Anal. Chem. 1988, 6 0 , 2127-2131 (43) Armstrong, D. W.; Seeman, J. I. et al., unpublished results, 1967. (44) Armstrong, D. W.; Splno. L. A.; Han. S. M.; Seeman, J. 1.; Secor, H. V. J. Chromatogr, 1987, 4 1 7 , 490. (45) Tsujlno, Y.; Shlbata, S.; Katsuyama, A.; Kisakl, T.; Kaneko, H. Heterocycles 1982. 19, 2151. (46) Hu, M. W.; Bondinell, W. E.; Hoffmann. D. J. Labelled Compd. 1974, 70, 79. (47) Seeman, J. I.; Secor, H. V.; Chavdarlan, C. G.; Sanders, E. 6.; Bassfield, R. L.; WhMby, J. F. J. Org. Chem. 1981, 4 6 , 3040. (48) Glenn, D. F.; Edwards, W. B., 111, J. Org. Chem. 1978, 43, 2860. (49) Cox, R. H.; Kao, J.; Secor, H. V.; Seeman, J. I. J. Mol. Struct. 1988, 740, 93. (50) Seeman, J. I.; Chavdarian, C. G.; Kornfeld, R. A,; Naworal, J. D. retrahedron 1985. 4 1 , 595. (51) Mattila, M.; Vartlalnen, A. Ann. Med. Exp. Biol. Fenn. 1964, 42, 27. (52) Abod, L. 0.; Reynolds, D. T.; Bldlack, J. M. Life Sci. 1980, 27, 1307. (53) Seeman, J. I. Synthesls 1977, 496. (54) Borch, R. F.; Bernstein, M. D.; Durst, H. D. J. Am. Chem. Soc.1971, 93, 2697. (55) Armstrong, D. W.; DeMond, W. J. Chromatogr. Sci. 1984, 22, 411. (56) Armstrong, D. W.; Alak, A.; BUI. K. H.; DeMond, W.; Ward, T.; Riehl, T. E.; Hinze, W. L. J. Incluslon Phenom. 1984, 2, 533. (57) Armstrong, D. W.; DeMond, W.; Czech, B. P.Anal. Chem. 1985, 57, 481. (56) Armstrong, D. W.; Ward, T. J.; Czech, A.; Czech, B. P.; Bartsch, R. A. J. Org. Chem. 1985, 5 0 , 5556. (59) Armstrong, D. W.; Ward, T. J.; Armstrong, R. D.; Beesley, T. E. Science (Washington,D . C . ) 1986, 232, 1132.
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(60) Hinze, W. L.; Armstrong, D. W. Anal. Left. 1980, 73, 1093. (61) Degowski, J.; Sybilska, D.; Juraczak, J. J. Chromatogr. 1982, 237, 303. (62) Sybllska, D.; Zukowskkl, J.; Bojarski, J. J. Lip.. Chromatogr. 1986, 9 , 591. (63) Seeman, J. I. Chem. Rev. 1983. 83, 83. (64) Seeman, J. I. J . Chem. Educ. 1986. 63, 42. (65) Seeman, J. I.; Whldby, J. F. J. Org. Chem. 1976. 47, 3624. (66) Love, R.; Cohen, R. 6.; Taft, R. W. J. Am. Chem. SOC. 1968, 90. 2455. (67) Perrln, D. D. Dissociation Constants of eganlc Bases In Aqueous Solutlon; Butterwmths: London, 1965. (66) Vlnogradov, S. N.; Linnell. R. H. Hvdrogen Bonding; Van Nostrand Reinhold: New York, 1971; p 14. (69) Boehm, R. E.; Martlre, D. E.; Armstrong, D. W. A m / . Chem. 1988, 60. 522.
RECEIVED for review August 17,1987. Resubmitted December 29, 1987. Accepted April 20,1988. Support of a portion (to D.W.A.) of this work by the Smokeless Tobacco Research Council, Inc. (Grant 0154), is gratefully acknowledged.
