Effect of Moile-Phase Composition on Pressure-Induced Shifts in Solute Retention for LC Separations using -Cyclodextrin Stationary Phases Moira C. Ringo, Christine E. Evans Chemistry Department, Uni ersity of Michigan, Ann Aror, MI 48109-1055, USA Received 27 May 1997; accepted 25 Feruary 1998 Astract: Modest pressure Ž 350 ars. has a significant impact on reversed-phase liquid chromatographic retention using -cyclodextrin stationary phase. Demonstrated here for separations of the positional isomers of nitrophenol and naphthol as well as the enantiomers of hexoarital and mephoarital, the effect of small pressure perturations on solute capacity factor ranges from 11 to 14%. These changes in retention arise from pressure-induced shifts in solute equiliria and are used to estimate the change in molar volume upon solute inding with -cyclodextrin Ž V.. In this article, the effect of the moile phase on pressure-induced shifts in solute retention is examined. Moile-phase osition is oserved to have a significant influence on the change in solute capacity factor with pressure and therefore on V. As the osition of water in the moile phase increases, the change in partial molar volume of lexation is oserved to ecome more positive for oth positional isomer and chiral separations. Although these studies imply that changes in solvent osition have a general effect on the oserved V and therefore on the change in pressure perturation with solvent osition, it is clear that more studies are needed to estalish the exact nature and readth of this effect. 1998 John Wiley & Sons, Inc. J Micro Sep 10: 647 652, 1998 Key words: -cyclodextrin; liquid chromatography; chiral separations; enantiomer INTRODUCTION The extensive use of -cyclodextrin in chemical separations has een driven y its aility to exhiit highly selective interactions with a wide variety of ounds. The -cyclodextrin molecule, osed of seven glucoside units in a toroidal geometry, possesses a rigid inner cavity capale of selective -cyclodextrin to a spherical silica gel support using a 3-glycidoxylsilane spacer arm 6. Inclusion lexation of solute with -cyclodextrin is elieved to e the dominant retention mechanism of this stationary phase in the reversed-phase mode 5. Although the temperature dependence of cyclodextrin onded phase separations has een insize and shape discrimination 1. In addition, the vestigated 7 9, pressure has only recently een larger rim of the cyclodextrin cavity contains chiral carons capale of enantioselective interactions 2. This comination of inding capailities has led to examined as a variale for retention 10 12. Even as density and viscosity of polar moile phases do not change significantly in this pressure range 13, its utilization in liquid chromatographic stationary modest pressures may impact the retention of solutes through shifts in equilirium. In this article we phases 3 5. Although the methods used to faricate the phase vary, the stationary phase used in this examine the role of modest pressure Ž 350 ars. investigation is synthesized y chemically attaching Presented at the Nineteenth International Symposium on Capillary Chromatography and Electrophoresis, Wintergreen, VA, May 1997 Correspondence to: C. E. Evans Contract grant sponsor: National Science Foundation; contract grant numer: CHE-9707513 Contract grant sponsor: Eli Lilly Company on solute retention in -cyclodextrin onded phases in the reversed-phase mode. These studies focus on the effect of changes in moile-phase osition on these pressure perturations of retention. EXPERIMENTAL METHODS Chemicals. The - and -naphthol as well as Ž R, S. -hexoarital Ž5-1-cyclohexenyl -1,5 dimethyl- Ž. J. Microcolumn Separations, 108,647 652 1998 1998 John Wiley & Sons, Inc. 647 CCC 1040-7685 98 080647-06
