method at molar ratios as high as 1000:1, whereas arsenate interferes significantly at molar ratios of 8.2:l. It is worth noting that a mole ratio of 8.2:1 hopefully reflects a larger arsenate-to-phosphate ratio than would be encountered normally in water samples. It should be pointed out that, in the present study, no effort was made to optimize the extraction to eliminate these interferences. The method presented here provides a simple X-ray fluorescence procedure for the determination of aqueous phosphate with high precision in the 20-ppb level. Lower detection limits may be achieved by the extraction of a larger aliquot of sample than used here. The use of an X-ray tube more efficient for the excitation of molybdenum such as rhodium, would significantly reduce the detection limit. The standard pellets may be stored for long periods of time, providing rapid calibration of the X-ray instrumentation. Although the method does not lend itself to automated analysis, it provides an alternate method convenient for a small member of samples requiring high accuracy. Such would be the case when confirmation was desired of samples which had been through a less precise screening analysis.
ACKNOWLEDGMENT The authors thank Dennis Revel1 of the Environmental Protection Agency, Athens, Ga, for his assistance in acquiring the data using the AutoAnalyzer. LITERATURE CITED R. I. Alakseyev, Zavod. Lab., 11, 123 (1945). J. Paul, Anal. Chim. Acta, 35, 200 (1966). S. V. Eisenreich and J. E. Going, Anal. Chim. Acta, 71, 2 (1974). A. Halosk, E. Pungor, and K. Polyak, Talanta, 18, 577 (1971). S. J. Simon and D. F. Boltz, Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, OH, 1974. (6) W. S. Zaugg and R . J. Knox, Anal. Chem., 38, 12 (1966). (7) G. Stork and H. Jeng, Fresenius'Z. Anal. Chem., 249, 161 (1970). ( 8 ) G. S. Smith, Chem. lnd. (London). 22, 907 (1963). (9) Dow-Corning Bulletin No. 03-023. (10) D. E. Leyden, G. H. Luttrell, and T. A. Patterson, Anal. Left., 8, 51 (1975). (1 1) A. Duca and T. Budiu, Rev. Roum. Chim., 11, 585 (1966). (12) "Methods for Chemical Analysis of Water and Wastes", Environmental Protection Agency, 1971, p 235. (13) M. G. Natrella. "Experimental Statistics", U.S. Government Handbook 91. Aug. 1, 1963.
(1) (2) (3) (4) (5)
RECEIVEDfor review December 16, 1974. Accepted February 20, 1975. This work was supported in part by Research Grant GP-38396X from the National Science Foundation.
Stereospecific Reactivity by Ion Cyclotron Resonance Spectrometry: Optimization of Reactivity Differences in Two Isomeric Esters Maurice M. Bursey' Venable and Kenan Chemical Laboratories, The University of North Carolina, Chapel Hill, NC 27514
J. Ronald Hass National Institute of EnvironmentalHealth Sciences, P.O. Box 12233, Research Triangle Park, NC 27709
Robert L. Stern Department of Chemistry, Oakland University, Rochester, MI 48063
Ion cyclotron resonance (ICR) spectrometry has been suggested as an analytical technique for the identification of isomers difficult to distinguish by conventional mass spectrometry (1-4). Differences do exist in the mass spectra of geometrical isomers ( 5 ) , but they are sometimes small and, in general, not predictable from the general theory of organic cracking patterns. The ion-molecule reactions of these isomers at low energies frequently do show significant differences. We now have optimized a set of conditions for distinguishing a pair of epimeric esters such that one undergoes a certain ion-molecule reaction to an easily measured extent while the other isomer does not. The reaction seems to be generally applicable, and we anticipate that it will be the prototype of many useful applications. We view our reactions in the ICR spectrometer as analogous to chemical ionization in which we ionize not by transfer of a proton but by transfer of the bulkier CH&O+ group, as for example in the reactions 1 and 2 (6). (CH,CO),*+ (CH,CO),'
+
+
M
M
+
--+
CH,COM+
CHsCOM'
+ CH,CO+ (CH,C0)2
(CH,C0)2*+ + (CH,CO)?
-
(CH,CO),'
+
0
Dcoco
(2)
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8, JULY 1975
CH,CO*
(3)
Using this sort of relative rate determination, it has been possible to show very clearly that steric factors are important in ion-molecule reaction rates. In a series of substituted cyclohexanones 1 taken as M in Equation 1, the rate of reaction decreases in a predictable fashion as R becomes larger ( 3 ) .
(1)
A u t h o r t o w h o m correspondence s h o u l d be addressed.