Cyclodextrin-Modified Solvent Extraction for Polynuclear Aromatic Hydrocarbons Lisa A. Blyshak, Thomas M. Rossi,l Gabor Patonay,2 a n d Isiah M. Warner*
Department of Chemistry, Emory University, Atlanta, Georgia 30322
The extractlon efflclencles of several polynuclear aromatlc hydrocarbons (PAHs) between Isopropyl ether/water and between Isopropyl ether:l-butanol (1:4)/water are measured In the presence of an aqueous ycyclodextrln (CDx) modHler at room temperature. The dlstrlbutlon of certaln PAHs into the aqueous phase Is Increased by the presence of M y-CDx. For compounds such as perylene and coronene, whlch show the most marked effects, the extractlon efflclencles into the aqueous phase from pure Isopropyl ether are 95.1 % and 93.7%, respectlvely, when the CDx modlfler Is used. I n the mlxed solvent system wlth 1-butanol, these values are 63.4% and 98.1 %, respectlvely. I n both systems, the Increased dlstrlbutlon Into water Is based In part on the slre relatlonshlp between the PAH and the CDx cavlty. I n the case of relatlvely small molecules llke anthracene, ilttle or no extractlon Is observed In the presence of the CDx modlfler. Thls type of extraction system may be useful for selectlve extractlon of large PAHs from mixtures. Extraction results for a varlety of PAHs are presented and dlscussed.
In recent years, the analytical utility of cyclodextrins has become increasingly evident. These compounds, which are cyclic oligosaccharides capable of forming inclusion complexes (1,2),have been evaluated for use in several analytical systems. The most commonly used oligosaccharides are a-,6-,and Present address: Smith, Kline & French Laboratories, Philadel hia, PA 19101. ‘!Present address: Department of Chemistry, Georgia State University, Atlanta, GA 30303. *Author t o whom correspondence should be addressed.
y-cyclodextrins that have approximate inner cavity diameters of 5.0, 7.8, and 9.5 A, respectively. The degree of complex formation between host and guest is closely related to the compatibility of the CDx cavity size with the size and steric arrangement of the potential guest and to the hydrophobicity of the potential guest (I). Since complex formation involves a stereoselective interaction that affords some measure of protection for the included species, these macrocycles have become useful in fluorescence and phosphorescence enhancement (3-5), stereoselective catalysis (6),and reversedphase chromatographic separations (7,8). In general, appreciable extraction of PAHs into the aqueous phase is not feasible because the solubilities of most PAHs in water are very low. In cases where extraction from a nonpolar organic phase to polar phase is desired, a polar organic solvent such as dimethyl sulfoxide (DMSO) is used to extract polycyclic aromatics from hydrocarbon solvents (9). Solvent extraction schemes requiring polar solvents such as these are simple and are useful for removing PAHs from organic matrices but are limited in their selective interactions with particular PAHs. The solvent extraction scheme described in this paper shows that the use of CDx as an aqueous phase modifier enhances the extraction of selected species into the aqueous layer while retaining other species in the bulk organic phase. The extraction efficiency is related to the cyclodextrih complexation and thus to the size and hydrophobicity of the compounds to be extracted. This method may be particularly useful for simplifying complex mixtures of organic material such as oil samples or air sample adsorbates, which are usually soluble in organic solvents. Such samples contain a variety of PAHs, which would make them amenable to separations by extraction into aqueous phase. The use of cyclodextrins would allow sim-
0003-2700/86/0360-2127$01.50/00 1988 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 19, OCTOBER 1, 19188
