Anal. Chem. 1988, 6 0 , 15-19 (4) Brown, D. A.; Qlasess, W. K.; Jan, M. R.; Mulders, R. M. W. Envlron. Technol. Len. 1986. 7 , 283-288. (5) Subramanlan, K. S.; Manger, J. C. I n t . J. Envlron. Anal. Chem. 1979, 7.25-40. (8) Tande, T.; Pattersen, J. E.; Torgrimsen, T. Chromafographia 1980, 73, 607-610. (7) Malissa, H.; Kotzlan, H. Talanta 1982, 9, 997-1002. (8) Schwedt, 0. Fresenius' 2.Anal. Chem. 1979, 2 9 5 , 382-387. (9) Bergmann, H.; Hardt, K. Fresenius' 2. Anal. Chem. 1979, 297, 38 1-383. (10) Annual Book of ASTM Standards; A.S.T.M.: Philadelphia, PA, 1981; Part 31, D1193-77, pp 29-31. (11) Vogel, A. I. A Textbook of Quantitative Inorganic Analysis, 3rd ed.; Longmans, Green: London, 1961; p 311.
15
(12) Subramanian, K. S.;Mbranger, J. C.; Wan, C. C.; Corsini, A. I n t . J. Envlron. Anal. Chem. 1985, 79, 261-272. (13) Halls, D. J.; Fell, G. S. J. Anal. At. Spectrom. 1988, 7 , 135-139. (14) Kayne, F. J.; Komer, G.; Labcda, H.; Van der Linde, R. E. Clln, Chem. (Winston-Salem, N . C . ) 1978, 2 4 , 2151-2154. (15) Ping, L.; Matsumoto. K.; Fuwa, K. Anal. Chim. Acta 1983, 747, 205-21 2. (16) Everson, R. J.; Parker, H. E. Anal. Chem. 1974, 4 6 , 2040-2042. (17) Cox, A. G.; Cook, I. G.; McLeod, C. W. Analyst (London) 1985, 170, 33 1-333.
RECEIVED for review March 19, 1987. Accepted September 10, 1987.
Use of 'H Nuclear Magnetic Resonance Longitudinal Relaxation Times in Structure Elucidation of Chlorinated Polyaromatic Compounds David L. Ashley,* Elizabeth R. Barnhart, Donald G. Patterson, Jr., and Robert H. Hill, Jr. Division of Environmental Health Laboratory Sciences, Center for Environmental Health, Centers for Disease Control, Public Health Service, US.Department of Health and Human Services, Atlanta, Georgia 30333
We describe a procedure for measuring relative longitudinal relaxatlon tlmes of protons In chlorinated polyaromatlc hydrocarbons. The results lndlcate that these times are dependent on Interproton dlstances and thus can be used to distlngulsh protons wlth ortho-proton neighbors from those wlthout ortho-proton nelghbors. I n addltlon, the longltudlnal relaxation tlme can also qualltatlvely descrlbe inter-rlng Interactkns under certain condnkns. The measurement is used for structural elucldatlon of three cases of unknown chlorlnated polyaromatlc hydrocarbons, and In each case an unambiguous amlgmnent Is possible. The use of relative proton longnudlnal relaxatkm tknes for stNctwal IdentMcatlonshould be broadly applicable to many structural problems.
Since the discovery of the chemical shift in the early 195Os, numerous nuclear magnetic resonance (NMR) techniques have been used to measure characteristic molecular properties. The use of the appropriate experimental design, coupling constants, relaxation times, and chemical shifts has yielded information about electronic environments, nuclear distances, and bond angles. Measurement of carbon-13 longitudinal relaxation times of organic compounds has been widely applied (I, 2), but the proton counterpart has not, even though it also has potential for molecular characterization (3). Polyaromatic halogenated hydrocarbons, including dibenzo-p-dioxins, dibenzofurans, biphenylenes, and pyrenes, are a significant public health concern. These compounds have been shown to be highly toxic to certain species, and health effects in humans have also been documented (4,5),although these findings are still controversial. In spite of the controversy, it is widely accepted that the toxicities of these compounds are extremely isomer-specific. Thus, assessing risk from exposure is contingent on identifying substitution patterns and quantitating each isomer. The technique most frequently used to determine levels of these contaminants is mass spectrometry following gas chromatography (6), but that technique does not provide direct isomer differentiation in
all cases. Independent methods must be available to characterize standards used with this technique. NMR is particularly suited for identifying substitution patterns because of the dependence of NMR spectra on molecular level properties. Thus, NMR can substantially aid in characterizing these isomers. We investigated the possibility of using proton NMR longitudinal relaxation times to differentiate certain substitution patterns in chlorinated polyaromatic compounds. This technique was then applied to three examples of unidentified polychlorinated compounds that could not be determined through recognition of peak splitting patterns.
