Anal. Chem. 1997, 69, 4608-4614
Characterization of 1,1,1,2-Tetrafluoroethane and Carbon Dioxide Binary Mobile Phases with Polymeric Columns Using Linear Solvation Energy Relationships John A. Blackwell* and Rodger W. Stringham
Dupont Merck Pharmaceutical Company, Chemical Process Research and Development, Chambers Works, PRF-1 (S-1), Deepwater, New Jersey 08023
In this study, the chromatographic properties of binary carbon dioxide/1,1,1,2-tetrafluoroethane (HFC-134a) mobile phases on a polymeric stationary phase are examined. Retention of various analytes, as a function of mobile phase composition, is dependent upon the physicochemical properties of the analyte. The specific intermolecular interactions between various solutes and the mobile and stationary phases responsible for the retention behavior are probed using linear solvation energy relationships. Using this methodology, it is apparent that solute hydrogen bond donating ability is the dominant factor dictating retention in these systems. Other solute properties, such as dipolarity/polarizability, hydrogen bond accepting ability, and excess molar refraction, have much smaller effects on retention. Recent studies have demonstrated the high apparent eluotropic strength of 1,1,1,2-tetrafluoroethane (HFC-134a) as a mobile phase under near-critical and supercritical conditions.1-3 Using HFC134a, polar and nonpolar analytes were more readily eluted from both nonpolar, open tubular capillary columns and bare silicapacked columns than with methanol-modified carbon dioxide mobile phases. In many cases, the extremely high eluotropic strength of HFC-134a resulted in capacity factors which were too low to be useful for reasonable separations. A straightforward approach to this “problem” is the mixing of carbon dioxide and HFC-134a as a binary mobile phase of adjustable eluotropic strength. Since carbon dioxide alone has very poor eluotropic strength, especially for polar analytes on polar supports, it is ideally suited as a “weak” eluent. Addition of variable amounts of HFC-134a would serve to increase the eluotropic strength of the mobile phase to the desired level. This methodology is the near-critical equivalent of normal phase chromatography, where the eluotropic strength of a nonpolar mobile phase is adjusted by the addition of a polar modifier. Binary mixtures of these two fluids have a number of advantages over conventional carbon dioxide-modifier combinations. Neither carbon dioxide nor HFC-134a has any appreciable absorbance in the UV region down to 192 nm, thus allowing detection of a wide range of analytes at very low wavelengths. Both fluids are completely miscible at 0 °C and pressures above (1) Blackwell, J. A.; Schallinger, L. E. J. Microcolumn. Sep. 1994, 6, 551-556. (2) Cantrell, G. O.; Blackwell, J. A. J. Chromatogr., in press. (3) Blackwell, J. A.; Stringham, R. W. Chirality, in press.
4608 Analytical Chemistry, Vol. 69, No. 22, November 15, 1997