1452
In Equations 1 and 2, the reagent ion is derived from biacetyl. The pathway is confirmed by the double resonance technique and, in all cases which we have examined so far, the product ion has not decomposed to other products. In these cases, then, it is possible to relate the amount of product formed to the rate of formation. In particular, we have often used a competitive technique in which the acylation of the substrate M in Equation 1 is compared with the acylation of the standard, biacetyl itself (Equation 3).
1
2
a
(I38
When the bulkier reagent 1,2-dicyclopropylethanedione(2) is used to carry out a similar set of reactions with the cyclohexanones (Equation 4), (C,H,C0)2*'
+
M
-
C3HSCOM'
+
C,H,CO*
(4)
then the rate of reaction of Equation 4 decreases even more sharply with increasing size of R (4). Another consequence of these studies was the observation that the stereoisomers 3 and 4 taken as M react at different rates in both Equation 1 and Equation 4.
m
0
0
A
4
3
The more reactive isomer is 3, and this fact can be rationalized on the basis of simple arguments of steric interference (3, 4 ) . The difference in reactivity has led us to explore other systems which might be taken as models for derivatives of stereoisomeric metabolites in which ICR spectrometry could eventually be used to distinguish isomers, and to study the dependence of relative reaction rates upon variables in the ICR experiment. The mass range of current instruments makes this eventual application feasible. As models for metabolite derivatives we have chosen the 4-tert- butylcyclohexyl acetates 5 and 6. These epimers were chosen because the oxygen-containing functional group is held in a different position in each compound.
H
H
OCOCH, 5
I!
H 6
Commonly a tert-butyl group attached to a cyclohexane ring has a very strong preference for the equatorial position (7). This preference is so great that a cyclohexane ring substituted with both a tert- butyl group and another substituent still adopts the conformation in which the tert- butyl group is equatorial, regardless of whether the other group is axial or equatorial, Consequently the two epimers adopt the conformations indicated. In 5, the acetoxy substituent is axial; in 6, it is equatorial. In general, the solution reactivities of substituted cyclohexane compounds are such as to lead to the general conclusion that an equatorial substituent is more accessible to an external species than the same substituent in the axial position. An explanation for this is that an axial substituent interferes with axial hydrogen atoms a t the 3 and 5 positions of the ring.
A reaction which leads to an increase in bulkiness about R a t the rate-determining step will increase this interference in the axial case, so that the reaction will be slower for the axial than for the equatorial substituent (8). Dipole moment studies indicate that this conformational preference is retained in the gas phase as well as in solution (9). Because six-membered rings are found in many drug systems, it was important to test whether this rule of thumb, whose proffered explanation is independent of solvent con-
Figure 1. ICR spectrum of cis-4-tert-butylcyclohexyl 2.3 X Torr pressure
acetate (5) at
Ionizing energy 13.0 eV, ionizing current 200 n A , total ion current 0.45 PA. Source voltages 4-0.38and -0.21 V, analyzer voltages +0.12 and -0.12 V. No molecular ion is observed; m/e 138 is [U - CH&OOH].+. and m/e 82 is CeHwf 1138
182
#bM P Figure 2. ICR spectrum of trans-4-tert-butylcyclohexyl acetate (6) at the same pressure and ionizing conditions as Figure 1 The spectra of 5 and 6 are virtually identical
siderations, could be used as an a priori guide to ion-molecule reactivity. If this sort of reasoning were found to be valid for this model system, then its extension to compounds of greater interest would be a practical consequence.
EXPERIMENTAL The isomers, cis- and trans-4-tert-butylcyclohexylacetate, were available from a previous study ( I O ) . New samples were also obtained by the method given in this reference. The sample of biacetyl was distilled before use. The polarizabilities were calculated according to Le FBvre (11); they are expected to be nearly identical for the esters because the compounds are isomeric. A value of 24.0 A3 was determined in this way. The dipole moments in benzene solution were determined from data obtained on a Weilheim Dipolmeter. The value of 5 is 1.87 D; that of 6,1.85 D. The ICR spectra were obtained on a modified Varian ICR-9 spectrometer a t room temperature. A standard flat cell was used with the following typical cell conditions: trap, 0.30 V; analyzer, split, -0.10 and +0.10 V; source, -0.25 and +0.25V. A typical residence time for the precursor ions is 1 msec. The emission current was operated between 100 and 200 nA. All samples were degassed by several freeze-pump-thaw cycles prior to use and were introduced through separate inlet ports. Typical pressures were in the range of 2 X lov6Torr ester and 4 X Torr biacetyl for exploratory experiments; results of pressure-variation experiments are described later. Reported pressures here are currents at the VacIon pump.
