Anal. Chem. 1997, 69, 4581-4585
Quadrupolar Effects on the Retention of Aromatic Hydrocarbons in Reversed-Phase Liquid Chromatography Qian-Hong Wan,* Lou Ramaley, and Robert Guy
Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J3
Because of their large molecular quadrupole moments, aromatic hydrocarbons can give rise to strong electrostatic interactions with polar molecules such as acetonitrile and water. Such interactions may have some significant effects on the retention and separation of the aromatic compounds in reversed-phase liquid chromatography. We report here an experimental investigation of these effects in which we studied the retention behavior of a series of aromatic hydrocarbons in comparison with that of aliphatic hydrocarbons which have no appreciable quadrupole moments. The elution order of benzene, cyclohexane, and n-hexane was determined and shown to follow inversely the increasing magnitude of the molecular quadrupole moments. A linear dependence of retention on molecular polarizability was observed for both the aliphatics and aromatics, the latter being consistently less retained than the former under the same chromatographic conditions. The molecular polarizability and quadrupole moment were regressed as independent variables against the logarithmic retention factors of both the aliphatic and aromatic solutes, and an excellent correlation was found with all five different chromatographic systems studied. These results clearly show that, while the retention of aromatic hydrocarbons is primarily governed by the dispersion interactions with the nonpolar stationary phase, the electrostatic interactions of the aromatic solutes with the polar mobile phase act to reduce the retention and thus contribute favorably to the control of the relative retention and selectivity in reversed-phase liquid chromatography. The partitioning of a solute between the stationary and mobile phases is controlled by a variety of noncovalent intermolecular interactions that operate between the solute and the molecules in each of the phases. Accurate prediction of the retention behavior of the solute requires a substantial understanding of such interactions, which are dominant in a given chromatographic system. Although the hydrophobic effects,1,2 hydrogen bonding,3,4 and charge-transfer interactions5,6 have been extensively studied and discussed in the context of liquid chromatography, there is * Corresponding author. Tel.: (902) 494-7079. Fax: (902) 494-1310. E-mail:
[email protected]. (1) Horvath, C.; Melander, W.; Molnar, I. J. Chromatogr. 1976, 125, 129-156. (2) Horvath, C.; Melander, W.; Molnar, I. Anal. Chem. 1977, 49, 142-154. (3) Minick, D. J.; Brent, D. A.; Frenz, J. J. Chromatogr. 1989, 461, 177-191. (4) Belsner, K.; Pfeiger, M.; Wiffert, B. J. Chromatogr. 1993, 629, 123-134. (5) Mourey, T. H.; Siggla, S. Anal. Chem. 1979, 51, 763-767. (6) Tanaka, N.; Kimata, K.; Hosoya, K.; Miyanishi, H.; Araki, T. J. Chromatogr. A 1993, 656, 265-287. S0003-2700(97)00337-5 CCC: $14.00
© 1997 American Chemical Society
another important but generally underappreciated molecular interaction. An aromatic molecule can undergo strongly attractive interactions with cations,7,8 polar molecules,9,10 and another aromatic molecule.11,12 These interactions, generally known as cation-π, polar-π, and π-π interactions, are widely believed to play an important role in a variety of molecular processes with applications ranging from molecular biology and molecular recognition to materials science. However, their role in influencing the chromatographic retention and selectivity for aromatic compounds remains to be explored and understood. Recently, we have shown that the retention of aromatic hydrocarbons in normal phase liquid chromatography is primarily governed by