33 bar, and at 25 °C and pressures above 60 bar.4 This allows high-pressure mixing to take place in the same manner as for conventional modified mobile phases without the problem of phase separation. Binary mixtures of carbon dioxide and HFC-134a have also been characterized with respect to their binary interaction parameter, enabling calculation of mobile phase densities and mixture critical temperatures using commercially available software.4 Preparative scale separation and isolation of analytes should be relatively straightforward since the “modifier” is extremely volatile and does not accumulate in the collection vessel in the same manner as conventional liquid modifiers. An interesting aspect of this combination of fluids lies in the ability of the modifier itself to reach a supercritical state under reasonable conditions. Unlike conventional liquid modifiers, HFC134a has a reasonable critical temperature of 101.2 °C and a relatively low critical pressure of 40.7 bar.4 If the operating pressure is above approximately 75 bar and the operating temperature is above approximately 110 °C, a modifier composition gradient may be maintained under supercritical conditions from 0 to 100% modifier. Other modifiers produce mixture critical temperatures far in excess of current column temperature limitations. Composition gradients using conventional modifiers run the risk of traversing into a two-phase region, resulting in irreproducible chromatographic results. In this study, the changes in mobile and stationary phase properties for a binary carbon dioxide/HFC-134a mobile phase using a polymeric reversed-phase support are characterized using linear solvation energy relationships (LSERs). LSERs have been used to characterize retention processes in gas chromatography,5-8 liquid chromatography,8-12 and supercritical fluid chromatography.13,14 In this study, an extensive set of well-characterized analytes was used to probe the changes in hydrogen bonding, (4) NIST Thermodynamic Properties of Refrigerants and Refrigerant Mixtures Database, version 4.0, NIST Standard Reference Database 23, November 1993. (5) Poole, C. F.; Kollie, T. O.; Poole, S. K. Chromatographia 1992, 34, 281302. (6) Abraham, M. H.; Whiting, G. S.; Doherty, R. M.; Shuely, W. J. J. Chem. Soc., Perkin Trans. 2 1990, 1451-1460. (7) Abraham, M. H. Anal. Chem. 1997, 69, 613-617. (8) Carr, P. W. Microchem. J. 1993, 48, 4-28. (9) Park, J. H.; Carr, P. W.; Abraham, M. H.; Taft, R. W.; Doherty, R. M.; Kamlet, M.J. Chromatographia 1988, 25, 373-381. (10) Sadek, P. C.; Carr, P. W.; Doherty, R. M.; Kamlet, M. J.; Taft, R. W.; Abraham, M. H. Anal. Chem. 1985, 57, 2971-2978. (11) Carr, P. W.; Doherty, R. M.; Kamlet, M. J.; Taft, R. W.; Melander, W.; Horvath, C. Anal. Chem. 1986, 58, 2674-2680. (12) Tan, L. C.; Carr, P. W.; Abraham, M. H. J. Chromatogr. 1996, 752, 1-18. S0003-2700(97)00398-3 CCC: $14.00
© 1997 American Chemical Society
Table 1. Calculated Properties for Carbon Dioxide/ HFC-134a Mixed Mobile Phases at 200 bar4 HFC-134a
Table 2. Physical Properties of Bulk Fluids4 fluid
TC (°C)
PC (bar)
dipole (D)
polarizability (cm3/mol)
carbon dioxide HFC-134a
30.9 101.2
73.8 40.7
1.46a 2.06
14.0 13.7
F (g/mL)
vol %
mol %
TC (°C)
at 75 °C
at 125 °C
0.0 20.0 40.0 54.2 60.0 62.2 80.0 100.0
0.0 13.1 29.0 41.8 47.7 50.0 70.6 100.0
30.9 51.0 64.7 75.0 79.4 81.1 94.9 101.2
0.629 0.933 1.065 1.141 1.169 1.179 1.250 1.147
0.386 0.576 0.796 0.932 0.979 0.996 1.107 0.985
a
Net dipole is 0.00 due to molecular symmetry.