648 arituric acid. and Ž R, S. -mephoarital Ž5-ethyl- 1-methyl-5-phenylarituric acid. standards were otained from Sigma Ž St. Louis, MO. while o-, m-, and p-nitrophenol standards were purchased from Aldrich Ž Milwaukee, WI.. Moile-phase solutions were prepared using distilled, deionized water Žmodel Milli-Q UV Plus, Millipore, Bedford, MA. and high-purity methanol ŽBaxter Healthcare, Muskegon, MI.. For the enantiomeric separations, the moile phases were uffered with high-purity acetic acid Ž Aldrich. and high-performance liquid chromatography Ž HPLC. grade triethylamine Ž TEA. ŽFisher, Fair Lawn, NJ. with ph reported as the apparent ph of the hydroorganic moile phase measured using a standard ph meter. All chemicals were used without further purification. Chromatography. A high-pressure syringe pump Ž model 260D, Isco, Lincoln, NE. equipped with a high-precision pressure transducer Ž 1 psi. was used to deliver the moile phase. The split-injection technique Ž split ratio 81 8; V 12 nl. inj was em- ployed with a 1- L internal volume injection valve Ž Valco Instruments, Houston, TX. to introduce analyte solutions Ž0.7 1.6 mm, dissolved in moile phase. onto the column. The analytes were detected utilizing an asorance detector Žmodel UVIS-205, Linear, Chicago, IL. fitted with a high-pressure capillary flow cell Ž76 m i.d. and 357 m o.d.; Polymicro Technologies, Phoenix, AZ.. The positional isomer and enantiomer studies each used single, packed capillary columns containing -cyclodextrin onded stationary phase ŽCyclo- ond I 2000, Advanced Separations Technology, Whippany, NJ.. These chromatographic columns were faricated using fused-silica capillary Ž251 m i.d. and 360 m o.d.; Polymicro Technologies, Phoenix, AZ. packed with a slurry of stationary phase and 80:20 Ž v v. acetone 20 mm aqueous NH NO under moderate pressure Ž 380 ars. 4 3. Both columns were terminated using a quartz wool frit at a final column length of 40.4 cm for the positional isomer studies and 37.5 cm for the enantiomer studies. The liquid chromatographic apparatus used in these studies maintains a constant column flow rate while varying the average pressure Ž P. av on the column. With no added restrictor at the column inlet or outlet, the average column pressure arises solely from the pressure necessary to create moilephase flow through the column. Therefore, the average column pressure may e approximated as simply one-half of the column inlet pressure since the column outlet is at atmospheric pressure. If restrictors producing equivalent ack pressures are added at the inlet and outlet, the column inlet and outlet pressures are increased to the same extent. Al- Ringo and Evans though the average pressure on the column is increased y the magnitude of the pressure change generated y the added restriction, the column flow rate and pressure gradient remain constant. In this way, the column flow rate was maintained at 1.5 0.04 L min throughout these studies, resulting in a column pressure gradient of 100 ars for 50 : 50 Ž v v. methanol water, 84.2 ars for 20 : 80 Ž v v. methanol water, 129 ars for 40 : 60 : 0.9 : 0.17 Ž v v v v. methanol water acetic acid TEA, and 104 ars for 30 : 70 : 0.9 : 0.17 Ž v v v v. methanol water acetic acid TEA moile phase. No correlation etween pressure and flow rate was oserved in these studies, yielding a relative standard deviation in the flow rate of less than 3% ased on replicate void time measurements. Solute retention reversiility after high pressure was evaluated y direct arison of capacity factors at low pressure after high-pressure perturation. RESULTS AND DISCUSSION Solute retention in liquid chromatographic separations has een shown to e well descried y equilirium thermodynamics. Although the role of temperature in separations has een widely examined, the other fundamental parameter in equilirium thermodynamics, pressure, has een long overlooked. The pressure dependence of the equilirium distriution coefficient of solute etween the stationary phase and the moile phase, K dist, can e derived from the change in Gis free energy for solute inding with the stationary phase Ž G.: Gdist ln Kdist ž / RT P ž P / T T dist nrt V S 1 where P is pressure and T is temperature. Since the isothermal change with pressure in G for an equilirium reaction is directly related to the change in partial molar volume Ž V. for that reaction, pressure-induced changes in the distriution coefficient may e directly related to V upon solute inding with the stationary phase. As Equation Ž. 1 indicates, the change in the equilirium distriution coefficient, K dist, with pressure is mediated y the nrt S term, which corrects for changes in molar concentration with pressure due to ression of ulk solvent. The magnitude of this term is therefore related to solvent isothermal ressiility Ž. S as well as the difference in the stoichiometric coefficients of products from reactants Ž n.. For the separations descried here, the ressiilities are 3.8, 3.9, 4.0, 5 1 and 4.2 10 ar for 20, 30, 40, and 50% Ž v v. methanol water solutions, respectively 14.
