Chlorine isotopic ratios by negative ion mass spectrometry

1 Graduate School Fellow, Summer, 1968; ...... Biochemistry Section, Southern Research Support Center, Veteran's Administration Hospital, Little Rock,...
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Chlorine Isotopic Ratios by Negative Ion Mass Spectrometry James W. Taylor and Eric P . Grimsrudl Department of Chemistry, The University of Wisconsin, Madison, Wis. 53706

For kinetic isotope effect studies the determination of the relative isotopic abundances of chlorine-35 and chlorine-37 can be accomplished directly on gaseous methyl chloride samples by comparison of the CI- ion intensities with a conventional isotope-ratio mass spectrometer equipped for negative ion operation. Procedures for the conversion of inorganic chloride samples to gaseous methyl chloride were examined to ensure isotopic identity of the methyl chloride from the chloride precursor. Because relative rather than absolute abundances are required for this application, rapid switching between sample and standard i s used to compensate for instrument drift and background. A comparison between positive and negative ion ratios revealed several advantages to be gained by the negative ion technique.

THE USE OF the stable chlorine isotopes in research problems dealing with kinetic isotope effects has been limited by the ability to analyze for isotopic differences readily. Bartholomew, Brown, and Loundsbury investigated the kinetic isotope effects (k35/ka7)for the reactions of t-butyl chloride with sodium hydroxide and with silver nitrate in aqueous alcohol in an attempt to describe the extent of bond breaking in the slow step ( I ) . In a somewhat similar fashion Howald used variations in chlorine isotopes to describe the extent of bond formation from ion pairs in solvents of low dielectric strength (2). Hill and Fry have demonstrated most effectively the information which may be revealed from chlorine kinetic isotope effects in nucleophilic substitution reactions by their study with benzyl and substituted benzyl chlorides using the nucleophiles water, cyanide ion, and thiosulfate ion (3). In this latter study the authors were able to demonstrate that reactions giving first-order kinetics gave kinetic isotope effects (kaslk37) near 1.0078, those giving second-order kinetics had values near 1.0058, and those reactions proceeding by borderline kinetics gave intermediate values between these two extremes. The implications of these conclusions became intriguing to us and led us initially to examine the possible techniques which might lead to measurement precision of the or better. chlorine ratios on the order of *0.01 Conductance ( 4 ) and titration techniques are capable of rendering sufficiently accurate kinetic data if the appropriate isotopically labeled reactants can be synthesized, but the isotope-ratio mass spectrometric determination of the isotopic abundances of the reactants or products does not suffer this limitation. The variation in isotopic composition of the reactant or product can be related to the fraction of reaction to yield the kinetic isotope effect through the use of several expressions described in the literature ( 5 , 6).

The problem with an isotope-ratio mass spectrometric determination, however, is the form in which the sample must be introduced. At the National Bureau of Standards (7) solid silver chloride is the sample. It is evaporated from an ammonical solution onto a sampling filament. A surface emission source is used to produce the C1- ions. This approach was used to give absolute and very accurate isotopic abundance ratios of the chlorine isotopes, but for the purposes of kinetic isotope effect studies where relative differences and not absolute values are required, this procedure is far too timeconsuming for the measurement of many samples. Previous investigators have tried more volatile derivatives such as HC1, Cls, and COClz but found troublesome memory effects (8,9). Methyl chloride has been employed with more success but the conversion from chloride to methyl chloride presents some difficulty. Howald ( 2 ) and Owens and Shaeffer (10) used the reaction of dimethyl sulfate with ammonium chloride to produce the methyl chloride (11). Hill and Fry (3) modified Langvad’s (12) procedure to give a better yield of methyl chloride. This less tedious approach involved the reaction of methyl iodide with silver chloride to produce methyl chloride in 89% yield. These authors were able to show conclusively that the isotopic identity was maintained during the conversion and measurement of the positive ion isotopic ratios (3). In our hands, however, this procedure led to fractionation of the isotopes. Our search for an answer to these different results led us to an investigation of the necessary conditions for the methyl chloride conversion and to an investigation of the use of negative ion intensities for the isotopic mass measurements. EXPERIMENTAL Apparatus. All isotopic measurements were made with a Nuclide Model RMS-6-60 isotope ratio mass spectrometer (Nuclide Associates, State College, Pa.) equipped with a rhenium ribbon (0.001 ” by 0.003 ’ I ) electron impact source. This instrument is equipped with dual, room temperature, balanced capillary inlets for sample and reference, respectively, with switching between the two samples accomplished by a solenoid arrangement. Before each run, small adjustments in the inlet pressure are made with a mercury piston arrangement to give identical Nuclide electrometer amplifier voltages for the sample and reference. The source and analyzer sections are differentially pumped with ion pumps. The ratio of the ion current of the beam focused on the Faraday cup to that falling on the collector plates is read from the 4-dial General Radio, 1454-A, Kelvin-Varley voltage divider with the last digit in the ratio interpolated from a recorder tracing of the remaining imbalance from true null. The Cary 401 is op-

