Anal. Chem. 1997, 69, 1223-1229
Separation of Polycyclic Aromatic Hydrocarbons by Nonaqueous Capillary Electrophoresis Using Charge-Transfer Complexation with Planar Organic Cations Joseph L. Miller,† Morteza G. Khaledi,*,† and Damian Shea‡
Department of Chemistry, North Carolina State University, Box 8204, Raleigh, North Carolina 27695, and Department of Toxicology, North Carolina State University, Box 8604, Raleigh, North Carolina 27695
In this study, we examine the use of charge-transfer interactions between polycyclic aromatic hydrocarbons (PAHs) and planar organic cations in nonaqueous capillary electrophoresis. Since the separations are performed in a purely nonaqueous medium, this method also facilitates the analysis of solutes that have low solubilities in aqueous or mixed media. Presented in this study are the separations of PAHs as well as the quantitative structuremigration relationships that assisted in achieving a better understanding of the forces through which the PAH molecules interact with the acceptor cation. It was found that, in addition to charge-transfer interactions, electrostatic and dispersive forces play an important role in PAH-cation binding. In recent years, capillary electrophoresis (CE) has undergone rapid development as a high-efficiency chemical separations technique.1,2 In addition to separating charged solutes, CE-based mechanisms have been used successfully in the separation of uncharged compounds. Because of their inability to migrate on their own in an electric field, these uncharged compounds must interact with a charged carrier added to the separation buffer. Polycyclic aromatic hydrocarbons (PAHs) constitute a major class of uncharged compounds. Due to their abundance in the environment and the adverse health effects to which they have been linked, these compounds have been the focus of considerable attention in a number of areas including chemical separations.3 The purpose of this study is to evaluate a new CE method that may be suitable for the separation of PAHs and other hydrophobic compounds. Several different techniques have been employed to induce the selective interaction of PAHs with charged additives. The most common technique has been micellar electrokinetic chromatography (MEKC),4 in which separation occurs based on the partitioning of solutes into a charged micelle. Both cationic and anionic micelles have been used to separate PAHs,5 but in many cases, especially those involving micelles containing highly hydrophobic cores, binding to the micelle is often so strong that †
Department of Chemistry. Department of Toxicology. (1) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298. (2) St.Claire, R. L., III Anal. Chem. 1996, 68, 569R. (3) Vo-Dinh, T., Ed. Chemical Analysis of Polycyclic Aromatic Compounds; Wiley and Sons: New York, 1989. ‡
S0003-2700(96)00734-2 CCC: $14.00
© 1997 American Chemical Society
it is difficult to achieve any degree of selectivity using the micelle alone. As a result, a modifier must be added to adjust the degree of interaction between the solute and micelle. The separation of PAHs has been successfully achieved using this approach with a surfactant such as sodium dodecyl sulfate along with such modifiers as cyclodextrins,6-12 urea,8,10 and organic solvents.8,13,14 More recently, it was shown that charged cyclodextrin derivatives could be used in the absence of micelles to separate hydrophobic solutes including PAHs.15,16 In this case, the solutes interact with the cyclodextrin molecules solely through the formation of inclusion complexes, and high-efficiency separations are obtained. The utility of micelles in electrokinetic chromatography (EKC) is limited by the instability of the micelle in the presence of large amounts of organic modifier. Nonaqueous and mixed media, nonetheless, appear to be promising alternatives to the use of aqueous buffers in separating hydrophobic compounds because of the increased solubility of such compounds in organic solvents. This results in improved detectability as solutions of greater concentration can be analyzed. Other advantages of using nonaqueous and mixed media in EKC separations center around the wide variety of physical and chemical characteristics that organic solvents possess.17 For example, as compared to aqueousmedia, greater electric field strengths and/or higher ionic strengths can be used with certain organic solvents. This generally yields higher efficiencies and increased sample-size capacity. Also, the wide range of dielectric and autoprotolytic (4) Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111. (5) Burton, D. E.; Sepaniak, M. J.; Maskarinec, M. P. J. Chromatogr. Sci. 1987, 25, 514. (6) Guttman, A.; Paulus, A.; Cohen, A. S.; Grinberg, N.; Karger, B. L. J. Chromatogr. 