parameters for each of the analytes are given in Table 3.15-18
EXPERIMENTAL SECTION Chromatographic System. The chromatographic system used in this study was a Gilson SF3 system (Gilson, Inc. Middleton, WI). Carbon dioxide mobile phase was pumped with a Gilson Model 308 pump with thermostated head. 1,1,1,2Tetrafluoroethane (HFC-134a) was pumped with a Gilson Model 306 pump. Mixing took place in a Gilson Model 811C dynamic mixer with a 1.5 mL mixing chamber. Fixed-loop injections (5 µL) were made using a Gilson Model 231XL sampling injector. Column thermostating was accomplished using a Gilson Model 831 temperature regulator. Detection was accomplished at 210 nm using a Gilson Model 117 variable wavelength UV detector with a 7 µL high-pressure flow cell. Column back pressure was maintained using a Gilson Model 821 pressure regulator. Columns. Jordi-Gel RP-C18 columns (250 mm × 4.6 mm; 5 µm particles) were obtained from Alltech Associates (Waukegan, IL). The packing material consists of highly cross-linked divinylbenzene with octadecyl ligands bonded via secondary arylamine linkages. Chromatographic Conditions. Various probe analytes were chromatographed using carbon dioxide/HFC-134a mixed mobile phases. Fluids were mixed at approximately 200 bar pressure at 25 °C. Total flow through the system was 1.0 mL/min (liquid flow). Back pressure was maintained at 200 bar. Table 1 gives the calculated critical parameters and densities for the mixed mobile phases, as calculated using NIST REFPROP v4.0.4 The relevant physicochemical properties of carbon dioxide and HFC134a are given in Table 2. Chemicals. Probe analytes listed in Table 3 were obtained from Aldrich (Milwaukee, WI) and were reagent grade or better. 1,1,1,2-Tetrafluoroethane (SUVA grade) was obtained from Dupont (Deepwater, NJ). SFC grade carbon dioxide (without helium headspace) was obtained from Scott Specialty Gases (Plumsteadville, PA). Samples. Solutions of the probe analytes were prepared in methanol to a final concentration of 1.0 mg/mL. The solvation
RESULTS AND DISCUSSION General Retention Characteristics. The primary motivation for mixing carbon dioxide with HFC-134a is to predictably control the eluotropic strength of the resulting binary mobile phase. Figure 1 shows the effect of mobile phase composition on retention for three types of analytes at two different temperatures. The retention behavior of toluene is representative of analytes which are either weak hydrogen bond acceptors or have no hydrogen bonding ability. At both temperatures, pure carbon dioxide shows slightly higher eluotropic strength than either pure HFC-134a or binary mixtures of the two fluids. None of the mobile phase combinations produce significant retention, though. The retention behavior of nitrobenzene, shown in Figure 1, is representative of that observed for strong hydrogen bond accepting compounds. At 75 °C, retention decreases as the proportion of HFC-134a in the mobile phase increases up to approximately 75% HFC-134a. Above this concentration, retention begins to increase as the proportion of HFC-134a increases. From the data in Table 1, this proportion of HFC-134a does not coincide with any anomalous occurrence, such as the molar equivalence point between the two fluids or the transition from subcritical to supercritical conditions. At 125 °C, increasing proportions of HFC134a produce lower retention factors with no observed minima in retention. Analytes which can act as both hydrogen bond donors and acceptors are represented by phenol in Figure 1. Here, as in the case for nitrobenzene, retention decreases as the proportion of HFC-134a in the mobile phase increases. At approximately 75% HFC-134a, retention reaches a minimum at 75 °C and begins to increase as the proportion of HFC-134a is further increased. At 125 °C, the retention factor appears to level off, with only a slight increase in retention as the mobile phase approaches pure HFC134a. One factor which is often cited as the primary driving force for elution in SFC is mobile phase density. As density increases, retention decreases due to the higher solvating ability of the mobile phase. Figure 2 shows the data from Figure 1 plotted versus mobile phase density. Although there is some correlation between mobile phase density and the logarithm of the retention factor, the correlations are poor. Retention for all of the analytes in 100% HFC-134a is very poorly represented. This suggests that specific molecular interactions between the mobile phase and the analytes may contribute significantly to the retention of these analytes. LSER Regressions. Analysis of the retention characteristics of a wide variety of analytes, in terms of specific molecular
(13) Pyo, D.; Li, W.; Lee, M. L.; Weckwerth, J. D.; Carr, P. W. J. Chromatogr. 1996, 753, 291-298. (14) Cantrell, G. O.; Stringham, R. W.; Blackwell, J. A. Anal. Chem. 1996, 68, 3645-3650.
(15) Weckwerth, J. D. Ph.D. Thesis, University of Minnesota, 1996. (16) Abraham, M. H.; McGowan, J. C. Chromatographia 1987, 23, 243-246. (17) Abraham, M. H. Chem. Soc. Rev. 1993, 73-83. (18) Havelec, P.; Sevcik, J. G. K. J. Phys. Chem. Ref. Data 1996, 25, 1483-1493.
dipolarity/polarizability, and other intermolecular interactions at 75 and 125 °C with varied binary mobile phase compositions ranging from pure carbon dioxide to pure HFC-134a. The results should help to rationalize the differences in apparent eluotropic strength for various types of compounds using these binary mobile phase systems.