1 Graduate School Fellow, Summer, 1968; NDEA Fellow, 1968-1969.

(1) R. M. Bartholomew, F. Brown, and M. Loundsbury, Can. J. Chem., 32, 979 (1955). (2) R. A. Howald, J. Amer. Chem. SOC.,82,20 (1960). (3) J. W. Hill and A. Fry, ibid., 84,2763 (1962). (4) A. J. Kresge, N. N. Lichtin, K. N. Rao, and R. E. Weston, Jr., ibid., 87,437 (1965). (5) J. Y. Tong and P. E. Yankwich, J. Phys. Chem., 61, 540 (1957). (6) C. J. Collins in “Advances in Physical Organic Chemistry,” Vol. 11, V. Gold, Ed., Academic Press, New York, N. Y., 1964..

(7) W. R. Shields, T. J. Murphy, E. L. Gardner, and V. H. Dibeler, J. Amer. Chem. Soc., 84,1519 (1962). (8) F. W. Aston, “Mass Spectra and Isotopes,” Longmans, Green, and Co., New York, N. Y., 1941. (9) A. 0. Nier, Phys. Reo., 50,722 (1936). (10) H. R. Owens and 0.A. Schaeffer,J. Amer. Chem. SOC.,71,898

(1955). (11) A. H. Blatt, Ed., “Organic Synthesis,” Col. Vol. 11, John Wiley and Sons, Inc., New York, N. Y., p 251. (12) T. Langvad, Acta. Chem. Scand., 8 , 526 (1954). VOL. 41, NO. 6, MAY 1969

805

h

plate 1

CUR

0 To Recordrr

Cary 401 Vibrating Rood Electromrter Amplifier Plate 2

Figure 1. Schematic of isotope ra ti0 mass spectrometer Electrome tor Amplifirr

4-diol Kelvin-Vorlry Voltage Divider

erated to measure the approach to null on the 30 and 100 mV full scale ranges. A separate voltage divider is used for the reference sample and is switched into the circuit when the sample and reference compounds are alternated. The switching time interval is approximately two minutes. Identical ratios are obtained on the reference when either divider is used and also when the reference is introduced from both inlets. To convert from positive to negative ion operation, the accelerating voltage is switched from +3,000 V to - 3,000 V, the leads to the electromagnet are reversed to change polarity, and the Cary Model 401 vibrating electrometer amplifier and the Nuclide electrometer amplifier are switched to read negative currents. The basic arrangement is shown schematically in Figure 1 except that the cup is actually a Faraday cage. The ion repeller circuit is not used for these samples. For positive ion ratios, mle 53 and 54 ions

Table I. Typical Operating Conditions of Mass Spectrometer with Methyl Chloride Positive ion, Negative ion, m/e = 35-37 Condition m/e = 50-52 3 x 10-8 2 x 10-7 Source pressure, torr 10l2ohms 1011 ohms Cary input resistor - 3 kV +3 kV Accelerating voltage 54 eV 59 eV Ionizing voltage 4.3 A 3.9 A Filament current 500 @a 100 I.ta Trap current 100 torr 10 torr Sample inlet pressure Nuclide electrometer voltage 1.O (m/e 35) 30 (m/e 50) on most abundant ion

