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two further advances have occurred in the equipment available for such work. The first of these was the development of the Hitachi/Perkin-Elmer RMH-2 mass spectrometer ( 3 ) , the first commercial instrument designed to give especially high sensitivity and high energy resolution for the study of metastable ions. This instrument enables a dynamic range of 105:1to be achieved routinely in the study of metastable peaks. The second was the design of special equipment giving high energy resolution using an arrangement of electric and magnetic fields in which the ion beam passes first through the magnetic sector ( 4 ) . Mass spectrometers of this design have been in use for many years ( 5 ) , but it is only recently that an instrument specially designed for studying metastable ions has been developed. The very large dynamic range of this massanalyzed ion kinetic energy (MIKE) spectrometer, even exceeding that of the RMH-2, coupled with its high energy resolution has led to the development of methods that enable organic compounds isotopically labeled a t natural abundance to be used in experiments that previously required the synthesis of samples specifically labeled with concentrated heavy isotopes. It should be emphasized that these measurements are not made possible merely by reversing the geometrical arrangement of the electric and magnetic sectors; they are primarily a consequence of the high sensitivity and energy resolution available. Given such sensitivity and, simultaneously, high energy resolution, then the IKES and MIKES techniques show complementary advantages. One example of the use of ion kinetic energy spectrometry to help sort out the fragmentation reactions of a complex mixture of isotopically labeled material was provided by the work reported on the rearrangement reactions of 1,2-13C2-3,4,5,6-d4 benzene (6). This has a molecular weight of 84 and had as its principal impurities less highly 1abele.d compounds of molecular weights 83, 82, and 81. Analysis was carried out a t a series of fragment ion masses. For example, a fragment ion of m/e 55 can be formed from metastable molecular ions of 84, 83, 82, and 81 by the loss of neutral acetylenes containing 3. 2, 1, and 0 labels, respectively. By analysis at a series of fragment ion masses, not only could the isotopic purity be assessed, but information concerning scrambling of the labels was obtained. This work was carried out using highly labeled material, but it pointed the way to carrying out useful studies on mixtures that are much less highly labeled. The experiments now reported were carried out on the MIKE spectrometer using n-butane labeled at natural abundance and serve to illustrate the performance of this instrument. Related experiments using a high energy resolution instrument of conventional geometry (the Hitachi RMH-2) are also described and the advantages of the MIKE spectrometer are emphasized. METHOD The essence of the new method is as follows. Every organic compound contains heavy isotopes at natural abundance. Taken over all molecules present, the isotopic labels are statistically distributed over all positions in the molecules. That is to say, we may list all the varieties of isotopically labeled compounds present and estimate their (3) J H. Beynon. W . E. Baitinger, and J. W. Amy, Int. J. Mass Spectrom. / o n Phys.. 3, 55 (1969). (4) J. H. Beynon. R. G. Cooks, J. W. Amy. W. E. Baitinger, and T. Y . Ridley, Anal. Chem., 45, 1023A (1973). ( 5 ) F. A . White, F. M . Rourke, and J . C. Sheffield, Appl. Spectrosc.. 12, 46 (1958). (6) J. H . Beynon, R . M . Caprioli, W 0. Perry, and W. E. Baitinger, J . Amer. Chem. Soc.. 94, 6828 (1972)
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relative abundances one to another. Each of the individual species listed constitutes a specifically labeled species. If we can design an experiment that responds only to this species and not to any of the others present, then we can carry out isotopic studies using naturally labeled material. One difficulty in carrying out such experiments arises because of the low level of isotopic incorporation a t natural abundance in organic compounds containing such elements as H, C, N , and 0. Label incorporation for each of these elements a t one particular atom is only about 1.6 x 1.1 X 3.8 x and 2.0 X respectively. However, using the high sensitivity of the RMH-2 or of the new MIKE spectrometer, this difficulty can be overcome and the extra specificity introduced when studying individual fragmentation reactions of metastable ions using the IKE or MIKE method sometimes enables the reactions of a specifically labeled species tcj be studied. Consider the case of n-butane and the reaction by which metastable molecular ions eliminate the elements of methane. We shall study this reaction both in unlabeled n-butane and in n-butane labeled specifically with a single atom of I3C in the terminal location of the chain. In terms of the masses involved the reactions are:
58' and
59'
-
-
42' 42'
+ 16 + 17
(1) (2)