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Table 3. Solvation Parameters for Probe Analytes15-17
a
analyte
R2
π2H
ΣR2H
Σβ2H
Log L16
biphenyl chlorobenzene bromobenzene iodobenzene nitrobenzene benzaldehyde methyl phenyl ether phenetole phenol benzyl alcohol naphthalene methylnaphthalene ethylnaphthalene phenylnaphthalene fluoronaphthalene chloronaphthalene bromonaphthalene iodonaphthalene nitronaphthalene cyanonaphthalene naphthaldehyde methoxynaphthalene ethoxynaphthalene naphthyl acetate naphthalenemethanol naphthaleneethanol (N,N-dimethylamino)naphthalene naphthylacetonitrile propiophenone acetophenone butylbenzene propylbenzene 1,4-dichlorobenzene ethyl benzoate benzyl chloride 1,2-dichlorobenzene methyl benzoate 1,3,5-trimethylbenzene diphenylmethane 2-nitrotoluene 3-nitrotoluene 4-nitrotoluene o-xylene m-xylene p-xylene o-cresol m-cresol p-cresol 2-nitrophenol
1.360 0.718 0.882 1.188 0.871 0.820 0.708 0.681 0.805 0.803 1.380 1.440 1.430 1.910 1.320 1.540 1.670 1.840 1.350 1.190 1.470 1.700 1.630 1.130 1.640 1.670 1.570 1.430 0.804 0.818 0.600 0.604 0.825 0.689 0.821 0.872 0.733 0.649 1.220 0.866 0.874 0.870 0.663 0.623 0.613 0.840 0.822 0.820 1.015
0.990 0.650 0.730 0.820 1.110 1.000 0.750 0.700 0.890 0.870 0.850 0.870 0.870 1.080 0.820 0.920 0.970 1.040 1.290 1.250 1.190 0.990 0.960 1.250 1.190 1.210 0.930 1.440 0.950 1.010 0.510 0.500 0.750 0.850 0.820 0.780 0.850 0.520 1.040 1.110 1.100 1.110 0.560 0.520 0.520 0.860 0.880 0.870 1.050
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.60 0.33 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.27 0.23 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.52 0.57 0.57 0.05
0.22 0.07 0.09 0.12 0.28 0.39 0.29 0.32 0.30 0.56 0.22 0.22 0.23 0.30 0.18 0.15 0.17 0.20 0.36 0.41 0.47 0.37 0.40 0.62 0.64 0.72 0.49 0.53 0.51 0.48 0.15 0.15 0.02 0.46 0.33 0.04 0.46 0.19 0.28 0.27 0.25 0.28 0.16 0.16 0.16 0.30 0.34 0.31 0.37
6.014 3.657 4.041 4.502 4.557 4.008 3.890 4.242 3.766 4.221 5.236a 5.719a 6.138a 8.532a 5.200a 6.024a 6.399a 6.890a 6.805a 6.414a 6.530a 6.377a 6.715a 7.284a 6.550a 7.052a 7.019a 6.961a 4.971 4.501 4.730 4.230 4.435 5.075 4.384 4.518 4.704 4.344 6.313 4.878 5.097 5.154 3.939 3.839 3.839 4.218 4.310 4.312 4.760
Value calculated using the method of Havelec and Sevcik.18
interactions, is readily accomplished using linear solvation energy relationships (LSERs). Since it is apparent that specific molecular interactions between the mobile phase and solutes contribute to retention, application of LSER methodology should help to elucidate the types and relative magnitudes of these interactions. In this study, we use the following equation to relate retention to the solvation properties of the probe analytes listed in Table 3:
log k′ ) SP0 + l Log L16 + sπ2H + aΣR2H + bΣβ2H + rR2 (1) where SP0 is the regression intercept, Log L16 is the gas-tohexadecane partition coefficient at 25 °C, π2H is the analyte dipolarity/polarizability, ΣR2H is the analyte effective hydrogen bond donating ability, Σβ2H is the analyte effective hydrogen bond accepting ability, and R2 is the analyte excess molar refraction. Using LSER methodology, the coefficients l, s, a, b, and r obtained via multilinear regression represent the differences 4610
Analytical Chemistry, Vol. 69, No. 22, November 15, 1997