are collected on plate 1, the m/e 52 ions (CHaa7C1)are focused in the cup, and ions of m/e 49-51 fall on plate 2. For negative ion operation, the m/e 37 ions ("Cl-) are focused in the cup and the m/e 35 ions (35Cl-) are collected on plate 2. A comparison of operating conditions for the positive ion measurements in the region m/e 50-52 (CH3a5Cl+and CH3a7C1+) and the negative ion measurements in the m/e 35-37 (asC1and 37C1-) region is shown in Table I. Table I1 shows the peaks, the presumed identity, and their intensities for both positive and negative ion operation. Conversion and Sample Purification. The conditions described herein were the optimum for the maintenance of isotopic identity. The variations are described in the Discussion. The inorganic chloride samples were precipitated by the addition of silver nitrate to a solution of the chloride at a pH of 6 containing 0.4M KNOI. The precipitate was filtered, washed with a slightly acid solution containing about 2 ml concentrated nitric acid per liter of water, and dried at 100 "C for two hours. The precipitate was then transferred to a reaction vessel designed for vacuum line manipulation and equipped with a break seal. In a typical reaction, 0.082 gram (0.57 mmole) AgCl was placed in the reaction vessel and 0.57 gram (4.0 mmole) methyl iodide (Eastman 164) was introduced and frozen with liquid nitrogen over the solid. The reaction vessel was then sealed and heated at 120 "C for 48 hours. After the methyl iodide and silver chloride had reacted, the vessel was attached to the vacuum line, opened via the break seal, and the contents transferred by distillation, using liquid nitrogen, to the sampling U-tube of the gas chromatograph. With helium as the carrier gas, the methyl chloride and the unreacted methyl iodide were swept into a 5 / ~ 6 1 1 i.d. glass gas chromatographic column

Table 11. Mass Scan of Methyl Chloride Reference Sample Positive Ions mle

Relative intensity

35 36 37 38 47

2.8 1.2 1 .o 0.4 7.7 3.2

48 49

50 51

52 53

806

0

9.6

100.0 3.4 31.4 0.5

ANALYTICAL CHEMISTRY

Major species

Negative Ions Relative intensity Major species 100.0

asci

H36Cl "C1 H *C1 C86C1 CH W l CHsW1 C"CI CHa36Cl CHWl "CHaW1 CHamCl CHa"Cl 13CHa"Cl

+ + +

...

32.4

...

0.25 0.20 0.50

0.10 0.14

...

3

~

1

*c1 CW l CH 8 C I CHtW1 CH "C1 CHa"C1

+ C"CI

x

prepared by depositing 5 by weight Apiezon-L (James G . Biddle and Co., Plymouth Meeting, Pa.) on 40/60 mesh Teflon T (Johns-Manville Products, screened from Teflon 6). The emerging purified methyl chloride was again trapped with liquid nitrogen and transferred to a mass spectrometer sampling bulb. The amount of methyl chloride produced was obtained from a calibration of peak area of the emerging sample from the gas chromatographic thermal conductivity detector. The yield of methyl chloride produced was then calculated from this value and the weight of silver chloride used for reaction. The gas chromatographic area measurements are estimated to have a mean error within 1 2 %. Controls, Calculations, and Calibration. By the use of the best procedure described above, the possibility of isotope fractionation in the conversion at high yields was examined in a manner similar to that of Hill and Fry (3). A sample of methyl chloride was converted to inorganic chloride by reaction with sodium and then subjected to the methyl chloride preparation procedure previously described. The read mean of five determinations of the negative ion ratio on the sample before decomposition was 0.36830 i. 0.00005 and that after the decomposition and conversion was 0.36830 =t0.00005, thus indicating no fractionation during the conversion. The 6, or parts per thousand, figures are calculated by taking the ratio obtained from a reference methyl chloride, subtracting the ratio obtained from the sample, multiplying the difference by 1000, and dividing this product by the ratio obtained from the reference. The ratios used are those obtained after approximately a fifteen minute stabilization of the mass spectrometer and represent the average of at least five determinations of the ratio as the sample and reference are alternately switched at approximately two minute time intervals. The positive ion ratios are obtained from the ratio of the intensity at mje 52 to the sum of the intensities at mje 54, 53, 51, 50, and 49. The negative ion ratios refer to the ion intensity at m/e 37 divided by that at m/e 35. The mass spectrometer readings could be calibrated by preparation of a series of standards using 99.0 1 0.05% enriched 35Clsodium chloride (Oak Ridge National Laboratories, Sample 150330, Lot 2012-1 16-B). This material was dried, weighed, and dissolved in a known volume of triply distilled water. Known volumes of this stock solution were then added to dried and weighed reagent sodium chloride (Mallinckrodt A.R. Crystals) to produce a range in 6 values up to 10. This limit would correspond to a maximum chlorine kinetic isotope effect of 1.010. The calculations of the 6 values assume natural abundance of the chlorine isotopes at 24.2295 % chlorine-35 and 75.7705 % chlorine-37 (7) in the reagent sodium chloride. RESULTS AND DISCUSSION