1988, 448, 41. (7) Snopek, J.; Jelinek, I.; Smolkova-Keulemansova, E. J. Chromatogr. 1988, 452, 571. (8) Terabe, S.; Miyashita, Y.; Shibata, O.; Barnhart, E. R.; Alexander, L. R.; Patterson, D. G.; Karger, B. L.; Hosoya, K.; Tanaka, N. J. Chromatogr. 1990, 516, 23. (9) Yik, Y. F.; Ong, C. P.; Khoo, S. B.; Lee, H. K.; Li, S. F. Y. Environ. Monit. Assess. 1991, 19, 73. (10) Nishi, H.; Matsuo, M. J. Liq. Chromatogr. 1991, 14, 973. (11) Copper, C. L.; Sepaniak, M. J. Anal. Chem. 1994, 66, 147. (12) Sepaniak, M. J.; Copper, C. L.; Whitaker, K. W.; Anigbogn, V. C. Anal. Chem. 1995, 67, 2037. (13) Janini, G. M.; Issaq, H. J. J. Liq. Chromatogr. 1992, 15, 927. (14) Cole, R. O.; Sepaniak, M. J.; Hinze, W. L.; Gorse, J.; Oldiges, K. J. Chromatogr. 1991, 557, 113. (15) Szolar, O. H. J.; Brown, R. S.; Luong, J. H. T. Anal. Chem. 1995, 67, 3004. (16) Brown, R. S.; Luong, J. H. T.; Szolar, O. H. J.; Halasz, A.; Hawari, J. Anal. Chem. 1996, 68, 287. (17) Sahota, R. S.; Khaledi, M. G. Anal. Chem. 1994, 66, 1141.
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behavior among organic solvents results in different acid-base chemistries and allows for greater versatility in dealing with solutes containing ionizable functional groups. The possibility of using nonaqueous and mixed media in the separation of PAHs by EKC has led to the investigation of other charged additives that are more compatible with organic solvents. One approach to the separation of hydrophobic solutes using EKC in nonaqueous and organic-rich media involves the use of unimicelles and charged polymers.18-21 The ionic triblock copolymer of poly(methyl methacrylate/ethyl acrylate/methacrylic acid) (Elvacite 2669) has been used in separating several hydrophobic solutes including PAHs.18 Other classes of organicsoluble polymers include polymerized micelles and starburst dendrimers,19,20 which were also employed in the separation of PAHs and other hydrophobic solutes.19-21 Another example of the use of nonaqueous and mixed media to separate hydrophobic solutes by CE was demonstrated by Walbroehl and Jorgenson, who studied the effects of a tetraalkylammonium cation on the electrophoretic mobilities of several neutral solutes.22 They found that separation occurred according to the degree to which an effective charge could be placed on each of the solutes. While this phenomenon was attributed to solvophobic interactions between the charged and uncharged species, it appears that the positively charged center of the tetraalkylammonium cation is primarily responsible for these interactions. This is supported by the observation that sodium dodecyl sulfate monomer, an ion of opposite charge, could not be used to separate a mixture of PAHs. In this case, all of the solutes eluted with the electroosmotic flow (EOF).23 The apparent affinity of certain solutes toward the tetraalkylammonium ion suggests that similar separation behavior occurs when these solutes are subjected to other cations. For example, the silver ion, through argentation (a type of charge-transfer complexation), was recently employed in the successful separation of nine sulfonamides.24 This separation, however, was achieved using only a small amount of organic modifier (15% acetonitrile). In separating highly hydrophobic solutes, it would be beneficial to use a cation capable of binding solutes in purely nonaqueous media. In such media, planar organic cations are known to form “charge-transfer” complexes with a number of uncharged compounds including PAHs.25 While the use of planar cations as stationary phase sorbents in liquid chromatography has been reported,26 there are no reports on the use of these cations in the area of CE separations. The tropylium ion (I) and the 2,4,6-triphenylpyrylium ion (II) are examples of cations whose charge-transfer properties have been documented,27,28 and these ions were selected to demonstrate (18) Yang, S.; Bumgarner, J. G.; Khaledi, M. G. J. High Resolut. Chromatogr. 1995, 18, 443. (19) Palmer, C. P.; Khaled, M. Y.; McNair, H. M. J. High Resolut. Chromatogr. 1992, 15, 756. (20) Tanaka, N.; Tetsuya, T.; Hosoya, K.; Kimata, K.; Araki, T.; Terabe, S. Chem. Lett. 1992, 959. (21) Tanaka, N.; Fukutome, T.; Hosoya, K.; Kimata, K.; Araki, T. J. Chromatogr. A, 1995, 716, 57. (22) Walbroehl, Y.; Jorgenson, J. G. Anal. Chem. 1986, 58, 479. (23) Ahuja, E. S.; Foley, J. P. J. Chromatogr. 1994, 680, 73. (24) Wright, P. B.; Dorsey, J. G. Anal. Chem. 1996, 68, 415. (25) Kampar, V. E. Russ. Chem. Rev. 1982, 51, 107. (26) Egly, J.-M.; Porath, J. J. Chromatogr. 1979, 168, 35. (27) Feldman, M.; Winstein, S. J. Am. Chem. Soc. 1961, 83, 3338. (28) Feldman, M.; Winstein, S. Tetrahedron Lett. 1962, 19, 853.