between the analyte-stationary phase interactions and the analyte-mobile phase interactions with respect to that particular property. Therefore, a positive s coefficient would indicate that the stationary phase dipolar/polarizable interactions with the analyte are dominant over the mobile phase interactions with the analyte. A negative b coefficient would, on the other hand, indicate that the mobile phase hydrogen bond donating ability is stronger than the donating ability of the stationary phase, due to the complementary nature of hydrogen bonding interactions. Using this equation for six different mobile phase compositions at two temperatures each results in an overall correlation coefficient of 0.991 ( 0.004, with a regression standard deviation of 0.061 ( 0.011. The regression coefficients and their standard deviations for each of the individual regressions are given in Tables 4 and 5 for the 75 and 125 °C data, respectively. Since the goal of this work is to elucidate the relative contributions of various intermolecular interactions using LSERs, emphasis is placed on the relative values of each of the regression
Figure 1. Retention of various analytes as a function of mobile phase composition at (a) 75 and (b) 125 °C. Analytes are (b) toluene, (9) nitrobenzene, and (2) phenol. Flow rate was 1.0 mL/min, with a back pressure of 200 bar on a 250 nm × 4.6 mm Jordi-Gel RP-C18 column.
Figure 2. Retention of various analytes as a function of mobile phase density at (a) 75 and (b) 125 °C. Analytes are (O) toluene, (0) nitrobenzene, and (4) phenol. Filled symbols indicate retention factors observed using 100 vol % HFC-134a. Flow rate was 1.0 mL/min, with a back pressure of 200 bar on a 250 mm × 4.6 mm Jordi-Gel RP-C18 column.
coefficients rather than the absolute values. In reversed-phase HPLC, for example, LSERs have been used to demonstrate that solute molecular volume and hydrogen bond accepting ability are the primary factors responsible for determining retention.8-12 Other factors, such as hydrogen bond donating ability, dipolarity/ polarizability, and excess molar refraction are relatively unimportant in determining overall retention. In order to properly emphasize this point, Figures 3-8, which depict the changes in LSER coefficients as a function of mobile phase composition, are all plotted with the same abscissa range. This range encompasses all of the coefficients and their standard deviations and should
allow for facile comparison of the relative effects of each intermolecular interaction. Error bars indicate the standard deviation for each of the coefficients determined with the multilinear regression. Log L16 Term. The Log L16 term in eq 1 represents the combination of two separate energetic processes. The first process is the energetically unfavorable formation of a cavity for the analyte to occupy in the stationary or mobile phase. The second process, which is the energetically favorable and dominant factor, is the dispersive interaction between the analyte and the stationary or mobile phase.19 Figure 3 shows that the coefficient Analytical Chemistry, Vol. 69, No. 22, November 15, 1997
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Table 4. LSER Regression Coefficients for HFC-134a/ CO2 Mixed Mobile Phases at 75 °C HFC-134a content (%) 0.0
20.0
40.0
60.0
80.0
100.0
r 0.51 0.58 0.64 0.69 0.77 0.86 SD 0.05 0.04 0.05 0.05 0.06 0.07 s 0.88 0.53 0.34 0.20 0.13 0.05 SD 0.06 0.05 0.06 0.07 0.08 0.09 a 1.88 1.77 1.79 1.77 1.75 1.62 SD 0.05 0.05 0.06 0.06 0.07 0.08 b -0.34 -0.38 -0.46 -0.53 -0.63 -0.68 SD 0.07 0.06 0.07 0.08 0.09 0.11 l 0.14 0.12 0.10 0.10 0.09 0.08 SD 0.02 0.02 0.02 0.02 0.02 0.03 intercept -1.54 -1.21 -1.10 -0.98 -0.90 -0.67 SD 0.04 0.04 0.05 0.05 0.06 0.07 correl coeff 0.995 0.994 0.992 0.990 0.987 0.983 regression SD 0.05 0.05 0.06 0.06 0.07 0.08
Table 5. LSER Regression Coefficients for HFC-134a/ CO2 Mixed Mobile Phases at 125 °C HFC-134a content (%) 0.0
20.0