In beginning our studies involving the use of chlorine isotopes in research applications, we were particularly concerned with the problem of measuring precisely any isotopic variation in the chlorine isotopes with reaction and not in the absolute abundance. A convincing case for the use of positive ion isotope-ratio mass spectrometry coupled with methyl chloride as the sampling gas had been advanced by Hill and Fry (3), and we sought to duplicate their approach. These authors were able to obtain a methyl chloride yield of 89% in their conversion of the chloride samples and demonstrated the lack of isotopic fractionation. In our hands, however, neither the yields nor the isotopic fractionation appeared to be under our control, even when we used the same chloride sample in repetitive experiments. The results shown in Table I11 illustrate the dilemma we faced. The values reported are the negative ion ratios, but similar results were obtained with the positive ion ratios as well. For example, Run 20a yielding 16 % methyl chloride gave a posi-

Table 111. Initial Results on Isotopic Analysis of a Single Sample of Chloride

Run 1Oa 10b 20a 20b 30a 30b

Yield, CH3Cl 67 8 16 21 45 99

Negative ion, 6 ValuesaP 0.00 +O. 97

+1.38 +1.10 +0.96 0.00

The reference here was a sample giving 92 yield of CH,CI. *The maximum range from the mean for the measurement of individual negative ion ratios was 10.156. Table IV.

Run 14b 14C 1oc 30

45 52

Variation in Yield of Methyl Chloride with Reaction Conditions 2 Yield, Condition CHiCl 25 Conversion at 110 "C, 22 hours Conversion at 49 140 "C, 5 days AgCl ground to 20 increase surface area AgCl precipitated in 45 0.2M KNOI AgCl precipitated in 99 0.4MKN0, at pH = 6 AgCl precipitated in 99 0.4MKN03at pH = 2

tive ion 6 of +1.19 compared to the listed negative ion 6 of +1.38. All the runs were on the same chloride sample and referenced to a sample which showed a high yield of CH3Cl. As closely as we were able to control it, all samples were treated identically. As can be seen from Table 111, there is fractionation of the isotopes and there appears to be some correlation between percentage yield of methyl chloride and positive deviation from the reference sample. No close correlation between the yield and the magnitude of the deviation, however, is obvious from these data. In an attempt to determine if the experimental conditions for precipitation or conversion had an effect on the yield, the data in Table IV were obtained. As can be seen from these data (runs 14b and 14c), an increase in temperature and duration of heating did increase the yield of CH3C1but not to the desired value of 90% or greater. Run 1Oc represents an abortive attempt to grind the precipitate to produce a greater surface area. Since even dried silver chloride is rather soft, what resulted was a compacted sample and a very low yield of CH3Cl. This experiment did give a clue, however, to the source of these low yields. That the density of the sample and the yields of methyl chloride might be related appears reasonable when it is recalled that the silver chloride-methyl iodide reaction likely occurs on the surface and that silver iodide is deposited on that surface during the course of the reaction. In Run 30 we attempted to prevent the crystallization of the silver chloride in large aggregates by raising the ionic strength of the solution and reducing the digestion period. These conditions did give increased yields of CH3C1, but much better results were obtained when 0.4M KNOB was used as shown for Run 45. The silver chloride, under these conditions, was microcrystalVOL. 41, NO. 6, MAY 1969

@

807

Table V.

