Anal. Chem. 1997, 69, 1414-1420
Factors That Control Successful Entropically Driven Chiral Separations in SFC and HPLC Rodger W. Stringham* and John A. Blackwell
CPR&D, The DuPont Merck Pharmaceutical Company, Chambers Works PRF (S1), Deepwater, New Jersey 08023-0999
With temperature increases, selectivity of chiral separations decreases until enantiomers coelute at an isoelution temperature. Above this temperature, elution order should reverse and selectivity will increase with temperature. In this region, separation is termed “entropically driven”. Entropically driven chiral separations hold the promise of being able to concurrently increase selectivity and column efficiency by means of increased temperature. The ability to achieve such separations is hindered by high isoelution temperatures. The isoelution temperature is determined by a balance of enthalpic and entropic contributions. A variety of mobile phase modifiers are evaluated for their ability to moderate these contributions. Results suggest that more use should be made of nonalcohol modifiers. The major barrier to entropically driven separations was found to be the nonspecific retention increase that is characteristic when the critical temperature is traversed. Use of hexane in place of CO2 shifts the position of the retention increase away from the temperature range used in this study, and dramatically successful entropically driven chiral separations are obtained. The feasibility of entropically driven chiral separations in SFC has recently been demonstrated.1 This phenomenon is predicted from thermodynamic relationships between retention and temperature. Chromatographic selectivity, defined as the ratio of capacity factors, may be related to temperature as
ln R ) -δ∆H°/RT + δ∆S°/R
(1)
where R is the ideal gas constant and T is absolute temperature. The δ∆H° term represents the difference between the enthalpy of the enantiomers’ interaction with the stationary phase, and δ∆S° represents the entropic differences. Plotting ln R vs 1/T should give a straight line with a slope of -δ∆H°/R and a y-intercept of δ∆S°/R. The x-intercept corresponds to R ) 1.0. At this temperature (Tiso), the enthalpic and entropic contributions to chiral recognition are balanced and the enantiomers coelute. Above this temperature, elution order of the enantiomers will switch and chiral recognition is said to be entropically controlled.2 Selectivity, now for the reversed elution order, increases with temperature. Column efficiency is also expected to increase with temperature as diffusion rates increase. This combination makes entropically driven separations especially attractive. The observa(1) Stringham, R. W.; Blackwell, J. A. Anal. Chem. 1996, 68, 2179-2185. (2) Schleimer, M.; Schurig, V. Analysis with Supercritical Fluids Extraction and Chromatography; Springer Verlag; New York, 1992; p 135.
1414 Analytical Chemistry, Vol. 69, No. 7, April 1, 1997
tion of entropically driven chiral separations in SFC has been thus far limited to two analytes,1 and the observed separations were insufficient to be useful. This paper details attempts to better understand the barriers to achieving successful entropically driven chiral separations. An obvious barrier to useful entropic separations is elevated isolelution temperatures. The isoelution temperature represents a balance of enthalpic and entropic contributions. At Tiso, ln R ) 0 and δ∆H°/RT ) δ∆S°/R. Solving for 1/T
1/Tiso ) δ∆S°/δ∆H°
(2)
Tiso ) δ∆H°/δ∆S°
(3)
Lowering Tiso would require an increase in δ∆S° or a decrease in δ∆H°. It is preferable to lower Tiso through δ∆S° since a decrease in δ∆H° would limit gains in selectivity with increasing temperature. Predicting parameters that will affect δ∆S° is difficult. Solvation differences and statistical concepts of molecular flexibility are commonly invoked when entropic terms are discussed. It is difficult to conceptualize a chiral character in entropy, but the results of molecular modeling are helpful. Lipkowitz3,4 reviewed the progression of the modeling of interactions between analytes and chiral stationary phases. For modeling to be successful, flexibility and solvation of both the analyte and chiral selector must be incorporated. Sabio and Topiol5 used molecular dynamics calculations to show entropic differences between enantiomers complexed with a chiral selector. While these authors were able to qualitatively compare entropic contributions to chiral recognition, they could only postulate that entropic