Effect of Moile-Phase Composition 649 In liquid chromatographic separations using cyclodextrin ound to a stationary support, the predominant solute retention mechanism is -cyclodextrin solute lexation 3 5. For this reason, the equilirium distriution coefficient of the solute etween the stationary phase and moile phase Ž K. dist is directly related to the fundamental equili- rium lexation constant Ž K. for -cyclo- dextrin lexation with the solute. This equilirium lexation constant can therefore e determined y measurement of the chromatographic capacity factor, k, the ratio of volumes of the stationary and moile phases Ž., and CD, which is the equilirium concentration of unound cyclodextrin: Kdist k K Ž 2. CD CD Therefore, sustitution of Equation Ž. 2 into the pressure dependence of Kdist descried in Equation Ž. 1 permits estimation of the change in partial molar volume of lexation directly from the measured change in capacity factor Ž k. with pressure, assuming that and CD do not change significantly with pressure: ž / ln k RT nrt V Ž 3. S P T For the modest pressure conditions encountered in liquid chromatography, the partial differential in Equation Ž. 1 can e accurately represented as a simple difference. The change in molar volume upon lexation Ž V. determined y Equation Ž 3. represents the difference in partial molar volumes of the products and reactants involved in the lexation interaction in their solvated states. Therefore, V ac- counts not only for differences in the partial molar volumes of the individual molecules ut for their solvation volumes as well. The oserved V may e viewed as the sum of partial molar volume changes for three distinct processes involved in -cyclodextrin solute lexation in liquid solution 15 : V V V V 4 desolv, solute desolv, CD inclus The first two terms of this expression account for the change in molar volume upon desolvation of that portion of the molecule involved in the inding interaction. Since the desolvation process generally leads to a loss of solvent ordering around the solute, these terms are expected to e positive Ž V desolv, solute 0; V 0.. The final term accounts for desolv, CD mutual solvation of the solute and cyclodextrin that occurs upon inclusion and generally results in a decrease in partial molar volume Ž V 0. inclus. Al- though modeling studies indicate the possile conformational flexiility of -cyclodextrin 16, 17, neutron diffraction measurements in the solvated state show that no significant change in structure is oserved 18 For this reason, it is assumed that no significant conformational changes occur upon lexation. As a result, the magnitude of the desolvation terms in Equation Ž. 4 relative to the inclusion term determines the direction and magnitude of the overall oserved change in solvated molar volume upon lexation. That is, if the desolvation terms are predominant Ž V 0., an increase in pres- sure is expected to yield a decrease in solute retention, and vice versa if the inclusion term is dominant Ž V 0.. In these studies, we focus on the influence of moile-phase osition on the pressure dependence of solute retention for liquid chromatographic separations using -cyclodextrin onded phase in the reversed-phase mode. Pressure has een isolated as the only perturation parameter y maintaining a constant column flow rate while controlling the average pressure on the chromatographic column. The packed capillary columns utilized for all measurements ensure effective temperature control under amient conditions Ž T 23 1 C.. Utilizing this approach, the oserved changes in capacity factor with pressure are expected to arise solely from shifts in the equilirium distriution of solute etween the moile phase and -cyclodextrin moieties in the stationary phase. Moreover, since this study employs positional isomers and enantiomers as proes, the effect of pressure on oth shape-selective and enantioselective separations is investigated under conditions commonly encountered y chromatographers. Pressure effects on positional isomer retention. As indicated in Tale I, modest pressure has a significant impact on capacity factor for solutes separated in the reversed-phase mode with -cyclodextrin stationary phase. For the nitrophenol separations, decreases in capacity factor up to 14% were oserved with a 280-ar increase in pressure. In contrast, the naphthol capacity factors increased 2 3% or did not change with pressure. The significance of these pressure-induced changes in capacity factor is highlighted y including the propagated error for triplicate measurements in Tales I and II. Not all solutes exhiit statistically significant changes in solute retention with pressure. Indeed, these positional isomers display the full range of pressure dependence, yielding either an increase, a decrease, or no change in solute capacity factors with pressure.