plification of such mixtures by extracting larger PAHs into the aqueous layer and leaving smaller components in the organic phase. Further extractions of the organic layer with cyclodextrins of varying size may make possible separations of smaller PAHs. A few studies on the use of cyclodextrins for extraction have been described (10, 11). In general, most extractions involve the use of low solvent volumes and require precipitation of the complex from aqueous solution. Then, the solid complex must be shaken with an organic solvent to remove the guest from the solid matrix. Although such extractions use minimal reagent volumes, the separation procedure is quite tedious. Patent work by Szejtli described the preparation of glass beads with covalently bonded CDx, which were used to extract organic materials from water (12). The work presented here describes the development of a straightforward solvent extraction scheme for PAHs using the complexation properties of cyclodextrins. In conventional solvent extraction, a solute is partitioned between two immiscible solvents. In this study, y-CDx is used as an aqueous phase modifier to increase the aqueous solubilities of several PAHs and thus to increase their aqueous phase extraction efficiencies. Only one study has been reported in which cyclodextrins have been used in a true solvent extraction (13). In that study, tri-0-methyl-P-cyclodextrin was used in the organic phase to increase the partitioning of p-nitrophenol into the organic layer. In most cases, the degree of extraction is related to the size and geometry of the potential guest in relation to the dimensions of the CDx cavity. Thus, the stereoselective behavior of the cyclodextrins gives them the potential to discriminate between organic phase solutes in solvent extraction. Following extraction, analytes of interest present in the aqueous phase may be studied without removal from the CDx cavity or may be back extracted into a suitable solvent such as cyclohexane. Clearly, the simplicity of this technique surpasses that of already existing CDx extraction methods. The scheme described here may enhance existing knowledge of CDx behavior in mixed solvent systems and may also have applications as a sample preparation technique for reversed-phase liquid chromatography. Modification of this technique to alter extraction efficiencies may be possible by controlling parameters such as solvent polarity and temperature. The effects of y-CDx modifier on the extraction efficiencies of several common PAHs are determined by using fluorescence analysis. The mechanism for the extraction and the degree of extraction observed for a series of compounds are described. In addition, the possibility of using this technique as a separation method for PAH mixtures is evaluated, and several examples of such extractions are provided.
EXPERIMENTAL SECTION Reagents. Perylene was obtained from Sigma Chemical Co. (St. Louis, MO). All other PAHs were supplied by Aldrich Chemical Co. (Milwaukee, WI) and were reported to contain less than 3% impurities. All PAHs were used without further purification. Isopropyl ether (99%) for extraction work was also purchased from Aldrich. Hydroquinone, a fluorescent stabilizer present in the ether, was removed prior to solution preparation by rotary evaporation of the ether. Fluorometric grade 1-butanol was supplied by Fisher Scientific Co. (Fair Lawn, NJ). All solutions for extractions of PAHs were prepared by evaporating portions of a stock cyclohexane solution and diluting to the appropriate volume with isopropyl ether or a 1:4 mixture of isopropyl ether:1-butanol. Fluorescence measurements were performed on 1:lO dilutions of the stock and final organic phase solutions. The effect of dissolved cyclodextrin on the fluorescence intensity of the organic phase PAH was minimized by dilution with original organic solvent. Cyclodextrin solution for extraction studies was prepared at M by dissolving solid y-CDx in a concentration of 1.0 x deionized water (Continental Water Systems, Atlanta, GA). Fresh
solution was prepared daily to prevent bacterial growth and CDx decomposition from interferingwith complexation and extraction. Cyclodextrin was purchased from Advanced Separation Technologies, Inc. (Whippany, NJ), and was used as received. Solid CDx from one lot number was used for all extractions. The polarity of the CDx cavity and of the organic solvents used was examined by using pyrene as a fluorescence probe. This method is useful for estimating the cavity polarity of CDx’s, as indicated by Street (14). The relative peak intensities of the pyrene fluorescence vibronic bands were used to estimate the polarity of the microenvironments of interest. The measurements made were compared to literature values that relate peak ratios to solvent polarity (15). Samples for studies of CDx effecta on fluorescence enhancement in organic solution were prepared by taking an aliquot of a pyrene stock solution in cyclohexane and evaporating the cyclohexane under a nitrogen purge. Samples were redissolved in a 1:4 mixture of isopropyl ether:1-butanol,which was saturated