EXPERIMENTAL SECTION Chlorinated dibenzo-p-dioxinswere prepared by reacting the dipotassium salts of chlorinated catechols with chlorinated benzenes or chlorinated nitrobenzenes in anhydrous dimethyl sulfoxide at 175 "C as previously reported (7).These mixtures were purified by chromatographyon silica gel with hexane as the eluting solvent. Chlorinated dibenzofurans were synthesized via palladium acetate cyclization of the appropriate chlorinated diphenyl ethers (8). Chlorinated pyrene and biphenylene derivatives were synthesizedby chlorinationof pyrene or biphenylene with S02C12reagent (reagent C, Perchlorination Kit, Analabs, Inc., New Haven, CT) (8). Polychlorinated biphenyl standards were acquired from Ultra Scientific (Hope, RI). Separation of mixtures of synthesis products sometimes required high-performanceliquid chromatography (HPLC). HPLC was carried out by using a Waters Associates M-6000 pump and Model 440 absorbance detector (254 nm) and a 25 X 2 cm i.d. Dynamax ODS column (Rainin Instrument Co., Woburn, MA). The derivatives were extracted into 50 MLof toluene, and this mixture was separated on the column with a flow rate of 9.5 ml/min. The eluting solvent varied from a 95% methanol/water mixture to 100% methanol. Purity and structural verification of standards were determined by using gas chromatography (GC) with flame ionization detection, GC coupled with mass spectrometry, and GC coupled with Fourier-transform infrared spectrometry. Proton NMR spectra were acquired on a Varian Associates XL-300 spectrometer that was equipped with a 7.0-T superconducting magnet and that employed the XL data system. Ap-
This article not subject to US. Copyrlght. Published 1987 by the American Chemical Society
16
ANALYTICAL CHEMISTRY, VOL. 60, NO. 1, JANUARY 1, 198 Table I. Longitudinal Relaxation Times
T,. s
compound .---w-w-
1,4,6,9-TCDD Hz,Hs,H,,HB 1,2,7,8-TCDD H3 H4 H,
8 0 sec
*li
1,4,7,8-TCDD
690 PPM
Figure 1. Stacked plot of proton NMR longitudinal relaxatlon spectra of 1,4,6,9-, 1,2,7,8-,and 1,4,7,8-TCDDat six different 7 values.
Dibenzodioxins
8 7 / u n
Dtbenzofurans
1
6'9rd: 10
1
8b9: 7 ' - A / 4
6
5
Pyrene Addition Products
5
4
Biphenylenes
*,
3,
44-
5
6
protons
6'5'
Biphenyls
Figure 2. Numbering schemes for polyaromatlc compounds examined
in the study. proximately 100 pg of the chlorinated derivatives was examined in deuteriated acetone. Samples were degassed by bubbling 99.999% Nz through them. All spectra were measured at 22 O C with a spectral width of 4OOO Hz and 30K data points. A 90° pulse of 9.4 ps was used, and a delay between acquisitions of at least 5 Tls was employed to allow relaxation. All spectra are reported relative to tetramethylsilane, with the residual acetone signal at 2.05 ppm as reference. Spin-lattice relaxation measurements were carried out by the inversion-recovery technique (9). Spectra at 11 different T values were measured and Tlvalues were determined by an exponential least-squares analysis of the peak heights versus time. A measure of how well the data agreed with the exponential model is shown by the error (standard deviation) in determination of these TI values, which averaged 7.4%. Studies aimed at determining reproducibility of Tlvalues by this technique indicated an average difference of 14% upon repeat measurement.