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the feasibility of using charge-transfer interactions to achieve CE separations of PAHs in nonaqueous media.
EXPERIMENTAL SECTION Apparatus. All separations were performed on a Beckman P/ACE System 2200 instrument (Fullerton, CA). The system was comprised of a 0-30 kV high-voltage built-in power supply, a selectable fixed-wavelength UV detector, and the GOLD software for system control and data handling. The capillaries were obtained from Polymicro Technologies (Tucson, AZ) and had an internal diameter of 53 µm and an outer diameter of 365 µm. The total length of the capillary ranged from 57 (50 cm effective length) to 97 cm (90 cm effective length). The temperature was controlled using a fluorocarbon-based cooling fluid obtained from 3M Industrial Chemical Products (St. Paul, MN). All experiments were performed at room temperature. The electropherograms were collected and processed on an IBM PS/2 Model 40 SX computer. Reagents and Chemicals. HPLC-grade acetonitrile was obtained from J. T. Baker (Phillipsburg, NJ), tropylium tetrafluoroborate and 2,4,6-triphenylpyrylium tetrafluoroborate were obtained from Aldrich Chemical (Milwaukee, WI), tetrahexylammonium perchlorate was obtained from Johnson Matthey (Ward Hill, MA), the PAHs were obtained from Aldrich Chemical and Supelco, Inc. (Bellefonte, PA), and formamide was obtained from Fluka Chemika-Biochemika (Buchs, Switzerland). Procedure. The run buffer was prepared by dissolving the appropriate amount of the salt in acetonitrile and filtering through a 0.45 µm Gelman nylon aerodisk filter. Concentrated stock solutions of individual PAHs were prepared in acetonitrile. The injected samples were prepared by transferring a measured aliquot of the stock solution containing the desired solute into an autosampler vial, adding formamide (the EOF marker) at a final concentration of 1% by volume, and diluting with acetonitrile. Prior to each run, the capillary was equilibrated with the run buffer for several minutes. All samples were injected for 2 s using the pressure injection mode. The applied voltage was 30 kV and was unchanged for the duration of each run; this yielded currents up to 60 µA. The absorbance detector wavelength was set at 254 nm. RESULTS AND DISCUSSION Figures 1 and 2 show the electrophoretic separation of 20 PAHs using the 2,4,6-triphenylpyrylium and tropylium ions. From these electropherograms, we observed that the PAHs separated more
Figure 3. Separation of perylene, benzo[a]pyrene, and dibenz[a,h]anthracene using an 80:20 2,4,6-triphenylpyrylium/tropylium ion solution (40 mM total concentration in acetonitrile).
Figure 1. Blank-subtracted electropherogram showing the separation of PAHs using 40 mM 2,4,6-triphenylpyrylium tetrafluoroborate in acetonitrile with a capillary length of 77 cm (70 cm effective length). 30 kV f 35 µA. Solute identification: (1) coronene, (2) naphtho[2,3a]pyrene, (3) benzo[ghi]perylene, (4) indeno[1,2,3-cd]pyrene, (5) perylene, (6) benzo[a]pyrene, (7) dibenz[ah]anthracene, (8) benzo[b]fluoranthene, (9) benzo[k]fluoranthene, (10) benz[a]anthracene, (11) chrysene, (12) pyrene, (13) fluoranthene, (14) naphthacene, (15) anthracene, (16) phenanthrene, (17) acenaphthene, (18) acenaphthylene, (19) fluorene, (20) naphthalene, (21) formamide.