RESULTS A N D CONCLUSIONS There are several forms of the physical theory of ionmolecule reactions. The first, due to Gioumousis and Stevenson (12), takes into account only the polarizability of the neutral molecule. The second ( 1 3 )adds a term for locking of the orientation of the dipole of the molecule by the ion. The most recent ( 1 4 , 15) recognizes that rotation prevents complete orientation of the neutral and permits an effect based on an average dipole orientation to be calculated. Because the polarizabilities and dipole moments of the two isomers 5 and 6 are nearly identical, the ratio of the rates of collision of these molecules with any ion is unity by any of the theories, within 1%.If no further parameters affect reaction, that is, if reaction occurs upon every collision, then the rates of reaction have a ratio of one also. If there are further parameters, the ratio may not be one, and if a steric parameter is overriding, k S / k 6 will be less than one. Indeed the low-pressure spectra of 5 and 6, shown in Figures 1 and 2, are so similar that one might be tempted to ANALYTICALCHEMISTRY, VOL.
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NO. 8, JULY 1975
1453
1129
Figure 3. ICR etate (2.5 X
spectrum of a mixture of cis-4-tert-butylcyclohexylacTorr) and biacetyl (50 X Torr)
Ionizing energy 13.0 eV, ionizing current 150 nA, total ion current 11.6 PA. Source voltages +0.33 and -0.21 V, analyzer voltages +0.12 and -0.12 V. The m/e 139 ion is not formed from biacetyl precursors: m/e 139 is a loss of acetic acid from the auasimolecuiar ion of the ester
extrapolate to reactions with biacetyl and predict that there would be no difference in reactivity. The low-pressure ICR spectra, however, are analogous to the mass spectra, in that they contain peaks produced only by ionization and unimolecular decompositions; there is no reason to anticipate large differences in their reactions under these conditions. On addition of Torr biacetyl to the system, a peak was found to appear a t rnle 241, corresponding t o the addition of acetyl ion to the neutral ester molecule. No other products appeared which could occur as a result of competitive channels for decomposition of the reaction complex leading to rnle 241, nor were products of the decomposition of m l e 241 itself observed. We concluded that the intensity of this ion is affected only by the rate of collision and the ratio of reactive decompositions of the complex to rate of formation of the complex, and not be competitive or consecutive decompositions. When we performed ion cyclotron double resonance experiments on m l e 241, we established signals from both rnle 86, (CH&O)y+, and mle 129, (CHsC0)3+. This situation is therefore more complex, on the surface, than many we have found before. A double resonance signal from m l e 86 in this situation cannot be taken as a safe indication of the relative contribution of Equation 1 to the overall acetylation process. An alternative explanation for the double resonance signal can be offered: changes in the concentration of rnle 86 ions, or in the rate constant for Equation 3, as a result of the irradiation of m l e 86, will affect the amount of m l e 129 produced by Equation 3. The change in the amount of rnle 129 may therefore affect the amount of rnle 241 produced through Equation 2. Hence, the indirect route will also contribute to the double resonance signal from m / e 86. This complex problem has been treated in detail (16). In order to demonstrate the importance of the two processes, the ratio [cis-mle 24l]l[trans-mle 2411 was measured a t constant ester pressure over a range of biacetyl pressures. The rationale for this is not obvious from the theoretical discussion of collision rates, because there should be no change in this ratio as a function of pressure if the reaction rates are the same, as the collision rates are from the table. But if k is affected by steric parameters, then we may predict that the ratio kcislktrans will be less than 1 for both Equations 1 and 2, and because (CH3C0)3+ is bulkier than (CH&0)2-+, the ratio will be smaller for Equation 2 than Equation 1. Therefore, increasing the pressure of biacetyl, which increases the relative contribution of Equation 2 to the overall formation of rnle 241, should lead to a decrease in the ratio. In fact, with a change in biacetyl pressure from 1.6 X Torr to 4.4 X Torr, the ratio kcislktransdecreased from approximately 1 by a factor of 0.57. Another variation could be performed to enhance this difference. As the pressure of the ester is lowered, Equation 1454
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8, JULY 1975
1"'