differences could result in separation. According to Lipkowitz,4 the analyte and chiral selector exist in contracted, solvated minimum energy conformations until they encounter one another. As a result of this encounter, the conformations tend to unravel somewhat to maximize the attractive dispersion forces between analyte and selector. This unraveling to a less stable form is offset by a gain in energy from complexation. Unraveling is also accompanied by solvation changes, an increased flexibility, and a gain in entropy. For weak complexes between analyte and selector (as would be required for chromatographic partitioning), the more stable complex (longer retained) shows the most unraveling. This complex would have the larger ∆H° of the two due to increased attractive forces, and the increased unraveling would give larger ∆S° as well. This presents a somewhat discouraging correlation between enthalpy and entropy, suggest(3) Lipkowitz, K. B. J. Chromatogr., A 1994, 666, 493-503. (4) Lipkowitz, K. B. In Theoretical and Computational Chemistry; Herndon, W., Parkanyi, C., Eds.; Elsevier: New York, 1996; Vol. 5. (5) Sabio, M.; Topiol, S. Chirality 1991, 3, 56-66. S0003-2700(96)00928-6 CCC: $14.00
© 1997 American Chemical Society
Table 1. Solvatochromic Properties of Selected Modifiersa
c e
modifier
π*b
Rc
βd
methanol ethanol 2-propanol 1-propanol 1-butanol tetrahydrofuran trifluoroethanol
0.60 0.54 0.48 0.52 0.47 0.58 0.73
0.93 0.83 0.76 0.78 0.79 0.00 1.51
0.62 0.77 0.95 n/ae 0.88 0.55 0.00
a See refs 7-10 for additional modifiers. b polarizability/dipolarity. hydrogen bond donating ability. d hydrogen bond accepting ability. Not available.
ing that it may be impossible to increase δ∆S° without also increasing δ∆H°. Lipkowitz states that solvation differences between enantiomer complexes can be significant, which also contributes to differences in ∆S°. This leads to the suggestion to test different solvents for their effect on entropic differences. The δ∆H° term is a function of the strength of interactions between individual enantiomers and the chiral stationary phase. The interaction between an analyte and a chiral selector is the sum of individual interactions between functional groups of the analyte and selector. Analyte enantiomers differ in the position of these functional groups relative to the selector, resulting in some interactions being of different strength. Using a three-point interaction model, two interactions between analyte and selector are the same for each enantiomer and one is different. This third interaction gives rise to δ∆H°. The other interactions do not contribute to chiral selectivity but do contribute to retention. The use of mobile phase conditions that weaken nonselective interactions sufficiently to allow elution may also attenuate the selective interaction. Ideally, selection of proper mobile phase modifiers that attenuate only the nonselective interactions would yield the highest δ∆H° values. Cantrell et al.6 reported that retention in packed-column achiral SFC can be well described in terms of solvatochromic equations which relate chromatographic behavior to modifier properties. From these data, modifiers may be ranked in terms of ability to modify hydrogen bond and other interactions. While the solvatochromic approach does not currently allow the prediction of chiral selectivity, it does offer suggestions for modifier selection. Extensive tabulations of modifier properties are available.7-10 Interactions with Pirkle type chiral columns typically involve hydrogen-bonding and π-electron interactions. Modifier selection should then be guided by evaluating modifiers with different abilities to interfere with these interactions. Alcohols are the most common modifiers in chiral SFC. Examination of Table 1 reveals that alcohols function as both hydrogen bond donors and acceptors. Trifluoroethanol (THE) is a potent hydrogen bond donor with no accepting ability. Tetrahydrofuran (THF) acts as an (6) Cantrell, G. O.; Stringham, R. W.; Blackwell, J. A.; Weckwerth, J. D.; Carr, P. W. Anal. Chem. 1996, 68, 3645-3650. (7) Kamlet, M. J.; Abboud, J. L. M.; Abraham, M. H.; Taft, R. W. J. Org. Chem. 1993, 48, 2877-2887. (8) Reid, R. C.; Prausnitz, J. M.; Poling, B. E. The Properties of Gases and Liquids, 4th ed.; McGraw-Hill: New York, 1987. (9) CRC Handbook of Chemistry and Physics, 74th ed.; Linde, D. R., Ed.; CRC Press: Boca Raton, FL, 1993. (10) Lange’s Handbook of Chemistry, 13th ed.; Dean, J. A., Ed.; McGraw-Hill: New York, 1985.