Figure 2. Electropherogram showing the separation of PAHs using 30 mM tropylium tetrafluoroborate in acetonitrile with a capillary length of 67 cm (60 cm effective length). 30 kV f 44 µA. (See Figure 1 for identification of solutes.)
or less according to size, with the largest PAHs eluting first, followed by the medium-sized PAHs, the smaller PAHs, and finally the EOF marker (formamide). This indicated that the largest PAHs had the greatest interaction with the cation and, in effect, became the most positively charged of the analytes (thus gaining the greatest electrophoretic mobility). Although each cation was capable of separating many of the PAHs, there were several PAHs
that coeluted, even under optimal conditions. In certain cases, however, a PAH could be isolated using a mixture of the two cations (whereas coelution occurred when either cation was used alone). For example, benzo[a]pyrene coeluted with dibenz[a,h]anthracene when the triphenylpyrylium ion was used, while in a system containing only the tropylium ion, it coeluted with perylene. However, when an 80:20 mixture of triphenylpyrylium/ tropylium was used, the three PAHs were completely separated, as Figure 3 shows. Not all of the PAHs could be isolated in this manner, though, as there were two PAH pairssbenz[a]anthracene/ chrysene and anthracene/phenanthrenesthat were never completely resolved by either cation (or any combination thereof). As Table 1 shows, the average peak efficiency obtained using the triphenylpyrylium ion (194 000 theoretical plates) exceeded that obtained with the tropylium ion (128 000 theoretical plates). In addition to providing greater efficiencies, the triphenylpyrylium ion also formed stronger complexes with the PAHs than did the tropylium ion; this led to greater selectivity (and thus better resolution). On the other hand, the analyte signal-to-noise ratio was greater for the tropylium ion than for the triphenylpyrylium ion due to less background absorbance at 254 nm. By comparison, Figure 4 shows the separation of PAHs that was obtained using 25 mM tetrahexylammonium perchlorate in 50:50 acetonitrile/water (the same run buffer used by Walbroehl and Jorgenson22). The PAHs eluted in roughly the same order as with the planar cations, but despite higher signal-to-noise ratios and comparable mobilities, poorer separations were obtained. Choice of Solvent. Three major factors were considered in selecting a solvent in which to perform the CE separation of PAHs using an organic cation as the charged carrier. The first of these factors was solubility; it was found that these cations were soluble at concentrations up to 50 mM in methanol, acetonitrile, acetone, formamide, dimethylformamide, methylene chloride, and chloroform, but insoluble in tetrahydrofuran, dioxane, ethanol, carbon tetrachloride, and other less-polar solvents. The second factor taken into consideration was the apparent reactivity of the cations with some of the solvents in which they were dissolved. When a small amount of tropylium tetrafluoroborate was added to a protic solvent, for example, precipitation was observed over the course of several hours. A probable cause for this behavior lies in the ability of the tropylium ion to act as a weak acid in water according Analytical Chemistry, Vol. 69, No. 6, March 15, 1997
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Table 1. Electrophoretic Mobility of PAH-Cation Complexes as a Function of Various Molecular Properties 2,4,6-triphenylpyrylium ion (30 mM in ACN)
tropylium ion (30 mM in ACN)
PAH
no. of fused rings
molecular polarizability (Å3)44
ionization potential (eV)45
µep (cm2 kV-1 min-1)
theor plates
µep (cm2 kV-1 min-1)
theor plates
naphthalene acenaphthylene acenaphthene fluorene anthracene phenanthrene fluoranthene pyrene benz[a]anthracene chrysene naphthacene benzo[b]fluoranthene benzo[k]fluoranthene perylene benzo[a]pyrene dibenz[a,h]anthracene indeno[123-cd]pyrene benzo[ghi]perylene naphtho[2,3-a]pyrene coronene
2 3 3 3 3 3 4 4 4 4 4 5 5 5 5 5 6 6 6 7
17.70 20.41 20.60 21.15 24.97 24.97 27.68 28.77 32.24 32.24 32.24 34.95 34.95 36.04 36.04 39.51 38.74 39.83 43.31 43.62
8.575 8.778 8.525 8.609 8.049 8.479 8.466 8.029 8.111 8.261 7.722 8.410 8.167 7.846 7.830 8.148 8.024 7.943 7.689 8.077
1.19 1.66 1.66 1.64 2.34 2.26 3.36 3.60 4.23 4.18 3.11 6.08 5.83 7.20 6.83 6.73 8.77 9.61 10.22 12.28
150 000 a a a a a 180 000 150 000 a a 200 000 210 000 230 000 210 000 a a 180 000 250 000 190 000 180 000
0.55 1.30 1.04 0.69 0.95 0.94 1.57 1.98 1.26 1.26 1.38 1.70 1.67 2.16 2.15 1.50 2.59 2.70 2.49 3.15
104 000 a a 100 000 95 000 a a 121 000 a 140 000 a a a a 139 000 a 155 000 153 000 a 142 000
a
Not measured.