40.0
60.0
80.0
100.0
r 0.36 0.45 0.51 0.54 0.60 0.69 SD 0.05 0.04 0.04 0.05 0.05 0.06 s 0.90 0.75 0.55 0.38 0.29 0.18 SD 0.06 0.05 0.05 0.06 0.06 0.07 a 1.35 1.28 1.28 1.33 1.43 1.42 SD 0.06 0.05 0.05 0.06 0.06 0.07 b -0.18 -0.27 -0.33 -0.41 -0.48 -0.53 SD 0.07 0.07 0.06 0.08 0.08 0.09 l 0.20 0.15 0.12 0.10 0.09 0.08 SD 0.02 0.02 0.02 0.02 0.02 0.02 intercept -1.57 -1.44 -1.22 -1.10 -1.04 -0.93 SD 0.05 0.04 0.04 0.05 0.05 0.06 correl coeff 0.994 0.994 0.994 0.989 0.989 0.986 regression SD 0.06 0.05 0.05 0.06 0.06 0.07
for this term is positive and rather small. In addition, there is very little effect of mobile phase composition on this term. At 125 °C, the coefficient obtained using pure carbon dioxide is slightly higher than that obtained with increasing concentrations of HFC-134a in the mobile phase. Overall, the change is minimal and relatively insignificant. These observations suggest that the dispersive interactions between the analytes and the stationary phase are stronger than the dispersive interactions between the analyte and the mobile phase, regardless of the percentage of HFC-134a. Under near-critical conditions, it is also very likely that the cavity formation process in the stationary phase is much more difficult than that in the mobile phase, based on density considerations alone. The magnitude of the coefficient indicates that the dispersive interactions between the analyte and the stationary or mobile phase are relatively small. Since both of the mobile phase components are excellent solvents for the alkyl ligands bound to the stationary phase surface, the bonded phase will contain a relatively high concentration of carbon dioxide and/or HFC-134a. This solvated ligand environment will not differ substantially, with respect to dispersive interaction properties, from the mobile phase. Although these coefficients are relatively small, this does not suggest that the Log L16 value of an analyte has little effect on (19) Abraham, M. H.; Whiting, G. S.; Doherty, R. M.; Shuely, W. J. J. Chromatogr. 1990, 518, 329-348.
4612 Analytical Chemistry, Vol. 69, No. 22, November 15, 1997
Figure 3. LSER coefficient for Log L16 at various HFC-134a/carbon dioxide mobile phase compositions at (b) 75 and (9) 125 °C. Chromatographic conditions are as in Figure 1. Error bars indicate the standard deviations for each coefficient.
Figure 4. LSER coefficient for dipolarity/polarizability at various HFC-134a/carbon dioxide mobile phase compositions at (b) 75 and (9) 125 °C. Chromatographic conditions are as in Figure 1. Error bars indicate the standard deviations for each coefficient.
retention in SFC using these mobile phases with this column. On the contrary, the Log L16 value of an analyte is very significant, primarily due to the fact that most compounds chosen for analysis using SFC have rather large Log L16 values. Dipolarity/Polarizability Term. The effect of mobile phase composition on the dipolarity/polarizability term is shown in Figure 4. Compared to the Log L16 coefficient, this term is much larger and shows a higher degree of mobile phase composition dependence. As the temperature is raised from 75 to 125 °C, the binary mobile phase coefficients increase in magnitude. Since this term describes the difference between the analyte-stationary phase and the analyte-mobile phase dispersive and polarizability
Figure 5. LSER coefficient for solute hydrogen bond donating ability at various HFC-134a/carbon dioxide mobile phase compositions at (b) 75 and (9) 125 °C. Chromatographic conditions are as in Figure 1. Error bars indicate the standard deviations for each coefficient.