with aqueous CDx solution. Samples of the same concentration were also prepared in which the organic solvent was not saturated with CDx solution. Fluorescence measurements were made on dilutions of these solutions. The solvent used to make up the final volume for dilutions was pure organic solvent. This procedure was followed in order to mimic the conditions used for the preparation of extraction samples for fluorescence analysis. Peak areas of the resulting fluorescence from both saturated and unsaturated samples were compared to determine the effect of CDx on PAH fluorescence in the organic phase. . Extraction Procedure and Apparatus. Extractions were performed by vigorously shaking equal volumes of aqueous M y-CDx and M PAH solutions for 2 min at room temperature and allowing the phases to separate. The two layers were separated in a separatory funnel, and the aqueous layer was discarded. Since fluorescence enhancement is often observed for luminophores in aqueous CDx media (3, 16-18), fluorescence measurements were made on the organic portion of the sample. Fluorescence in organic solvents is not expected to be strongly affected by CDx because of the generally low solubility of the oligosaccharides in these solvents (I). Extractions were also performed in the absence of modifier to examine the degree of extraction occurring in the absence of CDx. In addition,an organic phase volume change was noted for some extractions. These changes are reflected in the data calculations. To ensure that the concentration was linear with total fluorescence, dilutions of M organic phase solution and the organic phase the initial aftet extraction were made for all measurements to bring the concentration to lo4 M. The solvent used for dilution was the initial organic solvent. This dilution procedure diminishes any potential effect of cyclodexhin on fluorescence intensitiesof PAHs. Extraction efficiencies were evaluated by monitoring the fluorescence of the diluted organic samples. Since the peak area of fluorescence is proportional to concentration, these peak areas were substituted into the following equation for the distribution ratio:
where [PI, is the fiial PAH concentration in the aqueous phase and [PI,, is the final PAH concentration in the organic phase. Extraction efficiencies reported here were determined according to the equation E = lOOD/(D
+ V,/V,)
(2)
where D is the distribution ratio, V,/ V , is the volume ratio of the organic and aqueous phases, and E is the percent extracted into the aqueous phase. All samples were monitored with a Perkin-Elmer 650-10s fluorescence spectrophotometer. Fluorescence excitation and emission wavelengths for the PAHs in this study are provided in Tables I and 11. The resulting spectra were analyzed with an Apple 11+ computer by integrating peak areas to determine total changes in fluorescence. Fluorescence peak areas were determined because unlike single wavelength monitoring, total fluorescence peak areas are insensitive to peak shifts and peak ratio changes and thus more accurately reflect changes in a n a l e concentration. Since the fluorescence spectra for the components of the mixtures
ANALYTICAL CHEMISTRY, VOL. 60, NO. 19, OCTOBER 1, 1988
Table I. Instrument Parameters and Extraction Efficiencies (%) for Several PAHs in the Presence of y-Cyclodextrin Modifier excitation E from 1:4 waveemission E from isopropyl length," range, isopropyl ether: nm nmo ether, % 1-butanol, %
compd PVene benzo[e]pyrene perylene benzo[ghi]perylene coronene a
335 335
350-480 38.2 f 1.9 350-480 b
34.3 f 1.9
360 360
420-560 95.1 f 2.6 380-500 b
63.4 f 2.1 91.9 f 1.4
340
390-530 93.7 f 4.0
98.1 f 2.3
c
5-nm slits used. *Not measured. e See Table 11.
pasessed overlapping emission in some portions of each spectrum,
quantitation was performed with fluorescence intensity measurements in regions of sole emission for each compound in the mixture. These intensity values were compared to calibration curves also based on fluorescence intensity at the wavelength chosen for sole emission. The mixtures evaluated were anthracenelperylene, pyrenelperylene, and pyrene/coronene. The wavelengths chosen as regions of sole emission for these mixture studies were 385 nm for anthracene, 375 nm for pyrene, 475 nm for perylene, and 480 nm for coronene. Two-dimensional spectra of PAH mixtures were obtained by using a vidicon-based video fluorometer, which provides multiple emission spectra as a function of multiple excitation wavelengths (19).