whereas in methanol, the 2-methoxypyran forms
Figure 4. Electropherogram showing the separation of PAHs using 25 mM tetrahexylammonium perchlorate in 1:1 water/acetonitrile. The capillary length was 97 cm (90 cm effective length), the inner diameter was 53 µm, and the UV absorbance detector was set at 229 nm. 20 kV f 8 µA. (See Figure 1 for identification of solutes.)
to the equilibrium29
In methanol, similar behavior occurs:
The triphenylpyrylium ion, on the other hand, may react with water to form the corresponding pseudobase:30 (29) Kolomnikova, G. D.; Parnes, Z. N. Russ. Chem. Rev. 1967, 36, 735. (30) Balaban, A. T.; Dinculescu, A.; Dorofeenko, G. N.; Fischer, G. W.; Koblik, A. V.; Mazheritskii, V. V.; Schroth, W. Advances in Heterocyclic Chemistry Suppl. 2; Academic Press: New York, 1982.
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Consequently, when a solution of either cation in water or methanol was used as the CE run buffer, an extremely erratic baseline resulted and no recognizable peaks were seen. However, a solution of the cation in an aprotic solvent such as acetonitrile afforded a far more stable baseline. Finally, the UV cutoff wavelength of each solvent was considered. In using UVabsorbance detection, 254 nm was determined to be a suitable wavelength at which to monitor the separations. The cations strongly absorb at lower wavelengths, and at higher wavelengths, the signal-to-noise ratios for most of the PAHs diminish. Acetonitrilesbecause of its UV transparency and its ability to readily dissolve organic cations without reactingswas found to be the most suitable solvent in which to perform CE analyses using this technique and was the solvent of choice for our experiments. Effect of Cation Concentration on Electrophoretic Mobility and Electroosmotic Flow. The electrophoretic mobility (µep) of a solute is given by
µep )
(
)
LtLd 1 1 V tr teo
(1)
where Lt is the total length of the capillary, Ld is the effective length (i.e., distance between points of injection and detection)
Figure 5. Electrophoretic mobility (in cm2 min-1 kV-1) of selected PAHs as a function of 2,4,6-triphenylpyrylium ion concentration (square, coronene; triangle, benzo[a]pyrene; circle, naphthalene).
Figure 6. Electroosmotic mobility (in cm2 min-1 kV-1) as a function of 2,4,6-triphenylpyrylium ion concentration in acetonitrile.