650 Ringo and Evans Tale I. Effect of pressure on capacity factor k for re ersed-phase separations of the positional isomers of nitrophenol and naphthol on -cyclodextrin onded phase a o-nitrophenol m-nitrophenol p-nitrophenol -Naphthol -Naphthol Moile phase 50 : 50 methanol water Pav 55.2 0.585 0.309 1.528 0.498 0.362 ars Ž 0.0052. Ž 0.0017. Ž 0.0083. Ž 0.0021. Ž 0.0055. Pav 323 0.573 0.301 1.45 0.513 0.370 ars Ž 0.0092. Ž 0.0066. Ž 0.011. Ž 0.0038. Ž 0.0038. Percent change in 2 3 5.1 3.0 2 k Ž 1.8. Ž 2.2. Ž 0.9. Ž 0.9. Ž 1.8. V not significant not significant 3.7 4.2 3 Ž 0.86. Ž 0.89. Ž 1.9. Moile phase 20 : 80 Ž. methanol water Pav 42.1 1.16 0.99 3.34 5.14 3.47 Ž ars. Ž 0.018. Ž 0.022. Ž 0.036. Ž 0.053. Ž 0.055. Pav 318 1.07 0.89 2.876 5.13 3.43 Ž ars. Ž 0.015. Ž 0.016. Ž 0.0094. Ž 0.050. Ž 0.031. Percent change in 8 10 14 not significant not significant k Ž 2.0. Ž 2.8. Ž 1.1. V 6 9 12 not significant not significant Ž 1.9. Ž 2.5. Ž 1.0. a The error shown in parentheses for all capacity factor measurements is the standard deviation of 3 replicate measurements while the error for % change in k and for V is the propagated error. Nitrophenol data have een excerpted from ref. 11. The naphthol separations with 50 : 50 Ž v v. methanol water moile phase were performed in a separate assay for which the average column pressures were 49.0 and 285 ars, respectively. Tale II. Effect of pressure on capacity factor k for re ersed-phase chiral separations of hexoarital and mephoarital on -cyclodextrin onded phase a Hexoarital 1 Hexoarital 2 Mephoarital 1 Mephoarital 2 Moile phase 40 : 60 : 0.9 : 0.17 Ž. methanol water acetic acid TEA, ph 4.2 Pav 64.3 0.917 1.051 0.670 0.755 Ž ars. Ž 0.0039. Ž 0.0034. Ž 0.0007. Ž 0.0014. Pav 306 1.02 1.17 0.742 0.829 Ž ars. Ž 0.013. Ž 0.011. Ž 0.0062. Ž 0.0092. Percent change in 11 11 10.7 10 k Ž 1.5. Ž 1.1. Ž 0.9. Ž 1.2. V 12 12 11.5 10 Ž 1.4. Ž 1.1. Ž 0.9. Ž 1.2. Moile phase 30 : 70 : 0.9 : 0.17 Ž. methanol water acetic acid TEA, ph 4.0 Pav 52.2 2.46 2.84 1.97 2.24 Ž ars. Ž 0.048. Ž 0.068. Ž 0.017. Ž 0.021. Pav 307 2.60 3.07 2.07 2.38 Ž ars. Ž 0.043. Ž 0.015. Ž 0.017. Ž 0.024. Percent change in 6 8 5 6 k Ž 2.6. Ž 2.5. Ž 1.2. Ž 1.4. V 6 8 6 6 Ž 2.5. Ž 2.4. Ž 1.2. Ž 1.3. a The error shown in parentheses for all capacity factor measurements is the standard deviation of 3 replicate measurements while the error for % change in k and for V is the propagated error. The retention data for this moile phase have een excerpted in part from ref. 12.
Effect of Moile-Phase Composition 651 Moreover, these pressure-induced perturations in capacity factor are shown to e dependent on moile-phase osition. These moile-phase effects may e understood more clearly ased on changes in molar volume upon lexation Ž V. determined from the pressure dependence of solute capacity factor Equation Ž. 3. For these positional isomers, chromatographic retention measurements at different pressures indicate a more positive change in molar volume upon lexation with increasing water content in the moile phase Ž Tale I.. The nitrophenol isomers exhiit a negligile or slightly positive V in the 50 : 50 Ž v v. methanol water moile phase, with the V e- coming more positive upon increasing the water content of the moile phase to 20 : 80 Ž v v. methanol water. In contrast, the naphthol isomers show a negative V when separated using a 50% Ž v v. aqueous moile phase yet also exhiit a more positive V when the water content of the mo- ile phase is increased to 80% Ž v v.. Thus, regardless of whether V is positive or negative, an increase in water content over this range results in a more positive overall V. Demonstrated here for two methanol water moile phases, this dependence on solvent osition may not e consistent over the entire range of mixtures. Accordingly, further studies are needed to elucidate the exact dependence of the overall V on solvent osition. Since changes in the ulk ressiility of the moile phase with changing osition are already included in estimation of V from Equation Ž. 3, the oserved changes in V with moile-phase osition may e evaluated ased on expected contriutions to the partial molar volume during the lexation process. Based on Equation Ž. 4, shifts in the magnitude and sign of the change in molar volume upon lexation must arise from changes in the relative contriutions of desolvation and inclusion in the overall V. In order for more positive V to e oserved with increasing water content, the Vdesolv, solute and Vdesolv, CD contriu- tions arising from desolvation of the