RESULTS AND DISCUSSION One problem encountered in these studies was the choice of an organic solvent. Many of the common organic solvents form insoluble complexes with cyclodextrins, and thus, solvent choice was crucial in designing the CDx solvent extraction system. In preliminary studies, solvents such as cyclohexane and chloroform formed such stable complexes with y-CDx that upon shaking, the solvent42Dx complex precipitated from the aqueous phase with a corresponding decrease in the organic phase volume. When a PAH was present in these systems, it was completely extracted from the organic phase in the form of a solid ternary complex. This method, however, is not true solvent extraction, since a solid species forms during the separation. To avoid this problem, a small solvent molecule incapable of forming a 1:l complex was used. In these studies, isopropyl ether was used as the organic solvent. The ethers are known for their ability to form ternary complexes with CDx and another molecule; yet, in the absence of the other guest they do not complex well with CDx ( I ) . A mixture of isopropyl ether and 1-butanol was also used as the organic solvent to evaluate the effects of the organic phase solvent polarity on extraction efficiency. Although the solvent system chosen for these studies is an unusual one, it possesses some properties that are helpful in promoting the selective extraction observed for the PAHs. Since the organic solvent chosen has some degree of solubility in the aqueous phase, some organic solvent molecules can
reside in the CDx cavity. When a bulky molecule is extracted, it completely replaces the solvent in the cavity, and this results in an environment of different polarity for the PAH. If the included species does not completely fill the cavity, organic solvent molecules may remain in the cavity with the PAH to form a ternary complex. In such instances, the environment experienced by the PAH in the cavity is very similar to that in the bulk organic solvent. In such cases, the PAH is not extracted into the aqueous phase in the form of a cyclodextrin complex because the drive for complexation is not strong. Since the extraction efficiency for each PAH was determined by comparing the relative fluorescence values for the solution before and after aqueous CDx extraction, it was important to evaluate the effect of CDx on the fluorescence of PAHs in the organic solvent. Pyrene was chosen for these studies since it has an unusually long lifetime, which allows for changes in the fluorescence microenvironment to be easily observed (15). Based on peak area determinations, the ratio of total fluorescence in the presence of CDx to the total fluorescence in the absence of CDx was 1.006. This ratio suggests that the effect of CDx on the total fluorescence of the diluted solution is minimal and that a direct comparison of the fluorescence peak areas can be made. The extraction efficiencies for PAHs that extracted into aqueous CDx solution are given in Table I. Values for percentage of the PAH remaining mostly in the organic phase after extraction with CDx solution are shown in Table 11. The values are reported in this manner to reflect the fact that some of the PAHs did not possess appreciable extraction efficiencies. The reproducibility of each measurement is reflected in standard deviation units. Extraction was not observed in the absence of CDx. The compounds that extracted to a reasonable degree from the mixed solvent system were benzo[elpyrene, perylene, benzo[ghi]perylene, and coronene with respective extraction efficiencies of 34.3%, 63.4%, 91.9%, and 98.1%. In the isopropyl ether system, coronene, perylene, and pyrene possessed reasonable extraction efficiencies of 93.7 % , 95.1%, and 38.2%. Anthracene did not show an appreciable extraction efficiency in either solvent system. Relating this information to the relative dimensions of each compound given in Table I11 indicates that the extraction procedure is related to the bulkiness of the PAH and the tightness of its fit in the CDx cavity. The smaller molecules do not extract appreciably when aqueous y-CDx modifier is used. Anthracene is too small and tapered to adequately fill the cavity. About 97% of the anthracene remained in the mixed organic phase after extraction with CDx. In the pure isopropyl ether system, roughly 100% of the anthracene remained in the organic layer. Even compounds larger than anthracene such as 9-methylanthracene did not extract because they are more tapered and do not fill the cavity well. The data suggests that the compound needs to form a strong complex with CDx in order to provide reasonable complexation and transfer to the aqueous phase. This specific interaction may result from the possibility of ternary complex formation between small PAHs, organic solvent
Table 11. Instrument Parameters and PAH (%) Remaining in Organic Layer following Extraction with Aqueous y-Cyclodextrin % PAH remaining in 1:4
compd
excitation wavelength," nm
anthracene 340 9-methylanthracene 360 9-phenylanthracene 360 pyrene 335 a 5-nm slits used. *Not measured. See Table I.