their electrostatic interactions with the polar stationary phase.13 Benzene and other aromatic hydrocarbons are generally considered nonpolar molecules because they have either no or very small permanent dipole moments. They do, however, have fairly large quadrupole moments, which can be measured experimentally14-16 or calculated using quantum mechanical methods.17-19 Thus, the interaction energy contributing to the retention of aromatic compounds can be divided roughly into an electrostatic and a dispersion term. The dispersion term depends strongly on the molecular polarizability, whereas the electrostatic term depends on the magnitude and relative orientation of the dipole or quadrupole moments. In the case of normal phase liquid chromatography with a polar stationary phase and nonpolar mobile phase, the dispersion interactions of the solutes with the stationary phase are canceled out by the corresponding interactions with the mobile phase. Therefore, the electrostatic term arising from dipole-quadrupole interactions dominates the interaction and, hence, the retention. As a result, the relative retention of the aromatic hydrocarbons can be effectively predicted on the basis of magnitudes of their molecular quadrupole moments. (7) Dougherty, D. A. Science 1996, 271, 163-168. (8) Luhmer, M.; Bartik, K.; Dejaegere, A.; Bovy, P.; Reisse, J. Bull. Soc. Chim. Fr. 1994, 131, 603-606. (9) Suzuki, S.; Green, P. G.; Bumgarner, R. E.; Dasgupta, S.; Goddard, W. A., III; Blake, G. A. Science 1992, 257, 942-945. (10) Rodham, D. A.; Suzuki, S.; Suenram, R. D.; Lovas, F. J.; Dasgupta, S.; Goddard, W. A., III; Blake, G. A. Nature 1993, 362, 735-737. (11) Hunter, C. A.; Sanders, J. K. M. J. Am. Chem. Soc. 1990, 112, 5525-5534. (12) Williams, J. H. Acc. Chem. Res. 1993, 26, 593-598. (13) Wan, Q. H.; Ramaley, L.; Guy, R. Chromatographia, submitted. (14) Battaglia, M. R.; Buckingham, A. D.; Williams, J. H. Chem. Phys. Lett. 1981, 78, 421-423. (15) Craven, I. E.; Hesling, M. R.; Laver, D. R.; Lukins, P. B.; Ritchie, G. L. D. J. Phys. Chem. 1989, 93, 627-631. (16) Dennis, G. R.; Ritchies, G. L. D. J. Phys. Chem. 1991, 95, 656-660. (17) Chablo, A.; Cruicksbank, D. M. J.; Hinchliffe, A.; Munn, R. W. Chem. Phys. Lett. 1981, 78, 424-428. (18) Price, S. L. Chem. Phys. Lett. 1985, 114, 359-364. (19) Hernandez-Trujillo, J.; Vela, A. J. Phys. Chem. 1996, 100, 6524-6530.
Analytical Chemistry, Vol. 69, No. 22, November 15, 1997 4581
Table 1. Chromatographic Systems Used in This Work system
column
eluent
1 2 3 4 5
Vydac C18, 250 mm × 4.6 mm, 5 µm Zorbax SB-C18, 150 mm × 4.6 mm, 5 µm Supelcosil LC-C8, 150 mm × 4.6 mm, 5 µm Zorbax SB-Phenyl, 150 mm × 4.6 mm, 5 µm Supelcosil LC-DP, 150 mm × 4.6 mm, 5 µm
80% (v/v) acetonitrile/water 80% (v/v) acetonitrile/water 60% (v/v) acetonitrile/water 60% (v/v) acetonitrile/water 60% (v/v) acetonitrile/water
Table 2. Molecular Properties of 13 Hydrocarbons and Their Retention Factors Measured in Five RPLC Systems retention factor, k
solute
molecular polarizability, Ra (10-30 m3)
quadrupole moment, Q (10-40Cm2)
1
2
3
4
5
cyclohexane n-pentane n-hexane n-heptane n-nonane n-dodecane benzene naphthalene phenanthrene anthracene pyrene triphenylene perylene
11.0 9.95 11.8 13.6 17.4 22.7 10.0 16.5 24.7 25.4 28.2 31.1 37.8b
3.0c 0 0 0 0 0 -32.39d -52.76d -75.28d -74.43d -83.85d -97.45d -105.88d
0.88 0.82 1.24 1.75 3.91 15.36 0.27 0.47 0.94 1.12 1.76 2.18 5.61
3.05 2.65 4.02 6.00 13.47 48.29 0.75 1.26 2.29 2.41 3.35 3.92 6.34
4.22 3.99 6.14 9.20 23.00 85.11 1.24 2.22 3.91 4.28 5.64 6.77 10.84
2.59 2.54 3.33 4.86 9.23 30.03 1.21 2.04 3.41 3.65 4.63 5.90 9.86
1.65 1.59 2.02 2.82 5.07 14.92 0.88 1.42 2.26 2.39 2.95 3.57 5.46
a
Data taken from ref 20. b Calculated according to Muller and Svachik.20 c Data taken from ref 15. d Data taken from ref 18.