solute and cyclodextrin must ecome more positive and or the Vinclus contriution from the inclusion step must ecome less negative with increased water content of the moile phase. Unfortunately, these determinations yield the overall changes in molar volume upon lexation and do not allow isolation of the individual contriutions to the lexation process. Nonetheless, the osition of the moile phase has a significant effect on the pressure dependence of solute retention of positional isomers. Pressure effects on enantioselecti e retention. In addition to the separation of positional isomers, -cyclodextrin onded phase is also commonly employed for chiral separations in the reversed-phase mode. As shown in Tale II, modest pressure is shown to significantly affect enantioselective retention as well. For this study, the retention properties of the structurally similar sedatives hexoarital and mephoarital have een examined: Hexoarital CH Mephoarital CH 3 3 O N O O HC 3 HC NH 3 O N O NH Both enantiomers of hexoarital and mephoarital exhiit increases in capacity factor which range from 5 to 11% with an increase in pressure. In every case, these capacity factor shifts were used to estimate decreases in molar volume upon lexation Ž V. that were similar in magnitude for enantiomeric pairs. These pressure-induced perturations in enantiomeric capacity factor are also dependent on moile-phase osition. Consistent with the positional isomer separations, the enantioselective retention measurements at different pressures shown in Tale II indicate a change to more positive molar volumes Ž V. with increasing water content in the moile phase. Again, this trend indicates that the Vdesolv, solute and Vdesolv, CD contriutions aris- ing from desolvation ecome more positive and or the Vinclus contriution ecomes less negative with increased water content of the moile phase. These pressure-induced shifts in enantioselective retention y -cyclodextrin onded phase have important implications for pressure-induced changes in chiral resolution 12 for this challenging class of separations. At this point it is tempting to correlate the oserved changes in V with solvent osi- tion directly to interaction energetics. However, a greater change in the Gis free energy of interaction Ž G. and concomitant increase in solute capacity factor does not infer a greater change in solvated molar volume upon interaction Ž V..It is the change in G with pressure that gives rise to V Equation Ž. 2. Demonstration of the lack of a relationship etween the magnitudes of G and V can e seen in aring the nitrophenol and naph- O
652 thol capacity factors in the 20 : 80 Ž v v. methanol water moile phase Ž Tale I.. Although the naphthols exhiit larger capacity factors Ž higher G., the V upon lexation is significantly less for the naphthols than for the nitrophenols. However, the exact nature of the solvent-mediating effect on pressure-dependent retention remains unclear. Studies are presently underway to assess this pressure dependence over the full range of methanol water ositions as well as the impact for acetonitrileased moile phases. ACKNOWLEDGMENT The kind gift of the -cyclodextrin stationary phase from the Advanced Separations Technology Corp. is gratefully acknowledged. REFERENCES 1. M. L. Bender and M. Komiyama, Cyclodextrin Chemistry Ž Springer-Verlag, Berlin, 1978.. 2. W. L. Hinze, Sep. Purif. Methods 10, 159 Ž 1981.. 3. K. Fujimura, T. Ueda, and T. Ando, Anal. Chem. 55, 446 Ž 1983.. 4. Y. Kawaguchi, M. Tanaka, M. Nakae, K. Funazo, and T. Shono, Anal. Chem. 55, 1852 Ž 1983.. Ringo and Evans 5. D. W. Armstrong and W. DeMond, J. Chromatogr. Sci. 22, 411 Ž 1984.. 6. D. W. Armstrong, U.S. Patent 4,539,999, 1985. 7. M. Tanaka, Y. Kawaguchi, M. Nakae, Y. Mizouchi, and T. Shono, J. Chromatogr. 299, 341 Ž 1984.. 8. M. D. Beeson and G. Vigh, J. Chromatogr. 634, 197 Ž 1993.. 9. K. Carera and D. Luda, J. Chromatogr. 666, 433 Ž 1994.. 10. M. C. Ringo and C. E. Evans, J. Phys. Chem. B 101, 5525 Ž 1997.. 11. M. C. Ringo and C. E. Evans, Anal. Chem. 69, 643 Ž 1997.. 12. M. C. Ringo and C. E. Evans, Anal. Chem. 69, 4964 Ž 1997.. 13. M. Martin, G. Blu, and G. Guiochon, J. Chromatogr. Sci. 11, 641 Ž 1973.. 14. R. E. Gison, J. Am. Chem. Soc. 57, 1551 Ž 1935.. 15. Y. Sueishi, N. Nishimura, K. Hirata, and K. Kuwata, J. Phys. Chem. 95, 5359 Ž 1991.. 16. K. B. Lipkowitz, J. Org. Chem. 56, 6357 Ž 1991.. 17. B. Mayer and G. Kohler, THEOCHEM 363, 217 Ž 1996.. 18. C. Betzel, W. Saenger, B. E. Hingerty, and G. M. Brown, J. Am. Chem. Soc. 106, 7545 Ž 1984..