RESULTS AND DISCUSSION Figure 1shows a stacked plot of proton NMR longitudinal relaxation results for a mixture of 1,4,6,9-, 1,2,7,8-, and 1 , 4 , 7 , & t e t r a c h l o r o d i b e n ~ ~(TCDD). - ~ o ~ ~ The numbering schemes for all compounds examined in this study are given in Figure 2. The peaks in this spectrum can be assigned as follows: for 1,4,6,9-TCDD, H2, H3,H,,HB7.200 ppm; for 1,2,7,8-TCDD,H37.247, H46.998, H67.247, H97.322 ppm, and JS = 8.84 Hz;for 1,4,7,STCDD,Hz, H37.154, I-& & 7.341 , ppm. Figure 1 demonstrates the difference in longitudinal relaxation rates that can occur in these compounds. For example, in the spectrum obtained a t 20 s, six of the nine peaks have already obtained a positive value, whereas the other three are still negative. Calculation of Tlvalues from this data yielded the results presented in Table I, which indicate a dramatic difference in longitudinal relaxation times for protons in different environments. Specifically, protons that have no ortho-proton neighbors have larger T1values (33-36 s) than those with at least one ortho-proton neighbor (11-14 8 ) . This difference suggests that, under these experimental conditions, the intramolecular dipole-dipole interaction is chiefly responsible for longitudinal relaxation of the
H, Hz,H3 He..Ha
with without ortho-proton ortho-proton neighbors neighbors 14.07 11.46 10.72
33.27 35.16
14.23 36.02
protons in these molecules. The dipole-dipole mechanism is a function of the sixth power of the distance between dipoles, so only magnetic nuclei in the immediate vicinity of the nucleus being investigated will have a major relaxing effect. Carbon-12 nuclei do not possess angular momentum and are, therefore, nonmagnetic-even though chlorine nuclei do possess angular momentum, they are paramagnetic. The relaxation of paramagnetic chlorine nuclei is so rapid that they appear nonmagnetic and do not contribute substantially to longitudinal relaxation (IO). The longitudinal relaxation times of protons in these molecules are, thus, highly dependent on interproton distances. Another much more subtle effect is illustrated in these results. The longitudinal relaxation time for H, in 1,2,7,8TCDD is smaller than for H3.The TI for H6is also smaller than for Hg.These protons have an additional inter-ring interaction that their counterparts do not have. This effect is much smaller because of the larger distances involved, but it can be substantial under particular circumstances, as will be described later. Intramolecular dipole-dipole relaxation is not the only source of longitudinal relaxation. Intermolecular interactions can also contribute significantly, particularly due to the presence of molecular oxygen in the sample. To best exploit the differences in intramolecular contributions to relaxation and to compare values between different samples, we must either remove or minimize the effect of oxygen and keep it constant by degassing. Several methods have been used to deoxygenate samples for NMR experiments. The most popular is the freeze-pump-thaw method (3, 11, 12). Another technique sometimes used is the bubbling of pure nitrogen through the sample solution (13). With both of these techniques, the only successful way to demonstrate the removal of all oxygen from a sample is to repeat the process until the relaxation times no longer increase. Our experience indicates that achieving complete deoxygenation with either of these techniques requires a substantial amount of sample manipulation. Since compounds examined in this study are a significant, public health concern, only a minimum of sample manipulation could be performed. As an alternative approach to this problem, relative relaxation times were used to determine the isomer assignments. For the cases in which all isomers to be compared could be measured together, TI values could be directly compared with each other. Intersample comparisons presented a more complicated problem. To avoid sample irreproducibility, Grant et al. (3)have suggested the use of internal standards to monitor NMR relaxation times. For the comparison of multiple samples in our study, the relaxation times of standard compounds were determined along with those of the unknown samples. Relative TI values were then calculated so that these results could be related to other samples measured under slightly different conditions. Figure 3 illustrates the major problem with variability in the degree of deoxygenation among samples. As a sample is taken through successive stages of deoxygenation (shown by
NO. 1, JANUARY 1, 1988
ANALYTICAL CHEMISTRY, VOL. 00, 100,