Mass Spectral Ratios from asC Enriched Samples

2 Yield Run

CH3CI

Known 6a

34 33 32 31b

34 50

2.30 4.60 6.91 9.19

45 50

Error in 6b

+O .36 $0.20 +O. 63 $0.54 mean +0.43

0 Values calculated on the basis of known amounts of 99.0 f .OS% NaW1 added to normal NaCl assuming 75.7705% 35C1 and 24.22952 a7Cl. * Negative ion values referenced to sample giving a high yield of

CHjCI.

line or fluffy in appearance. The pH at which the precipitate was formed under these high ionic strength conditions did not appear to affect the yields of CHX1 for both Run 45 and Run 52 showed values of 99%. Because our method of determining this conversion by gas chromatography is probably good to k 2 % , we are unable to distinguish between these two conditions even if there is a difference. Once the cause of the low yields was understood, it was interesting to turn to a closer examination of the relationship between yield and isotope fractionation. We had prepared a series of enriched chlorine-35 samples using known amounts of 99.0 & 0.05% N a W l and salt of assumed natural abundance to give 6 values of between 2 to 10. These chloride samples were precipitated from solutions 0.2M in KNOBand treated identically through the isolation, conversion, and purification steps. It was hoped that all samples would show methyl chloride yields between 40-50Z. The mass spectral negative ion data from these experiments are shown in Table V (Runs 31b, 32-34). Because the error figure is obtained by subtracting the known 6 from the observed 6, a positive reading indicates an enrichment of chlorine-35 in the methyl chloride sample if the reference sample is assumed not to have undergone fractionation. These data do show considerable scatter, but in all cases the deviation is unidirectional with a mean error of f0.43. This positive deviation with low yields of methyl chloride and lack of deviation at higher yields rules out mass discrimination in the spectrometer as the probable cause. The yield and surface relationship do suggest that there may be an isotope effect in the reaction of the methyl iodide with the silver chloride to form the methyl chloride. If we assume the conversion occurs only on the surface of the silver chloride and that silver iodide covers this surface as it forms, there are two possibilities for the preferential incorporation of the chlorine-35 isotope in the methyl chloride. Either

the crystallization of the silver chloride occurs with a rate dependence on the isotopic chloride such that Ag*'Cl is deposited preferentially in the interior of the crystal, or there is a rate dependence on the formation of methyl chloride from the silver chloride such that the reaction rate of the chlorine-35 isotope is faster and the chlorine-37 isotope is, therefore, preferentially buried under the silver iodide. When we consider the nature of crystallization, it is difficult to see how the first possibility would be valid. The second possibility, however, would simply be an isotope effect in the breaking of the Ag-C1 bond. The studies of Howald (2) give some credibility to this explanation because of his finding that equilibrium isotope effects in ion pair formation fall in the series HgClz > SrClz > LiCl, with HgCl? forming relatively strong Hg-C1 bonds and yielding an equilibrium isotope effect of 1.0061 f 0.0009. If this explanation is valid, the variability in the fractionation with yield would have to be due to the number average of silver chloride crystals in the various forms. For example, a precipitate with one large crystal and a few small ones might give a low yield, yet the fractionation would be small because the smaller crystals might be converted without fractionation and the large one would be rapidly covered with silver iodide and not be completely reacted. The lack of reproducibility, then, could relate to the lack of control of the small crystals available for reaction. Because the precipitation from 0.4M KNOBgives very small crystals, the preceding explanation would require that the yields would increase and the fractionation decrease under these conditions. When the same series of enriched samples were converted to methyl chloride and analyzed, the data in Table VI were obtained. As can be seen from these data, the negative ion values cluster around the known values when the yield is 90% or greater. If a plot is made of the observed negative ion values versus the known values as shown in Table VI, including the origin, a straight line is obtained whose leastsquares slope is 1.002. The standard deviation of the slope is Zk0.014 and the standard deviation of the points from the least-squares line is +0.220. Both these calculations are for 95 confidence limits. Because of sensitivity problems with the negative ions we have a maximum range of k0.00005 from the mean of our read ratios. A difference of 0.00005 between the sample and reference for a reference ratio of 0.36000 gives a 6 value of k0.14, which is comfortably close to the standard deviation of h0.220. When an identical study was made on these same samples using the positive ion ratios, we obtained an observed leastsquares line with a slope of 0.936. The standard deviation is k0.350 (95% confidence limits including the origin as one data point). The sample from Run 47 had a known 6 of 10.06 but we observed a value of 9.38 and would calculate a