acceptor but is a poor donor. Use of these modifiers may offer the ability to modify nonspecific hydrogen bond interactions without attenuating hydrogen bonds involved in the chiral recognition process. Common alcohols along with trifluoroethanol and THF were evaluated for their effects on δ∆H°. In addition to barriers imposed by the limitations in the ability to control δ∆H° and δ∆S°, observation of useful entropically driven chiral separations may be obscured by the failure of the inherent assumptions contained in eq 1. One assumption is that retention mechanism does not change with temperature. Interactions between analytes and chiral selectors may occur in a multitude of orientations, and the preference of orientation may be temperature sensitive. A change from one preferred orientation to another should be manifested chromatographically by a split peak for a single enantiomer (or at least a significant broadening). Thus, the failure of this assumption would be observable. A second assumption made in the application of eq 1 is that δ∆H° does not change with temperature (constant heat capacity). The consequence of the failure of this assumption is shown to be the largest barrier to observation of entropically driven separations. Selection of mobile phase conditions that maintain the validity of this assumption result in dramatic separations. EXPERIMENTAL SECTION Columns. A 25 cm (S,S) Whelk-O (Regis Chemical Co., Morton Grove, IL) column end-capped according to the procedure of Pirkle and Readnour11 was used throughout this study. This is a brush-type column that has wide applicability, which has been shown to be stable to elevated temperatures.1 Chromatography. Chromatography was carried out using a Hewlett-Packard G 1205A SFC equipped with cryogenic cooling. The mobile phase consisted of a variety of modifiers entrained in carbon dioxide at 10-25 vol %, pumped at a flow rate of 1.5 mL/ min and column outlet pressures ranging from 120 to 300 bar. Column temperature varied from -20 to 190 °C. Injection volume was 5 µL, and detection was via UV absorbance at 210 nm. Modifiers included methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, TFE, and THF. Probe Samples. Test compounds were selected to represent a spectrum of resolution on the test column. These compounds include benzoin and (Z)-phenylalaninol, which were obtained from Aldrich (Milwaukee, WI) and Degussa (Ridgefield Park, NJ), respectively. Compounds 1-6 (Figure 1) were synthesized as part of process development at DuPont Merck. Individual enantiomers and artificial racemic mixtures were prepared at 1-2 mg/ mL in ethanol. Chromatographic Performance. Retention times (tR, taken at peak maximum), N, and resolution calculations were performed by the Hewlett-Packard G 1205A SFC Chem Station. t0 was estimated from the earliest baseline deflection or from injection of CCl4. Capacity factor, k′, was calculated from (tR - t0)/t0. Selectivity (R) was calculated as the ratio of capacity factors for individual enantiomers. Regressions of ln R data were performed by Microsoft Excel 4.0a giving the relationship ln R ) slope (1/T) + intercept. Tiso was calculated from the x-intercept. RESULTS AND DISCUSSION Effects of Modifier on δ∆S°, δ∆H°, and Tiso. All eight probe molecules were chromatographed with methanol, ethanol, (11) Pirkle, W. H.; Readnour, R. S. Chromatographia 1991, 31, 129-132.
Analytical Chemistry, Vol. 69, No. 7, April 1, 1997
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Table 2. Effect of Modifier Selection on Thermodynamic Parameters probe benzoin