Table 11. Relative Longitudinal Relaxation Times for Chlorinated Dibenzodioxins,Pyrene-Addition Products, and Biphenyls
compound
6
8
10
12
18
16
14
1,4,6,9-TCDD 1,2,7,8-TCDD
Deoxygenation (TI of Hpand Hgof 1.4,7.8-TCDD in aec)
Flgwe 3. Plot of proton longitudinal relaxation times (T,) of the protons of 1,2,7,8-TCDD (H,,H,, closed squares; H,,H,, open squares) over various stages of deoxygenation. The degree of deoxygenation is indicated by the T , of H, and H, of 1,4,7,ETCDD present in the sample as a relaxation time internal standard. 2.5
relative TI with without orthoorthoproton proton neighbors neighbors
protons Hz, H3, Hn Hs H3 H4
HB 2,3,7,8-TCDD HI, H4, H6, HB HZ 1,3,4,5,6,9-hexachloro4,5-dihydroH4 pyrene H5 H7 H 8
HlO H3’ H4’ H6
2,2’,3,4,5’-PCB
1.5 4
7
1.87 1.96 1.71 1.72 0.72 0.72 0.78 0.56 1.73 0.94 0.95 0.81 0.74
H6
4
0.99 0.87 0.84
H6
1
2.0
0.5
17
1.89 1.84 1.74
H6‘
2,2‘,4,4’,5-PCB
H3 H3’ 0.75
H5’
1.54
H6
0 1
6
’
8
,
10
12
,
14
,
,
18
,
0.70
H6’
I
18
Deoxygenation (T1 of H2and Hgof 1,4.7.8-TCDD in 8ec)
Flgure 4. Plot of relative proton longitudinal relaxation times of the protons of 1,2,7,&TCDD (H,,H,, closed squares; H,,H,, open squares) over various stages of deoxygenation. The degree of deoxygenation is indicated by the T, of H, and H, of 1,4,7,8-TCDD present in the sample as a relaxation time internal standard. The method for calculation of the relative T , values is described in the text.
increases in the T , values of Hzand H3of 174,7,8-TCDD),all longitudinal relaxation times increase but at different rates. For the protons with ortho-proton neighbors, oxygen removal has a smaller effect because other (intramolecular dipoledipole) mechanisms contribute significantly to their relaxation. Because of the lack of alternate mechanisms, the relaxation rates of protons without ortho-proton neighbors are highly dependent on the presence of molecular oxygen, and thus, its removal has a more pronounced effect on Tl values. Variability can be resolved by using relative T I values. 174,7,8-TCDDwas chosen as a standard for three reasons. First, it contains both types of protons of interest in this study. Hzand H3 are ortho and will have a substantial relaxation effect on each other. H6and H9are para and will thus have a much lower dipolar interaction. The second reason for using 1,4,7,8-TCDD is that the proton NMR spectrum of this compound contains only two singlets, which are found at a significant chemical shift difference. This occurs because of the equivalency of proton pairs and the distinctly different electronic environment between the pairs. Use of this compound will help prevent peak overlap with the resonances of the samples being investigated and make the mesurement of TI values possible. The third reason is that this particular dioxin isomer is considered less hazardous than some others in this class. Relative T1values can be calculated in a variety of ways. In this study, the T1of H2and H3 of 1,4,7,8-TCDD was set equal to 1.00 and the TI of H6and HBwas set equal to 2.00. The relative T1values of the unknown compound were calculated according to these values. An example of this transformation is shown in Figure 4, which is a recalculation of the results from Figure 3. As evident in this figure, the conversion produces relatively constant results over a wide
Table 111. Relative Longitudinal Relaxation Times for Chlorinated Dibenzofurans
compound 2,3,6,7-TCDF
1,2,7,8-TCDF
2,3,7,8-TCDF
protons Hl H4 Ha H9 H3 H4 HR Hi H1, HB HA. -. Hc Hl H5
relative TI with without ortho-proton ortho-proton neighbors neighbors 1.24 2.08 0.94 0.87 0.91 1.01 2.09 2.20 1.34 1.82 1.16
1
2,3,4,8-TCDF
H6
1,2,3,6,7&HxCDF
H9 H4 H9
0.99 0.97 1.11 1.95 1.79
range of deoxygenation levels. Only measurements with TI of H2 and H3 of 1,4,7,8-TCDDbetween 8 and 17 s were accepted as valid. Table I1 shows the results of the determination of relative longitudinal relaxation times for several polychlorinated aromatic compounds. Their numbering schemes are given in Figure 2. These results indicate that the relative T I values for these compounds are reasonably consistent once the presence or absence of ortho protons is taken into account. The T1values for protons with ortho-proton neighbors ranged from 0.56 to 0.99, and for protons without ortho-proton neighbors the T , values ranged from 1.54 to 1.96. Table I1 also illustrates a generally consistent effect among such different polyaromatic compounds as dibenzodioxins, pyrenes, and biphenyls. We believe this is a general method that can be applied with reasonable certainty to each of these polyaromatic classes and probably to other classes of compounds.