On going from the normal phase to the reversed-phase liquid chromatography (RPLC), the principal intermolecular interactions controlling the solute retention are expected to change, corresponding to a change in the relative polarity of the stationary and mobile phases. Given the nonpolar nature of the stationary phase typically employed in RPLC, it seems reasonable to assume that the dispersion interactions between the solute and the stationary phase would dominate the retention, whereas the electrostatic interactions between the solute and the mobile phase reduce the retention. Accordingly, the RPLC retention of solute might be effectively predicted by its molecular polarizability and electrical multipole moments. A straightforward test for this idea, and an extension of the general analysis, is to measure the retention data for aromatic and aliphatic hydrocarbons in the same RPLC system and to look for differential retention behaviors. We report here an experimental study of the elution order and retention behavior of the aliphatic and aromatic hydrocarbons and a correlation of the RPLC retention with molecular polarizability and quadrupole moment for these compounds. EXPERIMENTAL SECTION Chemicals and Columns. The aliphatic and aromatic hydrocarbons used as model compounds were obtained from the Aldrich Chemical Co. (Milwaukee, WI). The polyaromatic hydrocarbons are reportedly carcinogenic and should be handled and disposed of safely. HPLC grade acetonitrile was obtained from Fisher Scientific (Fair Lawn, NJ). All the columns used in this work were commercial products: Vydac C18 from The Separation Group (Hesperia, CA); Supelcosil LC-C8 and Supelcosil LC-DP from Supelco (Bellefonte, PA); and Zorbax SB-C18 and Zorbax SBPhenyl from Rockland Technologies Inc. (Newport, DE). The details of the column dimensions and eluent composition in each chromatographic system are listed in Table 1. HPLC Equipment and Measurements. The high-performance liquid chromatographic system consisted of a HP 1100 4582 Analytical Chemistry, Vol. 69, No. 22, November 15, 1997
quaternary pump (Hewlett-Packard, Waldbronn, Germany), a HP 8452 diode array UV-visible spectrophotometric detector (HewlettPackard) with a 30 µL flow cell (Hellma, Balden, Germany) or a Model R-401 differential refractometer (Waters Associates, Milford, MA), and a data acquisition systems based on IBMcompatible computers with software written in-house. All chromatographic measurements were carried out at room temperature (25 °C) with the flow rate of 1 mL/min and UV detection for the aromatics and RI detection for the aliphatics, unless otherwise indicated. The elution time of acetonitrile was used as t0 for the calculation of k (k ) (tr - t0)/t0, with tr the retention time of analyte). The reported data for the values of k are the means of triplicate determinations. RESULTS AND DISCUSSION Elution Order of Benzene, Cyclohexane, and n-Hexane. An investigation of the elution order for a series of simple compounds is useful to discern information about the nature of the intermolecular interactions governing a given chromatographic process. For this reason, we chose benzene, cyclohexane, and n-hexane for a detailed study because they have similar molecular polarizabilities but quite different quadrupole moments in magnitude and orientation (see Table 2). We separated these compounds and measured their retention factors (k) in five RPLC systems. A typical chromatogram is shown in Figure 1, and the observed retention factors are given in Table 2. As can be seen from Table 1, two types of reversed-phase packing were used: alkyl chain (octyl and octadecyl)- and aromatic ring (phenyl and diphenyl)-bonded silica gels. They represent a vast majority of the packing materials employed in current RPLC. Among the reversed-phase stationary phases studied, Vydac C18 is the only “polymeric” phase which has cross-linked silane polymers on the silica surface. Initially, 60% (v/v) acetonitrile/water was used as (20) Muller, K. J.; Savchik, J. A. J. Am. Chem. Soc. 1979, 101, 7206-7213.
Figure 1. Separation of benzene (1), cyclohexane (2), and n-hexane (3) by reversed-phase liquid chromatography. Conditions: column, Vydac C18, 5 µm, 250 mm × 4.6 mm; eluent, 80% (v/v) acetonitrile/ water; flow rate, 1 mL/min; detection, RI.