(Z)-phenylalalinol
1
2
3 Figure 1. Structures of probe molecules used in this study.
1-propanol, 2-propanol, 1-butanol, THE, and THF as modifiers at three temperatures to determine their effect on enthalpic and entropic differences and Tiso. Results are given in Table 2. As a general trend, 2-propanol gave the largest δ∆S°/R values. 2-Propanol is bulkier than the other alcohols tested, and this bulk could accentuate solvation differences. THF gave large δ∆S°/R values for benzoin and (Z)-phenylalaninol but only a moderate value for 2. TFE gave the largest δ∆S°/R values for 2 and the smallest for (Z)-phenylalaninol. These differences cannot be explained in terms of modifier properties alone. Entropic differences may also be interpreted in terms of molecular flexibility in the solvated chiral recognition complex. These results suggest using 2-propanol as a first choice to maximize entropic differences but that other modifiers should also be evaluated. There was variation among the alcohols in their effect on δ∆H°/R but no trends were apparent. Of the alcohols, 2-propanol tends to give the largest δ∆H°/R. THF and TFE gave intriguing enthalpic results. TFE gave the smallest δ∆H°/R values for (Z)phenylalaninol and 1 and the largest for 2. THF gave the largest δ∆H°/R value for (Z)-phenylalaninol. All modifiers gave straight ln R plots for benzoin, (Z)-phenylalaninol, and compounds 1-3. For 4, both THF and TFE gave flat ln R plots (Figure 2) with elution order the same as that of the entropically driven separation observed with the alcohols. A flat ln R plot indicates that δ∆H° ) 0 and that any such separation is already entropically driven. 5 gave no separation with alcohol modifiers, gave the expected temperature behavior with THF, but gave ln R values with TFE that did not change with temperature. 6 gave the expected temperature behavior with THF, separation only at 5 °C with the alcohols, and with TFE again gave ln R values that did not change with temperature. 1416 Analytical Chemistry, Vol. 69, No. 7, April 1, 1997
4
5
6
modifier
Tiso (°C)
slopea
y-intercepta
methanol ethanol 1-propanol 2-propanol 1-butanol trifluoroethanol tetrahydrofuran methanol ethanol 1-propanol 2-propanol 1-butanol trifluoroethanol tetrahydrofuran methanol ethanol 1-propanol 2-propanol 1-butanol trifluoroethanol tetrahydrofuran methanol ethanol 1-propanol 2-propanol 1-butanol trifluoroethanol tetrahydrofuran methanol ethanol 1-propanol 2-propanol 1-butanol trifluoroethanol tetrahydrofuran methanol ethanol 1-propanol 2-propanol 1-butanol trifluoroethanol tetrahydrofuran methanol ethanol 1-propanol 2-propanol 1-butanol trifluoroethanol tetrahydrofuran methanol ethanol 1-propanol 2-propanol 1-butanol trifluoroethanol tetrahydrofuran
171 193 197 193 189 188 171 106 143 132 130 135 119 109 120 130 132 132 138 108 dneb 169 204 216 206 218 187 211 216 296 360 293 373 232 dne 62 74 69 60 66 flat*c flat* e e e e e flatc 91 f f f f f e 72
423 529 453 658 460 540 605 126 129 122 184 117 83 146 250 305 257 356 251 234 dne 95 108 83 127 80 176 106 462 546 422 725 424 516 dne 56 106 72 124 77 flat* flat* e e e e e flat 45 f f f f f e 55
-0.951 -1.135 -0.962 -1.412 -0.995 -1.162 -1.362 -0.331 -0.352 -0.300 -0.454 -0.286 -0.212 -0.383 -0.635 -0.758 -0.636 -0.758 -0.609 -0.610 dne -0.214 -0.229 -0.169 -0.266 -0.162 -0.382 -0.217 -0.944 -0.958 -0.639 -1.280 -0.619 -1.013 dne -0.160 -0.303 -0.210 -0.372 -0.227 flat* flat* e e e e e flat -0.123 f f f f f e -0.158
a Slope has units of K arising from δ∆H° (cal/mol)/R (cal/mol K). y-Intercept is a dimensionless number arising from δ∆S° (cal/mol K)/R (cal/mol K). Tiso is the x-intercept and may alternately be calculated from δ∆H°/δ∆S°. b dne, did not elute. c flat*, ln R essentially unchanged with temperature, elution order reversed. d flat, ln R essentially unchanged with temperature. e No separation. f Separation only at 5 °C.