18
ANALYTICAL CHEMISTRY, VOL. 60, NO. 1, JANUARY 1, 1988
--_
c--J
+---.-,”---
y.wyw44 ’v* ~Un,
T
1
7 2
7 4
70
8 8 PPM
86
Proton NMR spectrum of an unknown tetrachhxobiphenylene with two possible structures. Figure 5.
76 Flgure 7.
7 5
74
73
7 2
7 1 PPM
Proton NMR spectrum of a mixture of 1,2,3,6,7,8- and
1,2,3,7,8,9-hexachlorodibenzodioxin with two structures.
product (14). The presence of three singlets and line width determinations (which indicated no inter-ring coupling) suggested that this compound could be either 1,3,4,5,6,8hexachloro-4,5-dihydropyreneshown in the figure or one of 14 possible nonachloropyrene addition products (protons in positions 1,4,6; 1,4,7; 1,4,8; 1,4,9; 1,5,7; 1,5,9;2,4,6; 2,4,7; 2,4,9; 2,4,10; 2,5,9; 2,5,10; 3,5,9; and 3,5,10). Gas chromatographymass spectrometry could not definitively distinguish the nonachloro and hexachloro compounds because pyrene addition products lose chlorine at the temperatures required for their LW__ .-.wdd1 separation. In the hexachloro compound H,, H,, H9, and Hlo 90 85 8’0 75 7’0 6 5 PPM 8 0 all have ortho-proton neighbors whereas H2 and H, do not. Figure 6. Proton NMR spectrum of an unknown chlorinated pyrene Therefore, two of the resonances should have relatively short addition product with 15 possible assignments. relaxation times compared with the third. In the nonachloro isomers, none of the protons have ortho-proton neighbors. The results of relative TI determinations on some selected chlorinated dibenzofuransare shown in Table 111. The results Thus, all of the protons should have very similar longitudinal relaxation times. Measurement of longitudinal relaxation of these measurements were consistent with the findings in times gave 10.6,10.7, and 67.0 s, indicating that the compound Table I1 except for four of the T , values. These four T1values were for protons without ortho-proton neighbors and were is the hexachloropyrene addition product. significantly lower than the normal range of results. This table The last example of this technique’s application involves shows that all of these cases are protons in either the 1 or 9 distinguishing two compounds created in the same synthesis position in dibenzofurans without chlorination in either of procedure. Because of the Smiles rearrangement, pairs of these positions. The aromatic rings in dibenzofurans are chlorinated dibenzodioxin molecules are formed with the bound by a carbon-carbon bond and an ether linkage; thus synthesis method used in this study (7). The proton NMR the protons in the 1and 9 positions are much closer to each spectrum of one such pair is shown in Figure 7. This pair is the 1,2,3,6,7,8-and 1,2,3,7,8,9-hexachlorodibenzo-p-dioxins other than the protons in the 4 and 6 positions. We discussed previously the inter-ring relaxation effects in the dibenzo(HxCDD). These compounds only differ in the relative oridioxins, and these dibenzofuran protons (1and 9) are even entation of the two aromatic rings. The protons in closer than the 1 and 9 positions in dibenzodioxins. Since 1,2,3,7,8,9-HxCDDlie directly across the ether linkage from each other, but in 1,2,3,6,7,8-HxCDD they are much farther dipole-dipole relaxation is a through space effect, their T , apart. The proton longitudinal relaxation times in values are decreased under these conditions. Figure 5 shows the proton NMR spectrum of one product 1,2,3,7,8,9-HxCDD should be shorter than in 1,2,3,6,7,8from the chlorination of biphenylene. This compound was HxCDD. Measurements of these values showed that the determined by mass spectrometry to be a tetrachlorinated resonance at 7.41 ppm gave a T , value of 92.8 s, and the biphenylene (TCBP). A single proton NMR resonance inresonance at 7.33 ppm had a T , of 61.7 s. Thus, these