Table VI. Mass Spectral Ratios from 36ClEnriched Samples at High Yields of CHBCl Negative ion values Positive ion values Run % Yield Known 6 Standar& Sample" 6, obs. Standardb Sampleb 6, obs. 41 93 9.19 0,36400 0,36070 9.07 0.33710 0.33424 8.49 43 84 4.60 0.36570 0,36400 4.66 0,33710 4.57 0.33556 44 91 2.30 0.36570 0,36490 2.20 0,33740 0.33670 2.08 45 99 7.75 0.36600 0.36315 7.79 0.34190 0.33940 7.32 46 98 8.51 0.36170 0.35860 8.58 0.34160 0,33885 8.00 41 93 10.06 0.36140 0,35775 10.11 0.34130 0.33810 9.38 a Ratios are the average of five determinations with the maximum range of ZkO.ooOo5 from the mean. b Ratios are the average of five determinations with the maximum range of 10.oooO3 from the mean. 6, corr. = 6, obs. X 1.056 to correct for fragmentation of the 37C1species. 0

808

0

ANALYTICAL CHEMISTRY

6, c0rr.C

8.96 4.83 2.20 7.73 8.45 9.90

value of 9.41 from the slope of the least-squares line. Because this sample was run using an ionizing voltage of 59 eV, it was interesting to see if fragments of the CHSSiC1caused the deviation from unity slope by adding to the ion current attributed to the W l species. At 13 eV where the effects of fragmentation are minimized but not completely eliminated, the W l species gives a molecular peak at m/e 50 and a carbon-13 isotope peak at m/e 51. The “Cl species gives corresponding peaks at m/e 52 and 53, respectively. Under these low voltage conditions, with maximum repeller voltage, the 6 value rose to 9.87. If further correction is made for the presence of the carbon-13 isotopes, the observed value becomes 9.91. This corrected observed value is still low by 1.5 but is reasonably close to the known value. When we take the fragmentation of the methyl chloride sample into account using the positive ion scan values in Table I, we can obtain a correction factor for the samples run at 59 eV. This correction factor is 1.056 and raises the Run 47 value from 9.38 to 9.90. This correction arises because the read ratio is the intensity of the m/e 5 2 peak divided by the sum of the intensities for the m/e 49, 50, 51, 53, and 54 ions. Other instruments with collectors of different design may be able to neglect the m/e 49, 53, and 54 peaks. We must include the m/e 53 and 54 unless we ground plate 1 of Figure 1. We could close the slits to exclude the m,’e 49, and may do this for subsequent samples. These calculations do show the necessity for correction for fragmentation. The residual difference between the known 10.06 and the observed 9.90 is within the standard deviation of the points from the least-squares line. It is possible that a small residual error could arise from carbon isotope effects in the reaction of the methyl iodide and the silver chloride. For the conversion to methyl chloride we used a molar ratio of 8 :1 methyl iodide to silver chloride and any carbon isotope effects from the methyl iodide might conceivably show up with these proportions. Unfortunately, our statistics are not precise enough to make any conclusion on this or other possibilities for any remaining error in the 6 values. In all of these calculations we have not tried to calculate absolutely the expected ratio for a given sample but the expected difference in 6 units from a reference. This approach allows us to neglect day to day small variations in the absolute ratio of the reference under the assumption that the drift would influence both sample and reference in the same manner. This point is easily illustrated by the data of Table VI where the negative ion ratio of the standard varies from 0.36140 to 0.36600 yet the observed 6 values fall quite close to the least-squares line. Because the negative ion measurements are being made directly on the two isotopes of interest and correction factors d o not appear to be necessary, the question of whether these ions might have been generated from reactions showing a kinetic isotope effect in the mass spectrometer deserves further comment. This examination becomes pertinent because of the observation of the negative ions in the region m/e 47-51 as shown in Table 11. Previous workers (13) did not report the observation of these peaks although they did see the m/e 35 and 37 peaks from methyl chloride. The mechanisms of negative ion formation as listed by Melton (14) are as follows:

x

(13) V. H. Dibeler and R. M. Reese, J . Research Natl. Bur. Standards, 54, 127 (1955). (14) C. E. Meiton in “Mass Spectrometry of Organic Ions,” F. W. McLafferty, Ed., Academic Press, New York, N. Y . , 1963,

Chapter 4.