Isoelution temperatures did change between modifiers. 2-Propanol’s large δ∆H° and δ∆S° values offset to give isoelution temperatures that are little different from those observed for other alcohols. Methanol consistently gives the lowest Tiso values, but the slope of ln R may be insufficient to allow observation of entropically driven separation. Reversal of elution order was
Table 3. Compound 4 Chromatographic Dataa
Figure 2. Plot of ln R vs 1/T for 4 with different modifiers. Common chromatographic conditions: 200 bar pressure; 1.5 mL/min flow rate. Key: (9) ethanol; (b) 1-butanol; (2) 2-propanol; (1) methanol; ([) 1-propanol; (+) trifluoroethanol; (×) tetrahydrofuran.
observed for 4 with all alcohols except methanol (Figure 2). Although no definitive conclusions may be drawn as to the best modifier for observation of entropically driven separations, these data demonstrate the value of testing modifiers other than the alcohols typically used in SFC. Validity of Single Binding Site Assumption. A major assumption that is made in eq 1 is that the retention mechanism does not change with temperature. The Whelk-O selector has a hydrogen bond donating site, a hydrogen bond accepting site, a π-basic region, and a π-acidic region.12 4 has three hydrogenbonding sites plus a π-basic region and a π-acidic region. Thus, multiple binding orientations are quite possible. The possibility of two competing binding sites for 4 raises the question as to whether entropically driven separation is in fact just a temperatureinduced change to a second binding site with the opposite elution order. Plotting ln R vs 1/T, ln R for one binding site would have a positive slope while ln R for the other binding site would have a negative slope. The observed ln R would still be a straight line with slope and intercept intermediate between the two. The only observable difference would be the lower slope and likely a broader flat region at ln R ) 0. In this flat region, binding to either site would be about equally likely. A single enantiomer would then give two peaks or at least a broader peak. 4 was chromatographed with 2-propanol from -20 to 200 °C in smaller temperature increments. Column efficiency obtained with a single enantiomer was measured across the entire range. Data are given in Table 3 and plotted in Figure 3. With increasing temperature (decreasing 1/T), ln R declines sharply to zero, stays flat over a range of 40 °C, and then declines again in the entropically driven region. With additional increases in temperature, ln R bottoms out and appears to rise again toward zero. As expected, column efficiency declines significantly at low temperatures. Efficiency does not decrease in the flat region of the curve. This strongly (12) Pirkle, W. H.; Welch, C. J.; Wilson, S. R. Chirality 1994, 6, 615-622.
temp (°C)
ln k′(R)
ln k′(S)
R
Nb
-20 -10 0 10 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 90 100 105 110 115 120 130 150 170 200
1.158 1.008 0.884 0.815 0.747 0.745 0.738 0.736 0.738 0.759 0.794 0.842 0.916 0.959 1.024 1.112 1.207 1.301 1.411 1.537 1.660 1.784 1.888 2.020 2.132 2.334 2.643 2.784 2.767
1.385 1.196 1.027 0.927 0.824 0.808 0.786 0.754 0.738 0.759 0.794 0.842 0.916 0.959 1.024 1.112 1.207 1.282 1.386 1.507 1.628 1.750 1.852 1.984 2.094 2.294 2.600 2.740 2.727
1.254 1.206 1.154 1.119 1.080 1.065 1.050 1.018 1 1 1 1 1 1 1 1 1 0.981 0.975 0.971 0.969 0.967 0.965 0.964 0.963 0.961 0.958 0.957 0.960
7 900 8 600 9 300 9 500 10 300 10 100 10 200 10 400 10 400 10 000 10 300 10 500 10 100 10 300 10 600 10 300 10 200 10 800 10 900 10 700 10 700 10 500 10 600 11 100 10 500 10 700 11 000 10 200 12 700
a Chromatographic conditions: 15% Isopropanol, 200 bar, 1.5 mL/ min. b N, number of theoretical plates for single enantiomer.
Figure 3. Plot of ln R vs 1/T for 4. Common chromatographic conditions: 15% 2-propanol; 200 bar pressure; 1.5 mL/min flow rate.
argues against the presence of a second binding site of the opposite elution order giving the perception of an entropically driven chiral separation. For this example at least, the assumption of a single retention mechanism appears to be valid. Validity of the Constant Heat Capacity Assumption. Equation 1 assumes that δ∆H° and δ∆S° do not change with temperature. It is readily apparent from the observation that retention increases dramatically near the critical temperature1 that Analytical Chemistry, Vol. 69, No. 7, April 1, 1997
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Figure 4. Effect of the nonspecific retention increase on ln R plots. Graphs on the left contain theoretical ln k′ vs 1/T plots with the retention increase (ln kn′ onspecific) superimposed to show the effect of the position of the isoelution temperature (dashed vertical line) relative to the retention increase on ln R plots. The nonspecific retention increase is incorporated into the theoretical ln R plots on the right. When the absolute value of a calculated ln R was less than 0.02 it was set equal to 0. In case A, the isoelution temperature is 45 °C and ln k′ at Tiso is 0.524. For case B, ln k′ at Tiso is kept at 0.524 and Tiso is changed to 13 °C. Tiso is changed by altering the y-intercepts of the ln k′ lines from case A. Slopes are retained from case A to maintain the same slope of a theoretical ln R plot. In case C, Tiso is changed to 130 °C.