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emission range," nm
% PAH remaining
360-500 370-520
100 f 2.5
in isopropyl ether
370-520
b b
350-480
C
isopropyl ether:butanol 97.1 f 97.4 f 96.9 f 91.1 f
1.5 2.6 1.1 3.3
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ANALYTICAL CHEMISTRY, VOL. 60, NO.
19,OCTOBER 1, 1988
Table 111. Estimateda Molecular Dimensions
Table V. Extraction Efficiencies (%) of PAH Mixtures
compound
dimensions, A*
anthracene 9-methylanthracene 9-phenylanthracene pyrene benzo[e]pyrene perylene benzo[ghi]perylene coronene
5.0 X 9.2
6.2 9.7 7.1
9.2 9.1 9.2 9.2
X X X X X X X
mixture E, % component 1 component 2 component 1 component 2
9.2 9.2
8.9 9.2
anthracene
perylene
pyrene
perylene coronene
pyrene
0.0 f 3.1 40.9 f 4.2 42.3 f 8.4
89.6 f 5.4 93.5 f 3.1 92.3 f 0.9
9.2
9.2
a
9.2
Estimated by using bond lengths. Table IV. Relative Peak Ratios of Pyrene Fluorescence in Various Solvents solvent
I
water y -cyclodextrin isopropyl ether mixed solvent
1.000
1.000 1.000 1.000
Deak number I11 0.634 0.974 1.030 0.904
3a
V 0.854
1.050 0.911 0.881
b
m
molecules, and CDx. Formation of ternary complexes changes the polarity of the CDx cavity to one that is less favorable for complexation. Molecules such as the more tapered ones studied here may be amenable to extraction by using the smaller a- and P-cyclodextrins, provided a suitable solvent system is used. Large, bulky guest molecules can form strong complexes with the cyclodextrin cavity, and this results in appreciable extraction efficiencies. Coronene, benzo[ghi]perylene, benzo[e]pyrene, and perylene are capable of fitting tightly in the cavity because their dimensions closely approximate the 9.5-A cavity diameter of y-CDx. The likelihood of ternary complex formation is decreased in this system because less room is available in the cavity for solvent molecules. Thus, larger PAHs possess larger formation constants and better extraction efficiencies than those of small molecules. In addition to the required geometry of the guest, the solvent choice also has an effect on the degree of extraction observed. As previously noted, ternary complex formation may, in part, influence the type of complexation and the degree of extraction observed for different sized PAHs. In addition, the solvent polarity also affects the possible interactions between the PAHs and the CDx cavity. I t is wellknown that formation of CDx complexes is related to the polarity changes experienced by the guest species (2). In this system, relative differences do exist between the polarities of the bulk organic solvent and the CDx cavity interior. The environments of the y-CDx cavity and of the organic solvents used in this study were examined by using pyrene as a probe to determine the polarity of the microenvironments (15). These results are provided in Table IV. The polarity is evaluated by examining the III/I peak ratios. This ratio increases when a decrease in the solvent polarity occurs. Pyrene experiences a slightly nonpolar environment when included in y-CDx. The mixed solvent system has a polarity very close to the CDx polarity, but the peak ratios in the presence of pure isopropyl ether indicate a much more nonpolar environment. If extraction efficiency is based partly on the relative change in polarity experienced by the PAH, the pure solvent system would be expected to offer greater extraction possibilities for PAHs. Inspection of the data suggests that this is true. With pyrene, the extraction efficiency decreases as the solvent polarity is increased with l-butanol. Perylene shows the same trend, with extraction efficiencies of 95.1% in isopropyl ether and 63.4% in the mixed solvent.