of the capillary, tr is the migration time of the solute, teo is the migration time due solely to EOF, and V is the applied voltage across the capillary. In our experiments, the electrophoretic mobilities of the PAHs increased in proportion to the cation concentration (Figure 5). This was expected since the electrophoretic mobility of a neutral solute depends on the degree to which it reversibly binds to the cation. At higher concentrations of the cation, such interactions are more frequent, and better separations are generally obtained. In our case, 30 mM tropylium and 40 mM 2,4,6-triphenylpyrylium in acetonitrile appeared to be the optimal concentrations at which to separate PAHs. At lower concentrations, the separations were inadequate, and at higher concentrations, erratic baselines were observed (possibly due to the accumulation of cations near the negatively charged capillary wall) and there was no further improvement in resolution. It is worth mentioning that the use of a laser-induced fluorescence detector (instead of UV absorbance) would likely result in a smoother baseline, thus allowing one to use a higher concentration of cation in the run buffer. The electropherograms shown in Figures 1 and 2 were obtained using the optimal concentration of each cation in acetonitrile. Also, we found that the EOF decreased with increasing salt concentration (Figure 6). This was to be expected since the ζ potential of the capillary wall is known to decrease as the concentration of the electrolyte increases. Therefore, by increasing the concentration of the cation, one may obtain better separations, not only due to the greater differences in solute-cation mobilities but also as a result of the decrease in EOF. A major problem that was encountered using the tropylium ion was the poor reproducibility of EOF between runs. Initially, the capillary (after being regenerated with sodium hydroxide solution) was filled with the tropylium solution prior to injection; between each run, the capillary was flushed with either water or acetonitrile and then refilled with tropylium solution. With each run, however, there was a marked increase in the migration times of the solutes and a decrease in peak efficiencies, especially when higher concentrations of the tropylium ion were used. In a 30
mM solution of tropylium tetrafluoroborate, for example, we observed that the time required for the EOF marker to elute increased by several minutes from one run to the next. This behavior was attributed to the interaction between the solutes and the capillary wall as a result of the accumulation of cations on the negatively charged wall surface, which led to variations in EOF and lower rates of mass transfer. To circumvent this problem, the capillary was rinsed with an aqueous acidic solution so as to induce protonation of the silanol groups, thus minimizing wall interactions. In our studies, the best reproducibility between runs using the tropylium ion was obtained when the capillary was regenerated with a sodium hydroxide solution followed by a lowpH rinse (in this case, the migration times for the EOF marker varied by less than 5%). This pretreatment was also applied to the runs involving the 2,4,6-triphenylpyrylium ion, although the problem of irreproducibility was much less apparent with this ion. Effect of Solute Properties on the Electrophoretic Mobility. The intermolecular forces that influence the stability of a charge-transfer complex have been the subject of debate for a number of years. In chromatographic studies, the identification of these forces is useful since it allows one to predict the separation behavior of other solutes in similar media. Quantitative structureretention relationships (QSRRs) based on the correlations between retention behavior and various molecular descriptors often assist in identifying the forces responsible for these interactions. In our case, the electrophoretic mobilities of the PAHs (which directly depend on their degree of interaction with the cation) were used in establishing quantitative structure-migration relationships (QSMRs) as detailed in the following paragraphs. The theory that attempts to account for the energetics of electron donor-acceptor complex formation centers around the application of perturbation methods to molecular orbital theory and was originally designed to relate molecular structure with physicochemical properties.31 Klopman and Hudson later used this theory to draw a relationship between the reactivity of an electron donor with an acceptor and the molecular orbital characteristics of the reacting species.32,33 More recently, other researchers have discussed the possible relevance of this theory to chromatographic processes involving charge-transfer phenomena.34 According to the theory, the perturbation energy that results from the interaction of two frontier orbitals is inversely related to the energy difference between the highest occupied molecular orbital (HOMO) of the donor and the lowest unoccupied molecular orbital (LUMO) of the acceptor. Therefore, if this theory were to account for the migration behavior of PAHs as they interact with a given cation, there should be an inverse relationship between the energy of interaction and the ionization potential (a HOMO-energy descriptor) of the PAH. Assuming that the mobility of solute-cation complex is size-independent and that the interaction energy alone determines the electrophoretic mobility, an inverse relationship should likewise be observed between the mobility and the ionization potential. As Figure 7 shows, inverse relationships were indeed observed as log µep was plotted against ionization potential (IP), but the correlations were somewhat low. (31) Coulson, C. A.; Longuet-Higgins, H. C. Proc. R. Soc. London 1947, A191, 39. (32) Klopman, G.; Hudson, R. F. Theor. Chim. Acta 1967, 8, 165. (33) Klopman, G. J. Am. Chem. Soc. 1968, 90, 223. (34) Nondek, L. In Complexation Chromatography; Cagniant, D., Ed.; Marcel Dekker: New York, 1992; pp 13-14.
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Figure 7. Electrophoretic mobility (in cm2 min-1 kV-1) of (a) PAHtriphenylpyrylium complexes and (b) PAH-tropylium complexes as a function of solute (donor) ionization potential.