∆H° changes with temperature. Chimowitz13-15 effectively modeled the retention increase by noting that ∆H° is composed of a ∆H°ads and a ∆H°sol. The ∆H°ads increases dramatically in this temperature region and results in the observed retention increase. To a first approximation, δ∆H° did not change traversing the critical temperature.1 Examination of Figure 3 suggests that this observation was misleading. The ln R ) 0 region persists well beyond what could be explained by the inability to differentiate (13) Kelley, F. D.; Chimowitz, E. H. AIChE. J. 1990, 36, 1163-1175. (14) Afrane, G.; Chimowitz, E. H. J. Supercrit. Fluids 1993, 6, 143-154. (15) Afrane, G.; Chimowitz, E. H. Fluid Phase Equilib. 1995, 111, 213-238.
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chromatographically low ln R values from zero. The cause of the eventual upturn is unclear. To evaluate the effect of the nonspecific retention increase on selectivity, linear regression was performed on the data from the five lowest temperatures in Table 3 using eq 4:
ln k′ ) ∆H°/RT + ∆S°/R + ln φ
(4)
This allows the estimation of ln k′ values at higher temperatures that would be expected if heat capacity were constant. The deviation between experimentally observed values and those
Table 4. Chromatographic Data Obtained with Hexane as Bulk Fluid temp (°C)
ln k′(R)
5 35 55 75 95 125
1.28 0.65 0.26 -0.05 -0.29 -0.54
ln k′(S) 10% 2-Propanol 1.28 0.62 0.20 -0.13 -0.38 -0.63
R
N
1.000 0.963 0.942 0.929 0.919 0.909
6300 11300 15000 16600 19600 18600
least-squares regression (R2 > 0.98) gave ln R: Tiso ) 2 °C; slope 89; y-intercept -0.326 5 20 35 55 75 95
1.88 1.55 1.16 0.87 0.56 0.35
5% 2-Propanol 1.84 1.47 1.07 0.76 0.43 0.21
0.955 0.927 0.909 0.889 0.878 0.876
4600 7800 10800 12800 14300 15200
least-squares regression (R2 > 0.98) gave ln R: Tiso ) 2 °C; slope 117; y-intercept -0.470
calculated by this equation were found to be the same for each enantiomer. Not surprisingly, addition of this deviation back to the regression calculated ln k′ values gave back the ln R plot shown in Figure 3 (with the stipulation that low values of ln R be misread as ln R ) 0). Isoelution temperatures vary between analytes and may depend upon the modifiers used. The position of the nonspecific retention increase is a property of the supercritical fluid.16-19 The position of the isoelution temperature relative to the retention increase may affect the shape of the ln R plots. The nonspecific retention increase generated from the data in Table 3 was used to generate additional ln R plots (Figure 4). In Table 3, Tiso is observed at 40 °C and a k′ of 2.12. This temperature is at the onset of the nonspecific retention increase. To evaluate the effect of a Tiso below the onset of the retention increase, regression equations for ln k′ were modified to give a Tiso of 13 °C with the same k′ of 2.12 at Tiso. Addition of the same nonselective retention increase to these modified equations gave a ln R plot with a very short flat portion followed by a breakthrough to the entropically driven region. With further increases in temperature ln R curved back toward ln R ) 0 where it again became flat. When regression equations for ln k′ were modified to give a Tiso in the midst of the retention increase (130 °C) with a k′ of 2.12 at Tiso ln R declined to ln R ) 0 and remained at zero. Incorporation of the nonselective retention increase into the thermodynamic equations will generate curves similar to all those observed. Use of a Different Bulk Fluid To Minimize Nonspecific Retention Increase. Clearly, the nonspecific retention increase associated with traversing the critical temperature interferes with the ability to observe and use entropically driven separations. The position of the increase is related to the critical temperature of the bulk supercritical fluid.16-19 The increase begins near the critical temperature and reaches a maximum above this temperature. Use of a bulk fluid with a critical temperature much higher (16) Novotny, M.; Bertsch, W.; Zlatkis, A. J. Chromatogr. 1971, 61, 17-28. (17) Leyendecker, D.; Schmitz, F. P.; Klesper, E. J. Chromatogr. 1984, 315, 1930. (18) Leyendecker, D.; Schmitz, F. P.; Leyendecker, D.; Klesper, E. J. Chromatogr. 1985, 321, 273-286. (19) Blackwell, J. A.; Schallinger, L. E. J. Microcolumn Sep. 1994, 6, 475-482.