Figure 2. Solute retention log k as a function of molecular polarizability for three alkyl-bonded silica phases: Vydac C18 (b, O), Zorbax SB-C18 (9, 0), and Supelcosil LC-C8 (2, 4). Solutes: aliphatic (filled symbols) and aromatic hydrocarbons (open symbols).
the mobile phase for all the columns studied. It was found that strongly retained solutes showed excessive peak broadening on octadecyl-bonded phases, making it difficult to measure the retention time accurately. For this reason, an increase in the volume percent of acetonitrile was made, corresponding to increasing retention strengths of these columns. The results with alkyl-bonded silica show that the elution order of these compounds in RPLC is characterized by kbenzene < kcyclohexane < kn-hexane. As can be seen from Table 2, the molecular polarizabilities of benzene, cyclohexane, and n-hexane are little different from each other, implying that these molecules should give rise to similar dispersion interactions with a given stationary phase. These molecular properties, therefore, cannot be a major factor contributing to the observed elution order. On the other hand, the quadrupole moments of these molecules differ greatly. While n-hexane has zero quadrupole moment, cyclohexane has the quadrupole moment of 3 × 10-40 Cm2. Furthermore, benzene has a quadrupole moment of -32 × 10-40 Cm2, one order of magnitude higher than that for cyclohexane. This means that, in the same electric field gradient, the interaction energy of benzene is approximately 10 times greater than the corresponding interaction energy of cyclohexane.8 In a typical RPLC, where the electrostatic interaction with the stationary phase is far from dominant, we anticipate that the retention of solute is weakened by the strong electrostatic solvation in the mobile phase, and, as such, the solute molecule of higher quadrupole moment is eluted first. Indeed, the observed elution order of benzene, cyclohexane, and n-hexane is consistent with the decreasing magnitudes of the molecular quadrupole moment. It appears that electrostatic interactions involving polar molecules in the mobile phase may play an important role in regulating the retention and selectivity in RPLC. This generalization is particularly useful in understanding the factors influencing the retention and separation of structurally related compounds such as structural and geometrical isomers. Considering an additional contribution arising from intermolecular interactions between aromatic quadrupole moments, it is not unreasonable to expect that the retention of benzene might
be enhanced relative to that of hexane when a phenyl bonded silica is used in place of an alkyl bonded phase, and thus a variation in the elution order might occur. As noted from the retention data, however, no change in the elution order was observed with the column packing going from alkyl to phenyl. The unchanged elution order suggests that the electrostatic interactions between benzene and phenyl stationary phase are relatively insignificant as compared to the predominant dispersion interactions, consistent with a similar conclusion drawn by Hunter and Sanders11 from their study of π-π interactions between porphyrins. Retention Behavior of Aliphatic and Aromatic Hydrocarbons. To clarify the role of dispersion and electrostatic interactions in determining the RPLC retention, it is necessary to study retention behavior for a wider range of aliphatic and aromatic hydrocarbons. Using the same chromatographic systems as above, we measured retention factors for 4 straight chain alkanes and 6 polycyclic aromatic hydrocarbons, in addition to those for benzene, cyclohexane and n-hexane. The results are given in Table 2, along with their molecular polarizabilities and quadrupole moments. The hydrocarbons with molecular weights higher than dodecane and perylene were not included because excessive peak broadening due to their poor solubility in the chosen mobile phase does not allow accurate determination of the retention data. Previously, it has been suggested that the retention of aromatic hydrocarbons can be predicted solely by their molecular polarizability.21 In Figure 2, the logarithmic retention factors obtained on the alkyl bonded phases are plotted as a function of molecular polarizability for aliphatic and aromatic hydrocarbons. Apparently, a linear correlation exists for these two classes of compound. This result is perhaps not surprising considering the predominant role of the dispersion interactions in determining the retention. However, there is an important difference between the two: the slopes are consistently smaller for the aromatic than for the aliphatic solutes. This means that for molecules of the same polarizability the retention of the aromatic hydrocarbons is always (21) Lamparczyk, H. Chromatographia 1985, 20, 283-288.