isomers dicates that the four protons in the molecule must be can be readily assigned. The protons in 1,2,3,6,7,8-HxCDD equivalent. Two possible isomers of TCBP fit this description: occur at 7.41 ppm, and the protons of 1,2,3,7,8,9-HxCDDoccur at 7.33 ppm. 1,4,5,8- and 2,3,6,7-TCBP. Because of the difference in substitution patterns, the protons in 1,4,5,8-TCBP have orthoThese applications illustrate three different problems that proton neighbors, and the protons in 2,3,6,7-TCBP do not. can be solved by using relative proton longitudinal relaxation If the unknown compound is 1,4,5,8-TCBP, the relative retimes. The first case distinguishes two possible isomers based laxation time should be in the range 0.56-0.99. If the comsolely on the differences in the substituents located ortho to pound is 2,3,6,7-TCBP, the relative relaxation time should the protons of interest. The second example shows how determining the number of protons with and without orthobe within the range 1.54-1.96. The relative relaxation time proton neighbors can be used to distinguish congeners. The for the biphenylene compound was determined to be 1.74, which indicates that the unknown biphenylene compound is last case illustrates that even inter-ring interactions can prove 2,3,6,7-TCBP. This is the product expected from the chlouseful for isomer differentiation in some cases. It must be rination since the 2, 3, 6, and 7 positions are less sterically pointed out however, that the relative interaction of relaxation hindered. In this compound, inter-ring relaxation effects may mechanisms and the effect of more than two spins in a system be a significant relaxation mechanism due to the proximity have not been completely investigated. of the 1 and 8 positions and the lack of ortho relaxation This method is a practical, simple way to differentiate interactions. certain c m s of isomers. The method is particularly valuable Figure 6 shows the proton NMR spectrum of an unknown when the protons that may interact are equivalent. In cases chlorination product of pyrene. The location of the resonance such as these, homonuclear decoupling cannot be performed, of 6.4 ppm indicates that the compound was a pyrene-addition and nuclear Overhauser enhancement (NOE) cannot be de~~
T
Anal. Chem. 1988, 60,19-22
termined. Thus, the method might be the most straightforward or, in some cases, the only way of determining chlorine substitution patterns short of X-ray diffraction procedures. In this paper, we describe the novel use of proton NMR longitudinal relaxation times for structural identification. We have found this method extremely useful in determining structures of various chlorinated polyaromatic compounds, and we believe this method has much broader application for structure elucidation problems where several products are possible. Registry No. 1,4,6,9-TCDD7 40581-93-9; 1,2,7,8-TCDD, 34816-53-0; 1,4,7,8TCDD,40581-94-0; 2,2’,3,4,5‘-PCB, 38380-02-8; 2,2’,4,4’,5-PCB, 38380-01-7; 2,3,6,7-TCDF, 57117-39-2; 1,2,7,8TCDF, 58802-20-3; 2,3,7,8-TCDF, 51207-31-9; 2,3,4,8-TCDF, 83704-32-9; 1,2,3,6,7,8-HxCDF, 57117-44-9; 1,3,4,5,6,9-hexachloro-4,5-dihydropyrene,110852-18-1.
LITERATURE CITED Levy, 0. C.; Nelson, 0. L. Carbon- 13 Nuclear Magnetic Resonance for Organic Chemists; Wiley-Intersclence: New York, 1972. Baklo, M.; Irgollc, K. J.; Nicolini, M.; Pappalardo, G. C.; Vni, V. J . Chem. SOC.,Faraday Trans. 11983, 79, 1633-1638. @ant, C. W. M.; bii. L. D.: Preston, C. M. J . Am, Chem. SOC.1973. 95, 7742-7747. Reggiani, G. I n Accidental Exposure to Dioxins. Human Health Aspects; Coulston, F., Pocchiari, F., Eds.; Academic: New York, 1983; pp 39-67.
19
(5) Bruul, P. I n Accidental Exposure to Dioxins. Human Health Aspects; (6) (7) (8)
(9) (10) (11) (12) (13) (14) . .