Flgure 1. Total luminescence spectra of a mixture of anthracene and perylene (a) prior to extraction and (b) following extraction with lo-' M y-cyclodextrin.
Thus, higher extraction efficiencies are observed when the polarity difference between the organic solvent and the cavity is the greatest. The utility of CDx solvent extraction for separating mixtures was evaluated by measuring the extraction efficiencies of several simple PAH mixtures. Several binary mixtures were extracted and quantified by using y-CDx modifier to see if this method would be useful for separations. The results of mixture analyses in isopropyl ether are shown in Table V. Of the three mixtures extracted, only one resulted in a relatively good separation of two PAHs. The anthracene/perylene mixture was easily separated, since anthracene does not extract into aqueous y-CDx solution while perylene possesses a reasonable extraction efficiency. Figure l depicts the total luminescence spectra of an anthracene-perylene mixture before and after extraction with aqueous low2M y-CDx. For those mixtures in which both species had a reasonable extraction into the modified aqueous phase, separation of the two compounds was not possible. Since excess CDx was present, the extraction of one compound did not interfere with the extraction of the other. Thus, PAH mixtures in which both compounds can extract well cannot be separated from one another effectively unless the CDx concentration is altered so that excess CDx would not be available for complexation of all species. Several modifications, if properly used, may allow the experimenter to control and alter the extraction efficiency of some PAHs. Since temperature affects the stability of some CDx complexes (I), lowering the temperature a t which the extraction is performed may increase the distribution of certain PAHs into the CDx phase. The control of solvent polarity may also be potentially useful for altering the equilibria in a solvent extraction scheme for PAHs. These and other studies on the utility of cyclodextrins as modifiers in solvent extraction are currently in progress.
Anal. Chem. 1900, 60, 2131-2134
CONCLUSIONS
Cline-Love, L. J.; Grayeski, M. L.; Noroski, J.; Weinberger, R. Anal. Chim. Acta 1985, 170, 3-12. Breslow, R.; Kohn, U.; Siegel, B. Tetrahedron Left. 1978. 1645-1646. Beesley. T. E. Am. Lab. (FaitfieM, Conn.) 1985, May, 78-87. Smolkova-Keulemansova, E. J . Chromatogr . 1982, 25 1 , 17-34. Natusch, D. F. S.; Tomkins, B. A. Anal. Chem. 1978, 50, 1429-1434. Matsunaga, K.; Imanaka, M.; Ishlda, T.; Oda, T. Anal. Chem. 1984, 56, 1980-1982. Harangi, J.; Nanasi, P. Anal. Chim. Acta 1984, 156, 103-109. Szejtli, J. German Patent 2927733, 1980; Chem. Abstr. 1980, 9 2 , 181902. Nakai, Y.; Yamamoto, K.; Terada, K.; Horibe, H. Chem. Pharm. Bull. 1982, 30(5), 1796-1802. Street, K. W. J. Liq. Chromatogr. 1987, 10(4), 655-662. Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. SOC. 1977, 9 9 , 2039-2044. Singh, H.; Hinze, W. L. Analyst(London) 1982, 107, 1073-1080. de Moreno, M. R.; Smith, R . V. Anal. Lett. 1983, 16(A20), 1633-1 .. - - .645. .... Hamai, S. Bull. Chem. SOC.Jpn. 1982, 5 5 , 2721-2729. Warner, I. M.; Fogarty, M. P.; Shelly, D. C. Anal. Chim. Acta 1979, 109. 361-372.