Let us list all the species of n-butane that are labeled with one, and only one, atom of a heavy isotope. There are only four, and they are: "CH,-CH,-CH,-CH, 1 CH,-'%H,-CH,-CH, 2 CH,D- CH*-CHY-CHJ 3 CH3-CHD-CH,-CH3 4 Only species 1 and 3 can undergo the reaction given in Equation 2 above. By adjusting the magnet of the MIKE spectrometer to pass only ions of mle 59, all reactant species of molecular weight 58, 60, etc. are eliminated; by setting the electric sector to collect only fragment ions of m / e 42, species 2 and 4 are eliminated. Similarly, only completely unlabeled n-butane can undergo reaction 1. All labeled species of molecular weights 59, 60. etc. are eliminated when the instrument is set to study this reaction. It is, of course, necessary to the above arguments that any contribution to reaction 1 due to the I3C isotope of C4Hg+([M - HIT in n-butane) and any contribution to reaction 2 due to a [M HI+ ion be negligible. This was, in fact, the case as discussed further herein. There are, thus, only two species, 1 and 3, in which reaction 2 cannot be distinguished. But these species are present in very different amounts in n-butane, the ratio of species 3 to species 1 being approximately 0.044:l. It is therefore permissible when studying the reactions of 1 to correct the observed peak heights for the reactions of 3 without appreciable loss of accuracy. The method just outlined can be applied to the study of kinetic isotope effects, by measuring metastable ion abundances, or it can be applied to the determination of isotope effects upon kinetic energy release. This latter information can be derived from accurate measurements of metastable peak widths given an instrument of adequate energy resolution. There is only a limited amount of data on such isotope effects in the literature (7, 8) but in sever-
+
(7) M . Bertrand, J H. Beynon, and R . G. Cooks, Org. Mass Spectrom., 7, 193 (1973) (8) M . Bertrand. J H . Beynon, and R. G . Cooks. I n t . J. Mass Spectrom. /on Phys., 9, 346 (1972).
1974
a1 of these cases the values found have been significantly different from unity (by factors of as much as 2). EXPERIMENTAL
Table I. Kinetic Energy Release (meV) for CH, Loss from C4Hla+a n d WC,H,a+. Run
The MIKE spectrometer was operated a t accelerating voltages of up to 7.4 kV. This corresponds to a n electric sector voltage (one plate to ground) of about 247 volts. An electron emission (trap) current of 100 pA was used and the bombarding electrons were of energy 70 eV. Indicated source pressure was maintained at approximately 1.0 X 10-5 Torr. Source and intermediate slits were 0.005 inch in width. Various collector slit widths were employed (see below) while slit height was always 0.22 inch. The detector system for the ion beam consists of a 16-stage electron multiplier (model R474 manufactured by Hamamatsu Company Ltd.) followed by a model 640 variable capacitor electrometer and amplifier manufactured by Keithley Instruments. Sensitivity changes between measurements of reactions of 58+ and 59+ ions were always made by switching the calibrated ranges on the electrometer amplifier. The calibration of this electrometer was checked in actual use by measuring ion beams of appropriate strength on adjacent scales which differ by factors of 3.000 or 3.333. The electrometer input resistor was never changed, nor was the voltage applied to the electron multiplier. In both the n-butane reactions, the product ions are the same species (CsHsf.) so that the only correction to be made for discrimination effects is that due to the kinetic energy of the ions impinging on the electron multiplier. Depending upon whether they are formed from 59+ or 58+, the daughter ions carry 42/59 and 42/58 of the initial kinetic energy. They therefore differ in kinetic energy by 1.7%, and this is therefore the magnitude of the difference in multiplier responses assuming a linear dependence upon kinetic energy ( 9 ) . Measured abundance data were therefore corrected by this factor. The RMH-2 mass spectrometer was operated a t an accelerating voltage of' 8 kV. Total electron emission was 1 mA. electron energy 70 eV, source pressure 1.3 X Torr. The width of the energy resolving slit was approximately 0.01 inch. Sensitivity changes were always effected by altering the range switch in the preamplifier head. These settings were calibrated analogously to those on the MIKE spectrometer. and the metastable peak heights were appropriately corrected, No correction for multiplier response as a function of ion energy is necessary with this instrument since the method of transmitting daughter ions depends upon their having a kinetic energy defined by the electric sector voltage and independent of their mass. It should be noted that no attempt was made to correct for the efficiency of the ion source at the different energies required to study reactions 1 and 2. Measurements were made of the peak heights and peak widths at half height for both transitions on both instruments. Peak width at half height is not sensitive to peak height, and so this measurement could be carried out for either transition at any time. For the comparative peak height measurements however, variation in sample pressure became an important factor. This pressure was therefore carefully monitored and the two metastable peaks were scanned alternately. In several sets of measurements, only the base line and the top 5-10% of each peak were plotted so as to make successive measurements quickly.