Coulston, F., Pocchiarl, F., Eds.; Academic: New York, 1983; pp 215-225. Tiernan, T. 0. I n Chlorlnefed Dbx/ns and Dbenzofurans in the Total Environment; Choudhary, G., Keith, L. H., Rappe, C., Eds.; Butterworth: Boston, MA, 1983; pp 211-237. Gelbaum, L. T.; Patterson, D. G., Jr.; Groce, D. 0. I n ChlorinatedDioxins and Ditmnzofurans in Perspective; Rappe, C., Choudhary, G., Keith, L. H.,Eds.; John Lewis: Chelsea, MI, 1986; Chapter 31. Barnhart, E. R.; Ashley, D. L.; Reddy, V. V.; Patterson, D. G., Jr. HRC CC,J. High Resolut. Chromatogr. Chromatogr. Common. 1968, 9 , 528-530. Farrar, T. C.; Becker, E. D. Pulse and Fourier Transform N M ;Academic: New York, 1971. Lambert, J. 8.; Shurvell, H. F.; Verbit, L.; Cooks, R. G.; Stout, G. H. Organic Structural Analysis; Macmlllan: New York, 1976; p 139. Nederbragt, G. W.; Rellly, C. A. J . Chem. Phys. 1958, 2 4 , 1110-1111. Martin, J.; Dalley, B. P. J. Chem. Phys. 1862, 37, 2594-2602. Tarpiey, A. R., Jr.; Goldsteln, J. H. J . Phys. Chem. 1971, 7 5 , 42 1-430. Ashlev. D. L.: Barnhart. E. R.: Patterson. D. 0..Jr.: Hill. R. H.. Jr. ADD/. Specirosc. 1967, 41. 1194-1199. .
I
RECEIVED for review April 30,1987. Accepted August 27,1987. use of trade names is for identification only and does not constitute endorsement by the Public Health Service or the Department of Health and Human Services. This research was funded by the Agency for Toxic Substances and Disease Registry.
Indicator Ligands in Metal Complexation Studies: Role of 4-(2=Pyridylazo)resorcinol in Europium Carbonate Equilibrium Investigations Shannon W.Thompson and Robert H. Byme* Marine Science Department, University of South Florida, St. Petersburg, Florida 33701
Spectrophotometric procedures utilizing the strongly compiexing indkator 4-( 2pyridylazo)resordnol (PAR) permlt determination of 1:2 metal-ilgand formatlon constants while substantially promoting metal solubiilty. The intrlcacles inherent in competltlve equilibria involving PAR can be welimanaged through use of low total metalhotel indicator (PAR) raUos and observations of comparative complexation at constant pH. The Influences of carbonate and oxalate complexation on the absorbance of Eu-PAR complexes are quantltathrely described in terms of Eu(CO,),-, Eu(OX),-, and ternary (Eu-PAR-ligand) complexes. Since PAR is a nonspeclflc colorlmetrlc complexant, the spectrophotometric procedures outlined In thls work are appllcabie to a wlde variety of metals.
The results of previous lanthanide carbonate complexation studies indicate that thermodynamic characterization of the species M(C03)2- is particularly challenging. While estimates of the EuC03+formation constant differ by nearly a factor of 6, estimates for the species E U ( C O ~ )differ ~ - by nearly 3 orders of magnitude (1-3). In the only case where lanthanide carbonate formation constants have been obtained independently by using a single technique (solvent exchange), formation constant results for the species EuC03+are in excellent agreement but differ for the species Eu(C03); by more 0003-2700/88/0360-0019$01.50/0
than a factor of 2 (2-4). Due to solubility constraints, potentiometric procedures are capable of examining MC03+ formation but exhibit no sensitivity to the species M(C03)2whatever (5, 6). In seeking procedures to further our investigations ( 3 , 4 ) of europium carbonate complexation, we were concerned by the lengthy equilibration times (7,8) and solid-phase composition ambiguities (8) inherent in solubility analysis. Following the observation that 4-(2-pyridylazo)resorcinol (PAR) is a useful colorimetric complexant for lanthanides (9, IO) and many other metals (II), we undertook the use of PAR in spectrophotometric investigations of Eu3+complexation. We report, in this work, procedures applicable to the study of lanthanide complexation within substantial solubility constraints. Our analyses are particularly well-suited to investigation of complexation constants beyond the first. As such, we expect that, in many instances, our methods will complement the procedures of Ohyoshi (9, IO),wherein PAR is used in examination of the first in a series of stepwise complexation equilibria. THEORY Investigations of lanthanide carbonate complexation typically require conditions that will support less than 1 pM of total dissolved metal (8). Sensitive spectrophotometric examinations of metal complexation equilibria generally require total metal concentrations on the order of 2-5 pM. Consequently, solubility constraints are an important consideration 0 1987 American Chemical Society