Figure 4. ICR spectrum of a mixture of trans-4-fed-butylcyclohexyl acetate (2.0 X Torr) and biacetyl (38 X Torr) Ionizing conditions same as Figure 3. Neither m/e 139 nor m/e 199 is formed from biacetyi precursors. Note the significant m/e 241 (M 43) and m/e 199 (M 4- 1) peaks under these conditions for this isomer. The intensity of m/e 199 is independent of biacetyi pressure but dependent strongly upon ester pressure: thus it is a protonation of the ester by ester ions. Note that this isomer is less hindered both for this reaction and the acetylation of interest
+
3 becomes more competitive with Equation 1,which has an effect equivalent to increasing [(CH3C0)3+]/[(CH3CO)2.+]. I t was found that this decrease in ester pressure also decreases the ratio kcislktrans, although one soon reaches a lower useful limit of pressure when the peak due to reactant can no longer be observed. By combination of these effects with the previously observed dependence of power absorption on cell potentials (which affect residence times of ions and therefore reaction extent) and ionizing voltage (17) (which affects rate constants because it influences ion internal energy), it has been possible to select conditions such that the trans isomer is about 7 times more reactive than the cis, and such that no product is observed for the cis isomer. From inspection of Figures 3 and 4, which illustrate the spectra obtained under these conditions, it is clear that the reaction conditions found allow a quick, simple diagnosis of the stereochemistry of the esters: the trans ester gives an acetylation product peak a t m / e 241; the cis gives no peak. Such simple, predictable, clean differences in reactivity are rarely found in conventional mass spectrometry. The ICR method seems considerably superior to the conventional method for distinguishing these isomers. We consider this result crucial to the assertion that ICR spectrometry is of analytical value. Whether it can be simply extended to problems of more immediate interest in metabolic studies is under current investigation. We are now examining whether other derivatives of alcohols have any greater promise than acetate esters of maximizing the difference in reactivity, and whether the voltage and pressure range found for these isomers is a suitable range for studying all compounds of this type. Since steric parameters have been required to explain the reactivities of ketones (3, 4 ) , alcohols ( 1 8 ) , phenols (19),and nitrogen bases (20), it seems reasonable to assume that the differences in reactivities of isomeric compounds of these classes may also be optimized to permit rapid identification of stereochemical environments of these functional groups too. ACKNOWLEDGMENT We thank Jorma Koskimies for measurement of the dipole moments.
LITERATURE CITED (1) J. M. S. Henis. Anal. Chem.. 41,( l o ) , 22A (1969). (2) M. L. Gross, P.-H. Lin, and S. J. Franklin, Anal. Chem.. 44,974 (1972). (3) M. M. Bursey. J.-L. Kao, J. D. Henion, C. E. Parker, and T.4. S.Huang, Anal. Chem., 46, 1709 (1974). (4) J. D. Henion, J.-L. Kao, W. 8. Nixon, and M. M. Bursey, Anal. Chem., 47,689 (1975). (5) S . Meyerson and A. W. Weitkamp. Org. Mass Spectrom., 2 , 603 (1969).
(6) M. M. Bursey. T. A. Elwood, M. K. Hoffman, T. A. Lehman, and J. M. Tesarek, Anal. Chem., 42, 1370 (1970). (7) E. L. Eliel, "Stereochemistry of Carbon Compounds", McGraw-Hill Book Company, New York, NY, 1962, p 219. (8) E. L. Eliel, "Stereochemistry of Carbon Compounds", McGraw-Hill Book Company, New York NY, 1962, p 222. (9) H. R. Nace and R. H.Nealey, J. Am. Chem. SOC., 88, 65 (1966). (10) R. L. Stern, J. S. Zannucci, and 8. L. Karger, Chem. Commun., 613 (1967). (1 1) R. W. J. LeFevre, Adv. fhys. Org. Chem., 3, 1 (1965). (12) G. Gioumousis and D. P. Stevenson, J. Cbem. fbys., 20, 294 (1958). (13) T. F. Moran and W. Hamill, J. Chem. Phys., 39, 1413 (1963). (14) T. Su and M. T. Bowers, J. Chem. fhys., 58, 3027 (1973). (15) T. Su and M. T. Bowers, Int. J. Mass Spectrom. /on Phys., 12, 347 (1973). (16) R. C. Dunbar, M. M. Bursey, and D. A. Chatfield, Int. J. Mass Spectrom. Ion Phys., 13, 195 (1974).
M. L. Gross and J. Norbeck. J. Chem. fhys., 54, 3641 (1971). M. K. Hoffman and M. M. Bursey, Can. J. Chem., 49,3995 (1971). S. A. Benezra and M. M. Bursey, J. Am. Chem. SOC., 94, 1024 (1972). J. D. Henion, M. C. Sammons, C. E. Parker, and M. M. Bursey, Tetrahedron Lett., 4925 (1973).
RECEIVEDfor review December 9, 1974. Accepted March 10, 1975. We are grateful for support from the National Institute of General Medical Sciences (GM 15,994). The instrument was purchased from funds donated by Hercules, Inc., the Shell Companies Foundation, the North Carolina Board of Science and Technology, and the National Science Foundation (GU 2059).