Figure 8. Electrophoretic mobility (in cm2 min-1 kV-1) of (a) PAHtriphenylpyrylium complexes and (b) PAH-tropylium complexes as a function of solute polarizability (in Å3).
30 mM 2,4,6-triphenylpyrylium ion in acetonitrile: log µep ) -0.681(IP) + 6.20
r ) 0.705 (n ) 20)
30 mM tropylium ion in acetonitrile: log µep ) -0.418(IP) + 3.57
r ) 0.646 (n ) 20)
on the binding force between solute and cation) and the polarizability of each solute. For each cation, we found that a plot of log µep vs solute polarizability (R) produced a linear curve (Table 1 and Figure 8). A plot of these parameters yielded the following equations:
30 mM 2,4,6-triphenylpyrylium ion in acetonitrile: The low correlations observed above would seem to cast doubt on the significance of charge-transfer contributions in the binding between the PAHs and cation. There is, however, an alternate explanation that may account for the observed PAH-cation mobilities. This explanation is based on the van der Waals forces of attraction (which include electrostatic, inductive, and dispersive contributions). Several authors have used this model in studies involving the separation of PAHs and have obtained good correlations.35,36 These forces occur as two or more atoms or molecules approach one another, and in the case of a neutral molecule approaching a cation, any or all of the following interactions may contribute to the overall attractive force: (1) iondipole interactions (electrostatic); (2) dipole-dipole interactions (electrostatic); (3) ion-induced dipole interactions (inductive); (4) dipole-induced dipole interactions (inductive); (5) induced dipoleinduced dipole interactions (dispersive). Since our study is concerned with the interaction of planar organic cations with PAHs (which possess no permanent dipole moment), only the forces involving induced dipole interactions (3-5 above) would be expected to contribute to total force of interaction. Since these forces are linearly related to the polarizability of the neutral species, one would expect to observe a correlation between the electrophoretic mobility (which depends (35) Lamparczyk, H. Chromatographia 1985, 20, 283. (36) Jinno, K.; Kawasaki, K. Chromatographia 1984, 18, 103.
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log µep ) 0.038R - 0.570
r ) 0.983 (n ) 20)
30 mM tropylium ion in acetonitrile: log µep ) 0.022R - 0.518
r ) 0.865 (n ) 20)
The strong correlation between log µep and polarizability for the interaction of PAHs with each cation (especially the triphenylpyrylium ion) suggests that van der Waals forces play significant role in solute-cation interactions and greatly determine the migration behavior in CE separations. On the basis of the one-parameter QSMRs discussed in the preceding paragraphs, it appears that while complexes involving charge transfer do in fact form between the PAHs and cation, the energetics of such formation are governed primarily by the van der Waals forces that are present among the interacting species. This is supported by the fact that the tetrahexylammonium ionswhich lacks a low-energy unoccupied orbital and thus cannot participate in the charge-transfer process to any significant degreesyields a similar PAH migration pattern. Also, it lends support to the findings of other researchers who have studied similar systems using nonchromatographic methods.37-41 (37) Dewar, M. J. S.; Thompson, C. C., Jr. Tetrahedron, Suppl. 1966, 7, 97. (38) LeFevre, R. J. W.; Radford, D. V.; Stiles, P. J. J. Chem. Soc. B 1968, 1297. (39) Mantione, M. J. Theor. Chim. Acta 1969, 15, 141.
Figure 9. Electronic distribution in the benzene molecule as viewed (a) edge-on and (b) from the top.