RESULTS AND DISCUSSIONS Under the conditions employed in this work, the ratio of the abundances of the main ion beams a t m / e 58 and mle 59 in n-butane as measured at the final collector of the MIKES was found to be 100:4.3. The 58+/57+ ratio was 100:54.7: hence, correcting the ion mle 58 for the 13C and 2H contributions from mle 57 (C4H9+),the ratio of the 1)+ ion becomes 100:4.41. In molecular ion to the (M the absence of any species contributing to m / e 59 except the 13C and 2H isotopes C4H10 the expected ratio is 100:4.48. The agreement between the experimental and calculated results justifies the neglect of ( M + H ) + and other impurities. The ion beam a t m / e 59 is therefore assigned as 13CC3Hlo + C4Hg2H in the calculated ratio of 23 parts to 1. The ion beam a t m / e 58 is assigned as C4H10 + 13CC3H9 in the ratio of41 parts t o 1.
+
( 9 ) C . La Lau in "Topics in Organic Mass Spectrometry," A . L. Burlingame. Ed , Wiley-lnterscience. New Y o r k , N Y . , 1970.
1 2 3 4
Average
C4Hio-'
'3CC3Hio * '
18.7
20.2
19.5 21.6 19.0 1 9 . 7 meV
19.7 22.1 19.6 20.4 meV
Difference
+1.5 +0.2 +0.5 +0.6
+ O . 7 meV
The metastable peak due to loss of 17 mass units from ions of rq/e 59 can be treated as if only 13CC3Hlo+. ions were involved. (The difference in isotope effects for 13C and 2H in the two mle 59 species would have to be very large indeed for this not to be a good approximation.) The metastable peak due to loss of 16 mass units from ions of m / e 58, however, contains contributions due to two reactions, CHI loss from C4Hlof' and CH4 loss from I3CC3Hgf (A third possibility I3CH3 loss from '3CC3Hs+ is ruled out since C4Hg+ does not give a metastable peak for CH3. loss). The relative ion abundances due to the two methane eliminations can be estimated from the metastable peak areas for methane loss from m / e 58 and m / c 57, which are in the ratio 12:l. Now the abundance ratio C4Hg+:13CC3Hg+ is 100:4.4 and of these I3CC3Hg+ ions only 314 will lose CH4 (?h will lose 13CH4). Hence, assuming negligible isotope effects, the contribution of the 13C isotope of mle 57 to the loss of methane from m/p 58 in a metastable ion decomposition will be 12:3.3/100 x 1, Le. 364: 1. This effect is small enough that it can be neglected; alternatively, this small isotopic contribution to the metastable peak abundance for C4Hlof' can readily be subtracted. The foregoing results allow the analysis of the ion abundance and peak width data for reactions 1 and 2. Let us first consider the kinetic energy release determined from the metastable peak width at half height and corrected for the main beam width in the usual way (I). This determination, if it is to be accurate, must be made using a narrow energy resolving slit, but a narrow slit cuts down on the already low abundance signal due to reaction of the labeled molecular ion. For this reason, ensemble averaging using the system described elsewhere ( I O ) was employed. Some runs were also taken by scanning very slowly with large (30 sec) time constants. The results of both methods were in good agreement. Table I gives kinetic energy releases measured on the MIKES for the reactions of the naturally labeled and the unlabeled n-butane molecular ions. Each run represents the average of several scans taken under identical conditions. The results show an isotope effect upon the kinetic energy release of 0.97 with 7'(12C) < T(13C). This is in agreement with the usual situation for hydrogen isotope effects where T(IH) < T(2H). The reaction was also studied using the RMH-2 with very similar results, uiz., the value of T for reaction 1 was 21.6 meV and for reaction 2 it was approximately 22 meV. Thus, the RMH-2 results also indicate an isotope effect which is very close to unity. The RMH-2 results for reaction 2 , taken with narrow slits, are subject to some uncertainty. Nor can this uncertainty be decreased by increasing the slit width as one would wish to for ion abundance measurements. The method of daughter ion selection and accelerating voltage scanning, used on the RMH-2, results in the metastable peaks for reactions 1 and 2 occurring together in a single accelerating voltage scan. Only a t slit (10) D. T. Terwilliger,
J. H. Beynon, Specfrom. / o n P h y s . . in press.
and R . G . Cooks,
I n f . J. Mass
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Table 11. Relative Abundances of Metastable Peaks for CH, Loss from C4Hl0+' and l3CC3Hlo+. Run
1 2 3 4
Instrument
C4HlO''
ICCaHio+'
MIKES RMH-2 MIKES MIKES
90 87 88 89 88.5
100
Average
100 100 100 100
widths of