The aqueous phase modifier y-CDx has been studied for its utility in separating PAH mixtures by solvent extraction. Preliminary results suggest that this technique has potential use for separating PAHs on the basis of molecular dimensions. If specific modifications are made, the possibility of separating mixtures may be enhanced. The best use for this system may be as a method for simplifying complex mixtures by partitioning groups of compounds between the organic and aqueous phases. Many applications for CDx-modified extraction may be found in the motor industry, in air sampling analysis, and in many types of industrial products that contain PAHs as primary components. Also, combining the extraction technique described here with data analysis techniques such as pattern recognition may allow for complete identification of all species present in complex mixtures.
LITERATURE CITED Szejtii. J. Cyclodextrins and Their Inclusion Complexes ; Akademiai Kiado: Budapest, 1982. Saenger, W. Angew. Chem., Int. Ed. Engl. 1980, 19, 344-362. Juks, 0.; Scypinski, S.; Cline-Love, L. J. Anal. Chim. Acta 1985, 169, 355-360. ... ... Scypinski, S.; Cline-Love, L. J. Anal. Chem. 1984, 5 6 , 322-327.
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RECEIVED for review March 14,1988. Accepted May 24, 1988. This work was supported in part by the National Science Foundation (CHE-8609372) and the Office of Naval Research. Isiah M. Warner acknowledges support from an NSF Presidential Young Investigator Award (CHE-8351675).
Separation of Deuteriated Isotopomers of Dopamine by Ion-Pair Reversed-Phase High-Performance Liquid Chromatography Carolyn F. Masters, Sanford P. Markey, and Ivan N. Mefford* Laboratory of Clinical Science, National Institute of Mental Health, Building 10, Room 2046, 9000 Rockville Pike, Bethesda, Maryland 20892
Mark W. Duncan Intramural Research Program, National Institute of Neurological and Communicative Disorders and Stroke, 9000 Rockville Pike, Bethesda; Maryland 20892
The ion-pair reversed-phase separation of dopamine and deuterium-substituted dopamine isotopomers is described. Chromatographlc parameters and deuterium isotope effects governlng the resolutlon are examined and compared to the factors reguiatlng the resolution of the chemically distinct entities dopamine, noreplnephrine, and epinephrine. The potential utility of the ['H,]dopamine isotopomer as an internal standard for the high-performance liquid chromatography analysis of dopamine is demonstrated by using aluminum oxide extraction prior to chromatographic Separation.
Stable isotope analogues have been extensively employed in the biological sciences. Quantitative methodologies based on mass spectrometric analysis have taken advantage of the fact that isotopomers are ideal mimics for the chemical behavior of a compound, better than either homologues or structural analogues, and the isotopomers can be distinguished and independently quantified. However, analysis methods incapable of mass discrimination require chromatographic separation for general application of stable isotopes. Whereas
* A u t h o r t o w h o m correspondence should b e addressed.
separation of deuterium-substituted compounds from their protium isotopomers has been possible by using gas chromatographic techniques for many years ( 1 , 2 ) , liquid chromatographic approaches have not commonly been used for this purpose. With the advent of high-resolution columns packed with hydrophobic stationary phases, it has been demonstrated that such separations are possible (3-6). The successful liquid chromatographic separation of deuteriated isotopomers makes possible their routine use as internal standards without introducing the need for mass specific detection or the application of radiolabeled isotopes. The ability to resolve deuteriated isotopomers of neurochemically active analogues would also make it possible to perform tracer and turnover studies in animals and humans without the safety hazard associated with the use of radioactive isotopes. Previously published separations of biochemically relevant isotopomers have generally not been useful for routine analyses for several reasons. Near complete resolution has been accomplished only for fully deuteriated molecules, some with more than 30 hydrogens exchanged (3-6). Separations of partially deuteriated compounds have only been accomplished on very long microbore columns (4.5 m X 1 mm i.d.) (6). These separations require exceedingly long analysis times as the flow rate must be kept low in order to maintain acceptable column back pressure (3, 4 , 6). Practical separation of carotenoid
This article not subject to U S . Copyright. Published 1988 by the American Chemical Society