Figure 7. LSER coefficient for excess molar refraction at various HFC-134a/carbon dioxide mobile phase compositions at (b) 75 and (9) 125 °C. Chromatographic conditions are as in Figure 1. Error bars indicate the standard deviations for each coefficient.
Figure 6. LSER coefficient for solute hydrogen bond accepting ability at various HFC-134a/carbon dioxide mobile phase compositions at (b) 75 and (9) 125 °C. Chromatographic conditions are as in Figure 1. Error bars indicate the standard deviations for each coefficient.
Figure 8. LSER regression intercept at various HFC-134a/carbon dioxide mobile phase compositions at (b) 75 and (9) 125 °C. Chromatographic conditions are as in Figure 1. Error bars indicate the standard deviations for each coefficient.
interactions, it is evident that the analyte-stationary phase interactions dominate over the analyte-mobile phase interactions. This is not surprising, since the divinylbenzene backbone of the polymeric stationary phase is expected to have a significant dipolarity/polarizability character. Ethylbenzene, for comparison, has a π2H value of 0.51.17 From the magnitude of the dipolarity/ polarizability coefficients, it is apparent that the carbon dioxide mobile phase is substantially lower in dipolarity/polarizability than the stationary phase, resulting in a large, positive s coefficient. HFC-134a, on the other hand, is relatively stronger in its dipolar/ polarizable interactions with the analytes, resulting in a smaller net coefficient. Inspection of Table 3 shows that carbon dioxide
and HFC-134a do not differ significantly with respect to their polarizabilities, but show larger differences in dipolarities. This suggests that the dipolar interaction between the analyte and the mobile phase may be more important than the analyte-mobile phase polarizability interactions. Hydrogen Bond Donating Ability. Figure 5 shows that one of the most significant factors governing retention in this system is the analyte hydrogen bond donating ability. There appears to be little mobile phase compositional dependence on this coefficient. Consistent with the exothermic nature of hydrogen bonding, the coefficient decreases as temperature increases. Due to the complementary nature of hydrogen bonding, this term Analytical Chemistry, Vol. 69, No. 22, November 15, 1997
4613
describes the difference in hydrogen bond accepting abilities between the stationary and mobile phases. The large, positive coefficients shown in Figure 4 indicate that the hydrogen bond accepting ability of the stationary phase far exceeds that of the mobile phase. Based on the nature of the stationary phase, this is not surprising. The secondary amine linker binding the alkyl ligand to the aromatic polymer backbone is expected to be a fairly good hydrogen bond acceptor, while the aromatic polymer backbone should contribute to the overall hydrogen bond basicity as well. N-Methylaniline, for example, has a β2H value of 0.47, while ethylbenzene has a β2H value of 0.15.20 It is apparent that neither carbon dioxide nor HFC-134a shows any appreciable hydrogen bond accepting ability. While this is not surprising based on the structure of HFC-134a, it is a bit surprising that carbon dioxide does not show any significant hydrogen bond accepting ability with its two electron-rich oxygen atoms. It is also interesting to note that the hydrogen bond donating ability of the analyte is not attenuated by the presence of HFC-134a. Due to the highly dipolar nature of HFC-134a, it may be anticipated to be a strong hydrogen bond donor. This donating ability would be expected to compete with the analyte hydrogen bond donating ability for the available hydrogen bond accepting sites on the stationary phase surface, thus attenuating this interaction. Pyo et al. found that methanol was very effective in this manner using a silica-based octadecyl stationary phase under supercritical conditions.13 Evidently, this competitive interaction is muted under the conditions employed in this study. Hydrogen Bond Accepting Ability. The effect of analyte hydrogen bond accepting ability is quite different from that of the hydrogen bond donating ability. As shown in Figure 6, the analyte hydrogen bond accepting ability coefficient is small and negative. This indicates that the hydrogen bond donating ability of the mobile phase is dominant over the donating ability of the stationary phase. While this makes sense with respect to the stationary phase (hydrogen bond donating ability of N-methylaniline is 0.17, ref 20), the carbon dioxide mobile phase