Application of Ion Exchange Membranes to Sampling and Enrichment: Interference of Metal Ion Binding Groups Walter J. Blaedel and Richard A. Niemann Department of Chemistry, University of Wisconsin, Madison, WI 53706
The identification or quantitation of cations present a t trace concentration levels in natural water systems has often involved an enrichment step prior to the analysis ( I ) . Ion exchangers are particularly well-suited for such enrichment, and many applications have been described in which the enrichment occurs into the ion exchanger phase. Materials have been particulate or membranous, and chelating resins also have been used (2-9). In principle, ion exchange membranes may be used in a different mode with particular advantage as sampling-enrichment devices, by concentrating the trace metal ions from a large volume of a dilute water sample (the donor solution) through the ion exchange membrane into a small volume of a concentrated solution (the acceptor solution). Measurement of the enriched cations in the acceptor solution would be simpler than in the original donor solution. The particular advantage of such a system for sampling and enrichment purposes is that the trace cation distribution depends only on the charge types of the ions, and is independent of the other physical and chemical properties of the ions or of the membrane. The theoretical basis of the distribution is well established, and it has been demonstrated experimentally (20-13). Exploratory attempts to utilize this approach for sampling and enrichment of Cu(I1) a t micromolar concentration levels required very long equilibration times, and led to the hypothesis that the membrane contained impurity groups capable of binding the metal ion very strongly by mechanisms other than the ion exchange mechanism. The work described in this note was undertaken to demonstrate unequivocally the occurrence of such binding, to establish the levels at which it operates for some of the commercially available membranes, and to seek infrared spectrometric evidence supporting the presence of impurity groups in the membranes.
EXPERIMENTAL Reagents. All chemicals used were reagent grade. All solutions were prepared from triply distilled water: once-distilled t a p water
was redistilled from alkaline permanganate, collected, made about 0.001M in HzS04 and distilled again. For making dilute solutions, an aqueous stock solution of 0.01M Cu(N03)2 was prepared and standardized by titration with EDTA. Solutions of lower concentration were prepared by appropriate dilution of aliquots of the stock solution. Radiotracer 64Cu was prepared in the University of Wisconsin nuclear reactor facility by thermal neutron irradiation of a weighed amount (about 30 mg) of pure metallic copper. After irradiation, the copper was dissolved in a few milliliters of 8M "03 and evaporated to near dryness over a low flame. The residue was evaporated with about 20-ml portions of water twice more to near dryness, and finally transferred with 0.10M NaCl to a 100-ml volumetric flask and made u p to volume with 0.10M NaC1. An aliquot of this stock solution was used in the first extraction of the successive extraction experiments. Three types of sulfonated cation exchange membranes were studied: AMF (2-103 (American Machine and Foundry Co., Stamford, CT, sulfonated styrene on a polyethylene backbone, 0.007 inch thick, 1.3 mequiv per dry gram of H-form); Nafion XR-170 (Plastics Dept., Du Pont and Co., Wilmington, DE, sulfonated fluorocarbon polymer, 0.0035 inch thick, 0.83 mequiv per dry gram of H-form); Permion PlOlO (RAI Research Corp., Hauppauge, Long Island, NY, T F E Teflon-sulfonated styrene copolymer, 0.0015 inch thick, 2.00 mequiv per dry gram of H-form). Before use, all membranes were conditioned and converted to the Na-form by the following steps: 1) wash for 0.5 hour in 95% ethanol; 2) three cycles of 5-minute rinses in 1M HCI, water, and 1M NaOH; 3) three half-hour washes in portions of 1M NaC1; 4) storage in 0.10M NaCl until needed. Copper(I1) Distribution Measurements. A weighed rectangular piece of membrane (about 0.1 mequiv total ion exchange capacity) was contacted with mechanical stirring for about 8 hours with a measured weight (between 150 and 500 g) of 0.10M NaCl solution that contained a known concentration of copper (about 15 ppm, or about 0.2mM), and that also contained r a d i ~ - ~ ~(activiCu ty greater than lo4 cpm/g solution). A significant fraction (5 to 10%) of the copper was taken up by the membrane, and the distribution ratio and copper content of the membrane were determined from the counting rate of the solution before and after extraction. The membrane was removed from solution, blotted dry with a tissue, and counted for 64Cu activity. T o ensure a reproducible counting geometry, the membrane, wrapped around a Plexiglas rod having a slit in one end, was inserted into the bottom of the counting tube where it was uncoiled from the rod and carefully positioned ANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
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