AB

+e AB + e

AB

-+

+e

+

A

+

AB- (resonance capture)

(1)

B- (dissociative resonance capture) (2)

A+

+ B- + e (ion-pair formation)

(3)

The electron energy regions where these three mechanisms predominate are expected to be different. The resonance capture process is expected in the region of 2 eV or smaller unless the electron captured is a secondary one emitted with this energy from another fragmentation process. The dissociative resonance capture reaction is expected to predominate in the region 2-10 eV and Mechanism 3 in the region above 10 eV. For methyl chloride the operation of Mechanism 1 could conceivably give rise to a kinetic isotope effect in the formation of CI- through the sequence: CHsCl e 4 CH,CI- + A + CI-, where A could be CHs’or CH3+ with the ejection of an electron. The operation of this sequence to give rise to the C1- ion would require the presence of a m/e 5 2 peak corresponding to a CHS3’C1- species. The only peak seen at this m/e is approximately 2 x of the m/e 51 and can adequately be explained, within experimental error at these low ion currents, as the carbon-13 isotope of the CHZa7C1-species. Thus the electron capture process does not contribute to any extent. Even if it did, all the species produced by resonance capture are converted to product and, therefore, cannot give rise to an isotope effect by this mechanism. If the intensity of the negative ion peaks in the region 47-51 had been sufficient, it might have been possible to determine the relative importance of Mechanisms 2 and 3 by varying the ionizing voltage and observing the changes in intensity. Because this could not be done with our instrument sensitivity, we might assume Mechanism 2 contributes to the formation of these ions provided we have some process for degradation of the electron energy from the nominal 54 eV down to the 10 eV or lower range as required for 2. Because the spread of electron energies expected from our filament would be on the order of 0.2 to 0.4 eV with 5 eV (15) as a maximum figure, one logical source of electrons of this low energy would be from secondary electron emission. If these ions are produced by dissociative resonance capture of the secondary electrons, the intensity of these ions should depend on at least the square of the pressure in the source (14). This would follow from the fact that one molecule of methyl chloride would be required to produce the secondary electron and a second molecule would be required for the dissociative resonance capture. Over the range 1 X torr to 5 X 10-0 torr the largest peak in the cluster, the m/e 49, was found to vary linearly with the pressure. We may, therefore, exclude secondary emission as the source of low energy electrons. Other physical sources of electron energy degradation such as wall collisions could not be adequately examined without changing the ionizing voltage. Any moderate reduction in this voltage caused these small peaks in the mje 47-51 region to merge into the background. This suggests, but is not adequate to prove, that dissociative resonance capture contributes negligibly to the formation of these peaks and leaves Mechanism 3 as the probable explanation for the appearance of these peaks. If the peaks in the region 47-51 are formed by ion pair formation, it is conceivable that fragmentation of these species may lead to C1- in a kinetically dependent reaction involving an isotope effect. The influence of this reaction

+

(15) R. W. Kiser, “Introduction to Mass Spectrometry and Its Application,” Prentice-Hall, Englewood Cliffs, N. J., 1965, p 33. VOL. 41, NO. 6, MAY 1969

809

sequence will have its greatest effect when a small percentage of these species fragments to give the C1-. Because the peaks due to CI- are approximately 150-200 times as intense as the grouping between 47 and 51, a small isotopic contribution from these species will produce a negligible addition to the C1- peaks. We must conclude, then, that the m/e 35 and 37 peaks used in the negative ion ratios are probably devoid of fragmentation isotope effects. Mass discrimination effects should be minimal because of the masses of the isotopes involved and the fact that a standard containing the two isotopes in approximately the same ratio as the sample is employed as the reference. In conclusion, we see several features of the negative ion ratios which recommend their use in future measurements. Background contributions to the measured peaks are virtually nonexistent and this may permit the use of less rigorously purified samples for the analysis. The second feature is that the measurement is being made directly on the two isotopes in question and this permits ease of comparison between samples of interest without extensive correction factors. Because the kinetic isotope effects reflect the change in isotopic content during the course of the reaction, the standard to which all reaction samples should be compared is the one representing complete reaction from the compound in question and not some arbitrary standard sample for which the ratios are known absolutely. In common with the positive ion ratios, the only sample