The 2,4,6-triphenylpyrylium ion forms stronger complexes with PAHs than does the tropylium ion, most likely as a result of the electronegative oxygen in the ring. However, Figures 1 and 2 show that the PAH elution order differs from cation to cation. This selectivity difference cannot be explained by merely considering the ionization potential or polarizability of each solute or by considering the electronegativity of the cation. It is quite obvious that the binding between the solute and cation is determined (at least in part) by the shape characteristics of the two species. One explanation that might account for the PAH shape-selective binding of the cations may be traced to the electrostatic forces in cation-π interactions. In the first QSMR discussed above, these forces were neglected under the assumption that, in uncharged molecules, only permanent dipole moments (which PAHs lack) could contribute to the overall polarity. However, one must realize that quadrupole (and even higher-order multipole) moments may also play a significant role in electrostatic binding. In a recent paper,42 Dougherty showed that the electron density is not uniformly distributed over a benzene ring but is concentrated above and below the plane of the ring (Figure 9a). When the ring is viewed from the top (Figure 9b), the electron density appears to be concentrated toward the center of the ring, with the ring edges remaining electropositive. The degree to which a cation interacts with the ring, therefore, depends on its accessibility to the electronegative ring center. Because this electronic distribution applies to PAHs as well as to benzene,43 the importance of the PAH shape to the binding process can easily be seen. We observed in several cases that the electrophoretic mobility of a given PAH differed greatly from that of another PAH with the same number of rings. For example, pyrene, a four-ring pericondensed PAH, appeared to bind strongly with the tropylium ion (as evidenced by its high electrophoretic mobility; see Table 1), whereas the four-ring catacondensed PAHs possessed lower mobilities. A likely explanation for this occurrence may involve the degree of overlap between the PAH and cation. Assuming a planar conformation, Figure 10 shows that the compact structure of a pericondensed PAH such as pyrene allows for complete overlap with the tropylium ion, whereas such overlap does not occur with a catacondensed PAH of similar size (benz[a]anthracene, in this case). This behavior was observed with all of the PAH-tropylium complexes and gave rise to an (40) Lippert, J. L.; Hanna, M. W.; Trotter, P. J. J. Am. Chem. Soc. 1969, 91, 4035. (41) Morokuma, K. Acc. Chem. Res. 1977, 10, 294. (42) Dougherty, D. A. Science 1996, 271, 163. (43) Price, S. L.; Stone, A. J. J. Chem. Phys. 1987, 86, 2859. (44) Molecular polarizabilities taken or calculated from: Miller, K. J. J. Am. Chem. Soc. 1990, 112, 8533. These values are based solely on molecular structure and do not account for the surrounding medium. (45) Ionization potentials taken from: Hites, R. A.; Simonsick, W. J., Jr. Calculated Molecular Properties of Polycyclic Aromatic Hydrocarbons; Elsevier: New York, 1987.
Figure 10. (a) Complete overlap of the tropylium ion by pyrene, thus maximizing electrophoretic mobility. (b) incompletely overlap the tropylium ion by benz[a]anthracene; therefore, reduced electrophoretic mobility.
interesting trend: the addition of the number of rings in a PAH molecule to the number of carbon atoms shared by three rings gave a total that we refer to as the number of “effective” rings (in the example above, pyrene has six effective rings, compared to four for benz[a]anthracene). The electrophoretic mobility of the PAH-cation complex correlated very well with the number of effective rings (r ) 0.973, n ) 20) and shows that both the size and shape of a PAH may play important roles in binding with a cation. CONCLUSION This research demonstrates the previously unreported technique of using planar organic cations in the separation of large hydrophobic molecules by CE. As a means of separating PAHs, this method compares well with other CE methods insofar as quick and efficient separations of a variety of PAHs may be obtained. Because this technique involves solute-cation interactions in a nonaqueous medium, it is especially advantageous in separating the larger (and more hydrophobic) PAHs. Under these conditions, such solutes provide sharp, early-eluting peaks, in contrast to MEKC methods in which these solutes elute much later. In this report, we refer to this method as “charge-transfer” CE (by analogy to other “charge-transfer” chromatographic methods that have been reported), but QSMR studies suggest that van der Waals forces of attraction play a significant (even perhaps a predominant role) in the migration behavior of the solutes. Moreover, it seems that the 2,4,6-triphenylpyrylium ion separates PAHs primarily according to size, whereas for the tropylium ion, separation occurs based on solute size and shape. Investigations are currently underway to compare the mobilities of various substituted PAHs as they interact with the cations. Also, additional cations will be examined as possible substitutes for the cations studied in this report. ACKNOWLEDGMENT Funding by the National Institutes of Health (GM 38738) and the U.S. Environmental Protection Agency is gratefully acknowledged.
Received for review July 23, 1996. Accepted December 16, 1996.X AC960734N X
Abstract published in Advance ACS Abstracts, February 1, 1997.
Analytical Chemistry, Vol. 69, No. 6, March 15, 1997
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