Analytical Chemistry, Vol. 69, No. 22, November 15, 1997
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Figure 3. Solute retention log k as a function of molecular polarizability for two phenyl-bonded silica phases: Zorbax SB-Phenyl (b, O) and Supelcosil LC-DP (9, 0). Solutes: aliphatic (filled symbols) and aromatic hydrocarbons (open symbols).
Figure 4. Correlation between predicted and observed log k for the 13 solutes studied with five RPLC columns: Vydac C18 (O), Zorbax SB-C18 (0), Supelcosil LC-C8 (4), Zorbax SB-Phenyl (3), and Supelcosil LC-DP (]).
less than that of the aliphatics and that the difference in retention increases with the polarizability. As shown in Figure 3, similar trends were also observed with phenyl bonded phases. These results show clearly that molecular polarizability alone cannot explain the differences between retention behaviors of aliphatic and aromatic hydrocarbons, indicating an additional factor contributing to the control of the overall retention. As mentioned previously, it seems likely that quadrupole moment arising from the π-electron distribution is such a factor that causes a reduction in the retention of the aromatic hydrocarbons due to the attractive dipole-quadrupole interactions with the polar mobile phase. This effect can be understood from the fact that the binding of a solute molecule to the stationary phase involves at least partial removal of the solute molecule from the surrounding polar solvent. The desolvation costs associated with this process is in general larger with polar than with nonpolar solute. Thus the increased difference in retention with polarizability as shown in Figures 2 and 3 is probably due to the corresponding increase in molecular polarity, namely, quadrupole moment. Correlation of Solute Retention with Molecular Properties. Further information about the retention mechanism can be obtained by studying the relationships between chromatographic retention of solutes and their molecular properties. It has been suggested that chromatographic retention can be expressed as a linear function of molecular properties which are dominant in a given retention process.22 In the case of aliphatic and aromatic hydrocarbons considered, molecular polarizability and quadrupole moment appear to be of paramount importance to the overall retention. Thus we suggested a simple relation model as follows:
of the quadrupole moment needs to be considered, regardless of its orientation. This is because the solvent dipole will adjust its orientation relative to the quadrupole to maximize the attractive interaction energy. For aliphatic hydrocarbons with Q ) 0, the equation reduces to
log k ) a + bR + cQ
(1)
where R is the solute molecular polarizability and Q is the solute quadrupole moment; a, b, and c are the regression coefficients. It should be noted that, in the above equation, only the magnitude (22) Kaliszan, R. J. Chromatogr. A 1993, 656, 417-435.
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Analytical Chemistry, Vol. 69, No. 22, November 15, 1997
log k ) a + bR
(2)
This is of the same form as the equation previously proposed by Lamparczyk21 for aromatic hydrocarbons. Table 3 shows the results from a multiple linear regression analysis of the experimental retention data given in Table 2. Five linear regression equations corresponding to five RPLC systems were obtained using the Minitab statistical package (Minitab Inc., University Park, PA). R2 is the coefficient of multiple correlation, s is the standard error of estimate, and F is the value of the F-test for the statistical significance of the regression. The t-test values and significance level p-values of each coefficient are given in parentheses. Figure 4 shows a plot of the predicted versus the observed retention for 13 hydrocarbon compounds in all five RPLC systems. Several trends are immediately clear upon inspecting the data in Table 3. First, the retention model fits the data well, with R2 g 0.97 in all the cases. The F-test values for five equations derived are all considerably greater than 7.56, the critical F-value for the 99% confidence level. This close correlation is further supported by an excellent agreement between the measured and predicted retention, as shown in Figure 4. Second, the t-test results show that all three terms in each of the five equations are statistically significant, at least at the 99% confidence level. Third, the coefficients of the individual variables in all five equations have the expected signs: positive with the solute polarizability and negative with the quadrupole moment. The positive signs on the R terms indicate that attractive dispersion interactions between a solute molecule and the stationary phase are stronger than the corresponding dispersion interactions of the solute with the polar
Table 3. Results from Multiple Linear Regression Analyses of Experimental Retention Data by log k ) a + br + cQ system
a (t-ratio, p-value)
b (t-ratio, p-value)
c (t-ratio, p-value)
R2 a
sb
Fc
1 2 3 4 5
-1.086 ( 0.067 (-16.18,