Figure 5. Entropically driven chiral separations obtained with hexane as the bulk fluid. (A) Plot of ln k′ vs 1/T for 4. 2 and 1 represent ln k′ for the R and S enantiomers at 10% 2-propanol, respectively. 9 and b represent ln k′ for the R and S enantiomers at 5% 2-propanol, respectively. (B) Plot of ln R vs 1/T for 4. 9 represent data generated at 10% 2-propanol and b data generated at 5% 2-propanol.
Figure 6. Overlaid chromatograms of entropically driven chiral separations of 4 obtained at different temperatures, with hexane as the bulk fluid, containing 5% 2-propanol. Chromatograms are labeled above the peaks with the temperatures at which they were obtained.
than that of CO2 (Tc ) 31 °C, Pc ) 74 bar)8 should move the retention increase well away from the entropically driven temperature range. A very simple choice is hexane (Tc ) 234.5 °C, Pc ) 30bar)8. Equipment requirements for the use of hexane as the bulk fluid are limited to a pressure restrictor, required to prevent boiling, and a flow cell capable of withstanding this pressure. The Hewlett-Packard G 1205A SFC described above was used without modification although it was necessary to fully vent CO2 from the system. Compound 4 was chromatographed at 10 and 5% Analytical Chemistry, Vol. 69, No. 7, April 1, 1997
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Figure 7. Ttheoretical plate height as a function of flow rate at four temperatures. The separation is the same as shown in Figure 6: (9) 15 °C; (0) 35 °C; ([) ) 55 °C; (]) ) 75 °C.
2-propanol in hexane at 1.0 mL/min with a 100 bar back pressure. Data obtained are presented in Table 4 and in Figure 5. When hexane is used as the bulk fluid plots of ln k′ vs 1/T (Figure 5) do not show the retention increase that plagues CO2. (20) Antia, F. D.; Horvath, C. J. Chromatogr. 1988, 435, 1-15.
1420 Analytical Chemistry, Vol. 69, No. 7, April 1, 1997
As predicted by thermodynamic arguments, ln k′ declines smoothly with decreasing 1/T. Plots of ln R decrease steadily with decreasing 1/T, and the entropically driven separation of 4 is obtained. The isoelution temperature (2 °C) is considerably lower than that determined for 2-propanol in a CO2 system (60 °C). The cause of this difference is not clear at this time. Not only does selectivity increase with temperature, column efficiency does as well (Table 4). This combination, along with the decline in retention, gives rise to the dramatic entropically driven chiral separation shown in Figure 6. A separation at 5 °C that is unusable with a 25 min run time, at 75 °C becomes an excellent separation completed in 8 min. It is acknowledged that this is no longer supercritical fluid chromatography but high-temperature HPLC, which was theoretically examined by Antia and Horvath.20 These authors predicted increased column efficiency, lower pressure drops, shorter analysis times, and flatter van Deemter curves when high temperatures are used. A brief experiment where the separation shown in Figure 7 was repeated at flow rates between 0.5 and 3.5 mL/min at four temperatures demonstrated the validity of these predictions. The shortcoming of using higher temperatures in HPLC has always been the price that is paid in terms of selectivity. With entropically driven separations, this shortcoming is overcome. Received for review September 12, 1996. January 23, 1997.X AC9609283 X
Abstract published in Advance ACS Abstracts, March 1, 1997.
Accepted