limitation appears to be the requirement that it can be converted into inorganic chloride for subsequent precipitation by silver nitrate. The lower sensitivity in the negative ion intensity does cause a loss in measurement precision, but this appears not to be a severe limitation for isotope effects over the range 1.000 to 1.010 where we have shown a standard deviation leading to an isotope effect uncertainty of 10.00022. The sample preparation time is not unduly long except for the heating period. Changing samples to the mass spectrometer involves nothing more than pumping out the previous sample and balancing the sample and reference currents. This can be done in 15 minutes or less. ACKNOWLEDGMENT The authors are indebted to Robert C. Williams for the computer programs used to perform the statistical analysis of the experimental data. RECEIVED for review November 1, 1968. Accepted March 11, 1969. This research was supported by the Wisconsin Alumni Research Foundation and the National Science Foundation through Grant GP-8369. Portions of this work were presented at the 155th Meeting of the American Chemical Society, San Francisco, April 1968. Use of the University of Wisconsin Computer Center was made possible through support of the Wisconsin Alumni Research Foundation (WARF) through the Wisconsin Research Committee.

Role of Induction Forces Interactions in Effecting Gas Chromatographic Separation of Cardiac Glycoside Trimethylsilyl Ethers W. E. Wilson’ and J. E. Ripley Biochemistry Section, Southern Research Support Center, Veteran’s Administration Hospital, Little Rock, Ark. 72206

Trimethylsilyl (TMSi) ether derivatives of digoxin, digitoxin, and gitoxin have been shown to be resolvable on a gas chromatographic column packing containing a phenyl-methyl siloxane polymeric liquid phase (OV-17). At 340 O C , separation of the glycoside derivatives was effected by permitting differing degrees of dipole-induced dipole interaction between solute and liquid phase. In the cases of digoxin TMSi ether and gitoxin TMSi ether, the sphere of influence of the dipole of the lactone ring in the aglycone was sterically hindered, resulting in varying effectiveness of induction of electron delocalization in the T electron system of the aromatic groups of the liquid phase.

THEAGLYCONES of cardiac glycosides of plant origin possess several common structural characteristics which are illustrated in Figure 1. Fusion between rings AB, B C , and C D is cis, trans, cis, respectively. A tertiary alcohol is substituted at carbon 14. The 3p alcohol is bonded to digitoxose in the glycosides used in this study. A five-membered lactone ring is substituted at the 17p position ( I ) . 1 To whom correspondence should be sent at the following address: Analytical and Synthetic Chemistry Section, National Institute of Environmental Health Sciences, National Institutes of Health, P. 0. Box 12233, Research Triangle Park, N. C. 27709.

810

ANALYTICAL CHEMISTRY

The lactone ring of the biologically important plant glycosides possesses a,p unsaturation which confers a rather extensive a-electron system to the ring. In this regard, the conjugated lactone of the plant aglycones has been shown to be capable of enolization to a substituted a-hydroxyfuran derivative (2). Consideration of geometrical configuration and of charge distribution reveals that crotonolactone differs significantly from y-butyrolactone. The respective dipole moments of crotonolactone and y-butyrolactone have been reported to be 4.62 debyes and 4.13 debyes (3). Trimethylsilyl (TMSi) ether derivatives of the cardiac glycosides were demonstrated to be eluted during gas-liquid chromatography (GLC) using a relatively nonpolar methyl siloxane polymeric liquid phase (4). That the TMSi ether derivatives were not separable under the reported conditions (1) Ch. Tamm, First International Pharmacological Meeting,

Vol. 3, “New Aspects of Cardiac Glycosides,”W. Wilbrandt, Ed., The Macmillan Company, New York (1963) pp. 11-26. (2) B. Maume, W. E. Wilson, and E. C. Horning, Anal. Letters, 1, 401 (1968). (3) K. Sukigara, Y.Hata, J. Kurta, and M. Kubo, Tetrahedron, 4, 337 (1958). (4) W. E. Wilson, S. A. Johnson, W. A. Perkins, and J. E. Ripley, ANAL.CHEM., 39,40(1967).