has no hydrogen bond donating ability. The small coefficients obtained for pure carbon dioxide are, therefore, likely to be artifacts of the regression technique. As the proportion of HFC-134a increases, the coefficient becomes larger, which is consistent with the presumed hydrogen bond donating ability of HFC-134a. Surprisingly, the magnitude of the change in the coefficient, going from 0 to 100% HFC-134a, is rather small. This, combined with the observations made regarding analyte hydrogen bond donating ability, suggest that, under these conditions, HFC-134a is not a very strong hydrogen bond donor despite its large dipole. Overall, the hydrogen bond accepting ability of the analyte has little effect on retention in this system. Excess Molar Refraction. The ability of the analyte to interact with the stationary phase or mobile phase through interactions between π electrons is measured, in part, by the excess molar refraction coefficient. As shown in Figure 7, this factor is relatively significant under the conditions employed in this study. In pure carbon dioxide, the coefficient is positive, indicating that the interactions between the analyte and the stationary phase are dominant over those between the analyte and the mobile phase. Although the aromatic nature of the polymeric backbone is expected to interact strongly with the analyte, (20) Li, J.; Zhang, Y.; Dallas, A. J.; Carr, P. W. J. Chromatogr. 1991, 550, 101134.
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Analytical Chemistry, Vol. 69, No. 22, November 15, 1997
interactions between the analyte and the π electrons of carbon dioxide attenuate the overall effect. When the proportion of HFC-134a is increased, the coefficient increases. This reflects the dilution of carbon dioxide with a mobile phase component which lacks the ability to interact via π electrons. The result is that the stationary phase becomes even more dominant over the mobile phase. When the temperature is increased from 75 to 125 °C, the coefficient uniformly decreases at all mobile phase compositions. Regression Intercept. From a theoretical standpoint, the regression intercept represents retention in a reference system.8 From a practical standpoint, the regression intercept is a collection of various unaccounted intermolecular interactions, changes in phase ratio, and a variety of other undefined factors. Figure 8 shows that the regression intercepts obtained in this study are both large and variable. Although there is a relatively small temperature dependence on the intercepts, there is a relatively large mobile phase compositional dependence. Pure carbon dioxide produces very large intercepts which are unaffected by operating temperature. HFC-134a produces smaller intercepts which decrease as the temperature decreases. While little chemical information may be derived from these intercepts, it should be noted that they are very significant factors which govern the resulting log k′ data. Among these factors are the Lewis acidities of carbon dioxide and HFC-134a. CONCLUSIONS The apparent eluotropic strength of binary carbon dioxide/ HFC-134a mobile phases is very dependent upon the structure of the analyte and the operating temperature. Many analytes show decreased retention in a mixed mobile phase compared to that of either pure mobile phase component. Using LSER methodology, it is apparent that the hydrogen bond donating ability of the analyte is the dominant factor governing retention on this stationary phase using carbon dioxide, HFC-134a, or a combination of these two fluids. Dipolarity/ polarizabilty and excess molar refraction also contribute significantly to retention, although the effect of analyte dipolarity/ polarizability decreases as the proportion of HFC-134a in the mobile phase increases. As the proportion of HFC-134a in the mobile phase increases, the effect of analyte excess molar refraction becomes more significant. The effect of analyte hydrogen bond accepting ability also increases as the proportion of HFC-134a in the mobile phase increases, although the effect of this factor is relatively small. The effect of cavity formation/ dispersive interactions, as measured by the Log L16 coefficient, is relatively small and insensitive to either operating temperature or mobile phase composition. As temperature is increased, the effect of dipolarity/polarizability increases, while all of the other factors decrease.
Received for review April 15, 1997. Accepted September 3, 1997.X AC970398Q X
Abstract published in Advance ACS Abstracts, October 1, 1997.
